U.S. patent application number 11/513079 was filed with the patent office on 2007-08-02 for fhit proteins and nucleic acids and methods based thereon.
Invention is credited to Carlo M. Croce, Frances Kay Huebner.
Application Number | 20070178105 11/513079 |
Document ID | / |
Family ID | 27083164 |
Filed Date | 2007-08-02 |
United States Patent
Application |
20070178105 |
Kind Code |
A1 |
Croce; Carlo M. ; et
al. |
August 2, 2007 |
FHIT proteins and nucleic acids and methods based thereon
Abstract
The present invention relates to nucleotide sequences of FHIT
genes and amino acid sequences of their encoded proteins, as well
as derivatives and analogs thereof, and antibodies thereto. The
FHIT gene sequence is mutated in diseases involving cell
overproliferation, particularly malignancies of the digestive
tract. The present invention further relates to the use of FHIT
genes and their encoded proteins as diagnostic and therapeutic
reagents for the detection and treatment of disease states
associated with cell overproliferation.
Inventors: |
Croce; Carlo M.;
(Philadelphia, PA) ; Huebner; Frances Kay;
(Philadelphia, PA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Family ID: |
27083164 |
Appl. No.: |
11/513079 |
Filed: |
August 30, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10889934 |
Jul 13, 2004 |
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11513079 |
Aug 30, 2006 |
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09717054 |
Nov 21, 2000 |
6774217 |
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10889934 |
Jul 13, 2004 |
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08605430 |
Feb 22, 1996 |
6242212 |
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09717054 |
Nov 21, 2000 |
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08598873 |
Feb 9, 1996 |
5928884 |
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08605430 |
Feb 22, 1996 |
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Current U.S.
Class: |
424/155.1 ;
530/388.8; 530/391.1 |
Current CPC
Class: |
C07K 14/4703 20130101;
A61P 11/00 20180101; A61P 13/12 20180101; A61P 35/00 20180101; A61P
15/00 20180101; A61P 13/00 20180101; A61K 38/00 20130101; C07K
16/18 20130101; A61P 19/00 20180101; C07K 2319/00 20130101; A61P
1/00 20180101 |
Class at
Publication: |
424/155.1 ;
530/388.8; 530/391.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/30 20060101 C07K016/30 |
Goverment Interests
[0002] This invention was made in part with government support
under Grant numbers CA51083, CA39860, CA21124, and CA56336 awarded
by the National Cancer Institute. The government has certain rights
in the invention.
Claims
1-21. (canceled)
22. An isolated antibody that binds to at least one member selected
from the group consisting of SEQ ID NO:2 and a protein having at
least 60% identity to SEQ ID NO:2.
23. The antibody of claim 22, wherein the antibody binds to SEQ ID
NO:2.
24. The antibody of claim 22, wherein the antibody binds to the
protein having at least 60% identity to SEQ ID NO:2.
25. The antibody of claim 22, wherein the antibody binds to a
protein selected from the group consisting of a protein having at
least 70% identity to SEQ ID NO:2, a protein having at least 80%
identity to SEQ ID NO:2, a protein having at least 90% identity to
SEQ ID NO:2 and a protein having at least 95% identity to SEQ ID
NO:2.
26. The antibody of claim 22, wherein the antibody includes a
monoclonal antibody.
27. The antibody of claim 22, wherein the antibody includes a
polyclonal antibody.
28. The antibody of claim 22, wherein the antibody includes a
chimeric antibody.
29. The antibody of claim 22, wherein the antibody includes a human
antibody.
30. The antibody of claim 22, wherein the antibody includes a
single chain antibody.
31. The antibody of claim 22, wherein the antibody includes an
antibody fragment.
32. The antibody of claim 28, wherein the antibody fragment
includes an Fab fragment.
33. The antibody of claim 28, wherein the antibody fragment
includes an Fab' fragment.
34. The antibody of claim 28, wherein the antibody fragment
includes an F(ab').sub.2 fragment.
35. The antibody of claim 22, further including a detectable
label.
36. The antibody of claim 35, wherein the detectable label is
selected from the group consisting of an enzyme, a radiolabel, a
fluorescent molecule, a chemiluminescent molecule, a cofactor, an
enzyme substrate, an enzyme inhibitor, and a magnetic particle.
37. A kit comprising an isolated antibody that binds to at least
one member selected from the group consisting of SEQ ID NO:2 and a
protein having at least 60% identity to SEQ ID NO:2.
38. The kit of claim 37, wherein the antibody binds to SEQ ID
NO:2.
39. The kit of claim 37, wherein the antibody binds to the protein
having at least 60% identity to SEQ ID NO:2.
40. The kit of claim 37, wherein the antibody binds to a protein
selected from the group consisting of a protein having at least 70%
identity to SEQ ID NO:2, a protein having at least 80% identity to
SEQ ID NO:2, a protein having at least 90% identity to SEQ ID NO:2
and a protein having at least 95% identity to SEQ ID NO:2.
41. The kit of claim 37, further including a labeled binding
partner to the isolated antibody.
42. The kit of claim 37, wherein the antibody further includes a
detectable label.
Description
[0001] This is a continuation of U.S. patent application Ser. No.
09/717,054, filed Nov. 21, 2000, which is a continuation of U.S.
patent application Ser. No. 08/605,430, filed Feb. 22, 1996, which
issued as U.S. Pat. No. 6,242,212 on Jun. 5, 2001, which is a
continuation-in-part of U.S. patent application Ser. No.
08/598,873, filed Feb. 9, 1996, which issued as U.S. Pat. No.
5,928,884 on Jul. 27, 1999, the entire disclosure of which is
incorporated herein by reference.
1. INTRODUCTION
[0003] The present invention relates to nucleotide sequences of the
tumor suppressor FHIT genes and amino acid sequences of their
encoded proteins, as well as derivatives and analogs thereof and
antibodies thereto. The present invention relates to the use of
nucleotide sequences of FHIT genes and amino acid sequences of
their encoded proteins, as well as derivatives and analogs thereof
and antibodies thereto, as diagnostic and therapeutic reagents for
the detection and treatment of cancer. The present invention also
relates to therapeutic compositions comprising Fhit proteins,
derivatives or analogs thereof, antibodies thereto, nucleic acids
encoding the Fhit proteins, derivatives or analogs, and FHIT
antisense nucleic acids.
2. BACKGROUND OF THE INVENTION
[0004] Cancer remains one of the most severe health problems in
America, accounting for substantial fatality and health costs in
society. Tumorigenesis in humans is a complex process involving
activation of oncogenes and inactivation of tumor suppressor genes
(Bishop, 1991, Cell 64:235-248). Tumor suppressor genes in humans
have been identified through studies of genetic changes occurring
in cancer cells (Ponder, 1990, Trends Genet. 6:213-218; Weinberg,
1991, Science 254:1138-1146).
[0005] There is a close association between particular chromosomal
abnormalities, e.g., chromosomal translocations, inversions, and
deletions, and certain types of malignancy, indicating that such
abnormalities may have a causative role in the cancer process.
Chromosomal abnormalities may lead to gene fusion resulting in
chimeric oncoproteins, such as is observed in the majority of the
tumors involving the myeloid lineage. Alternatively, chromosomal
abnormalities may lead to deregulation of protooncogenes by their
juxtaposition to a regulatory element active in the hematopoietic
cells, such as is observed in the translocation occurring in the
lymphocytic lineage (Virgilio et al., 1993, Proc. Natl. Acad. Sci.
USA 90:9275-9279). Deletions may cause loss of tumor suppressor
genes, leading to malignancy.
[0006] Nonrandom chromosomal translocations are characteristic of
most human hematopoietic malignancies (Haluska et al., 1987, Ann.
Rev. Genet. 21:321-345) and may be involved in some solid tumors
(Croce, 1987, Cell 49:155-156). In B and T cells, chromosomal
translocations and inversions often occur as a consequence of
mistakes during the normal process of recombination of the genes
for immunoglobulins (Ig) or T-cell receptors (TCR). These
rearrangements juxtapose enhancer elements of the Ig or TCR genes
to oncogenes whose expression is then deregulated (Croce, 1987,
Cell 49:155-156). In the majority of the cases, the rearrangements
observed in lymphoid malignancies occur between two different
chromosomes.
[0007] The TCL-1 locus on chromosome 14 band q32.1 is frequently
involved in the chromosomal translocations and inversions with the
T-cell receptor genes observed in several post-thymic types of
T-cell leukemias and lymphomas, including T-prolymphocytic
leukemias (T-PLL) (Brito-Babapulle and Catovsky, 1991, Cancer
Genet. Cytogenet. 55:1-9), acute and chronic leukemias associated
with the immunodeficiency syndrome ataxia-telangiectasia (AT)
(Russo et al., 1988, Cell 53:137-144; Russo et al., 1989, Proc.
Natl. Acad. Sci. USA 86:602-606), and adult T-cell leukemia
(Virgilio et al., 1993, Proc. Natl. Acad. Sci. USA
90:9275-9279).
[0008] In 1979, a large Italian-American family in Boston was
observed to be transmitting a constitutional reciprocal
t(3;8)(p14.2;q24) chromosome translocation (Cohen et al., 1979, N.
Engl. J. Med. 301:592-595; Wang and Perkins, 1984, Cancer Genet.
Cytogenet. 11:479-481) which segregated in the family with early
onset, bilateral and multifocal clear cell renal carcinoma (RCC).
Follow-up cytogenetic studies in several familial tumors
demonstrated that the tumors had lost the derivative 8 chromosome
carrying the translocated 3p14-pter region; consequently, the
tumors were homozygous for all loci telomeric to the 3p14.2 break
(Li et al., 1993, Annals of Internal Medicine 118:106-111). It was
suggested that the translocation affects expression of a tumor
suppressor gene (Cohen et al., 1979, N. Engl. J. Med. 301:592-595)
and several investigators have sought candidate suppressor genes.
We had suggested the protein tyrosine phosphatase gamma gene
(PTPRG) as a candidate tumor suppressor gene (LaForgia et al.,
1991, Proc. Natl. Acad. Sci. USA 88:5036-5040), and that the
majority of clear cell RCCs exhibit loss of heterozygosity of a 0.5
Mb region flanking the translocation (Lubinski et al., 1994, Cancer
Res. 54:3710-3713; Druck et al., 1995, Cancer Res. 55:5348-5355),
although we did not observe aberrations in the remaining PTPRG
gene. The 3p14.2 region is also included in deletions in numerous
other tumor types, including nasopharyngeal carcinomas (Lo et al.,
1994, Int. J. Oncol. 4:1359-1364).
[0009] The t(3;8) translocation breakpoint was cloned and a 3 kb
transcript of a candidate tumor suppressor gene was detected using
a probe from near the breakpoint (Boldog et al., 1993, Proc. Natl.
Acad. Sci. USA 90:8509-8513); further details concerning this
transcript have not been reported in spite of a later publication
from this group relating to this subject, and reporting a YAC
contig of approximately 6 Mb DNA spanning the 3p14.2 3;8
translocation breakpoint (Boldog et al., 1994, Genes, Chromosomes
& Cancer 11:216-221). It has also been suggested that there may
not be a suppressor gene at 3p14.2, that in fact the t(3;8)
translocation was a mechanism for losing the von Hippel-Lindau
gene, a tumor suppressor gene at 3p25 (Gnarra et al., 1994, Nature
Genet. 7:85-90).
[0010] Another cytogenetic landmark in chromosome region 3p14.2 is
the most common of the constitutive aphidicolin inducible fragile
sites, FRA3B, which is cytogenetically indistinguishable from the
t(3;8) translocation (Glover et al., 1988, Cancer Genet. Cytogenet.
31:69-73). Fragile sites, of which over 100 have been described in
human (for review, see Sutherland, 1991, Genet. Anal. Tech. Appl.
8:1616-166), are regions of the human genome which reveal
cytogenetically detectable gaps when exposed to specific reagents
or culture conditions; several folate sensitive, heritable,
X-linked and autosomal fragile sites have been localized to
unstable CCG or CGG repeats (Yu et al., 1991, Science
252:1179-1181; Kremer et al., 1991, Science 252, 1711-1714; Verkerk
et al., 1991, Cell 65:905-914; Fu et al., 1991, Cell 67:1047-1058),
and for one of these, the FRA11B at 11q23.3, the CCG repeat is
within the 5' untranslated region of the CBL2 gene, a known
protooncogene (Jones et al., 1995, Nature 376:145-149). Also this
fragile site, FRA11B, is associated with Jacobsen (11q-) syndrome,
showing a direct link between a fragile site and in vivo chromosome
breakage (Jones et al., 1994, Hum. Mol. Genet. 3:2123-2130).
Because the induced fragile sites resemble gaps or breaks in
chromosomes, it has frequently been speculated that fragile sites
could be sites of chromosomal rearrangement in cancer (Yunis and
Soreng, 1984, Science 226:1199-1204). Previously identified fragile
sites have also been shown to be hypermethylated (Knight et al.,
1993, Cell 74:127-134); thus methylation of a fragile site in a
tumor suppressor gene regulatory region might cause loss of
transcription of the suppressor gene, serving as one "hit" in the
tumorigenic process, as pointed out previously (Jones et al., 1995,
Nature 376:145-149). These authors also suggested that an important
contribution of fragile site expression in tumorigenesis might be
to increase the incidence of chromosome deletion during
tumorigenesis.
[0011] The FRA3B region has been delineated by studies of several
groups using rodent-human hybrids; hybrid cells retaining human
chromosome 3 or 3 and X, on a hamster background, were treated with
aphidicolin or 6-thioguanine (to select hybrids which had lost the
X chromosome) and subclones selected. Subclones retaining portions
of chromosome 3 with apparent breaks in region 3p14-p21 were
characterized for loss or retention of specific 3p markers to
determine the position of 3p14-21 breaks (LaForgia et al., 1991,
Proc. Natl. Acad. Sci. USA 88:5036-5040, LaForgia et al., 1993,
Cancer Res. 53:3118-3124; Paradee et al., 1995, Genomics
27:358-361).
[0012] Alterations in oncogenes and tumor suppressor genes in small
cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) have
been described, the most frequent target being alterations of p53
(Takahashi et al., 1989, Science 246:491-494; Chiba et al., 1990,
Oncogene 5:1603-1610; Mitsudomi et al., 1992, Oncogene 7:171-180)
and retinoblastoma (Harbour et al., 1988, Science 241:353-357; Xu
et al., 1994, J. Natl. Cancer Inst. 86:695-699) genes and allelic
deletions of the short arm of chromosome 3 (3p) (Kok et al., 1987,
Nature 330:578-581; Naylor et al., 1987, Nature 329:451-454;
Rabbitts et al., 1989, Genes Chrom. Cancer 1:95-105). In addition
to cytogenetically visible deletions (Whang-Peng et al., 1982,
Science 215:181-182; Testa et al., 1994, Genes Chrom. Cancer
11:178-194), loss of heterozygosity (LOH) at loci on 3p has been
reported in nearly 100% of SCLC (Kok et al., 1987, Nature
330:578-581; Naylor et al., 1987, Nature 329:451-454; Brauch et
al., 1987, N. Engl. J. Med. 317:1109-1113; Yokota et al., 1987,
Proc. Natl. Acad. Sci. USA. 84:9252-9256) and in 50% or more of
NSCLC (Brauch et al., 1987, N. Engl. J. Med. 317:1109-1113; Yokota
et al., 1987, Proc. Natl. Acad. Sci. USA. 84:9252-9256; Rabbitts et
al., 1990, Genes Chrom. Cancer 2:231-238; Hibi et al., 1992,
Oncogene 7:445-449; Yokoyama et al., 1992, Cancer Res. 52:873-877;
Horio et al., 1993, Cancer Res. 53:1-4), strongly suggesting the
presence of at least one tumor suppressor gene in this chromosomal
region.
[0013] However, the observation that allelic losses often involve
most of the 3p has hampered the isolation of the involved gene(s).
Candidate loci have been identified such as the von-Hippel Lindau
gene, located at 3p25, which was subsequently found to be rarely
mutated in lung cancer cell lines (Sekido et al., 1994, Oncogene
9:1599-1604). Other loci located in a region within 3p21 were
reported to be sites of recurrent homozygous deletions in SCLC
(Daly et al., 1993, Oncogene 8:1721-1729; Kok et al., 1993, Proc.
Natl. Acad. Sci. USA 90:6071-6075; Kok et al., 1994, Cancer Res.
54:4183-4187). In addition, transfer of subchromosomal fragments of
the region 3p21.3-p21.2 to tumor cell lines has suggested tumor
suppressor activity (Killary et al., 1992, Proc. Natl. Acad. Sci.
USA 89:10877-10881; Daly et al., 1993, Oncogene 8:1721-1729). More
proximal deletions in the 3p12-14 region have also been reported
(Rabbitts et al., 1989, Genes Chrom. Cancer 1:95-105; Rabbitts et
al., 1990, Genes Chrom. Cancer 2:231-238; Daly et al., 1991,
Genomics 9:113-119).
[0014] Lung cancer is a major cause of mortality worldwide and the
overall survival rate has not improved significantly in the last 20
years. Despite the success achieved by primary prevention, lung
cancer is still an overwhelming medical and social problem. Even in
the cohort of ex-smokers lung cancer incidence remains high for
several years, as a consequence of the accumulated damage, and
there is an objective need for strategies aimed at reducing cancer
mortality in individuals who have stopped smoking.
[0015] There remains an unfulfilled need to isolate and
characterize the genes associated with digestive tract and other
cancers for use as a diagnostic and therapeutic/prophylactic
reagent in the detection, treatment, and prevention of such
cancers.
[0016] Citation of a reference hereinabove shall not be construed
as an admission that such reference is prior art to the present
invention.
3. SUMMARY OF THE INVENTION
[0017] The present invention relates to nucleotide sequences of
PHIT genes, and amino acid sequences of their encoded FHIT
proteins, as well as derivatives (e.g., fragments) and analogs
thereof, and antibodies thereto. The present invention further
relates to nucleic acids hybridizable to or complementary to the
foregoing nucleotide sequences as well as equivalent nucleic acid
sequences encoding a FHIT protein. In a specific embodiment, the
FHIT genes and proteins are human genes and proteins.
[0018] Mutations (in particular, deletions) of FHIT gene sequences
are associated with esophageal, gastric, colon, kidney, and other
cancers.
[0019] The present invention also relates to expression vectors
encoding a FHIT protein, derivative or analog thereof, as well as
host cells containing the expression vectors encoding the FHIT
protein, derivative or analog thereof. As used herein, "FHIT" shall
be used with reference to the FHIT gene, whereas "Fhit" shall be
used with reference to the protein product of the FHIT gene.
[0020] The present invention further relates to the use of
nucleotide sequences of FHIT genes and amino acid sequences of
their encoded Fhit proteins as diagnostic reagents or in the
preparation of diagnostic agents useful in the detection of cancer
or precancerous conditions or hyperproliferative disorders, in
particular those associated with chromosomal or molecular
abnormalities, in particular at 3p14.2, and/or decreased levels of
expression, or expression of dysfunctional forms, of the Fhit
protein. The invention further relates to the use of nucleotide
sequences of FHIT genes and amino acid sequences of their encoded
Fhit proteins as therapeutic/prophylactic agents in the
treatment/prevention of cancer, in particular, associated with
chromosomal or molecular abnormalities at 3p14.2, and/or decreased
levels of expression, or expression of dysfunctional forms, of the
Fhit protein.
[0021] The invention also relates to Fhit derivatives and analogs
of the invention which are functionally active, i.e., they are
capable of displaying one or more known functional activities
associated with a full-length (wild-type) Fhit protein. Such
functional activities include but are not limited to antigenicity
[ability to bind (or compete with Fhit for binding) to an anti-Fhit
antibody], immunogenicity (ability to generate antibody which binds
to Fhit), and ability to bind (or compete with Fhit for binding) to
a receptor/ligand or substrate for Fhit, and ability to multmerize
with Fhit.
[0022] The invention further relates to fragments (and derivatives
and analogs thereof) of Fhit which comprise one or more domains of
a Fhit protein, e.g., the histadine triad, and/or retain the
antigenicity of a Fhit protein (i.e., are able to be bound by an
anti-Fhit antibody).
[0023] The FHIT gene and protein sequences disclosed herein, and
antibodies to such protein sequences, may be used in assays to
diagnose cancers, e.g., digestive tract and airway tumors,
associated with chromosomal or molecular abnormalities at 3p14.2,
and/or decreased Fhit protein levels or activity by detecting or
measuring a decrease in FHIT wild-type mRNA from a patient sample
or by detecting or measuring a decrease in levels or activity of
Fhit protein from a patient sample, or by detecting an aberrant
Fhit DNA, mRNA, or protein.
[0024] The Fhit protein, or derivatives or analogs thereof,
disclosed herein, may be used for the production of anti-Fhit
antibodies which antibodies may be used diagnostically in
immunoassays for the detection or measurement of Fhit protein in a
patient sample. Anti-Fhit antibodies may be used, for example, for
the diagnostic detection or measurement of Fhit protein in biopsied
cells and tissues.
[0025] The present invention also relates to therapeutic
compositions comprising Fhit proteins, derivatives or analogs
thereof, antibodies thereto, and nucleic acids encoding the Fhit
proteins, derivatives or analogs, and FHIT antisense nucleic
acids.
[0026] The present invention also relates to therapeutic and
diagnostic methods and compositions based on Fhit proteins and
nucleic acids. Therapeutic compounds of the invention include but
are not limited to Fhit proteins and analogs and derivatives
(including fragments) thereof; antibodies thereto; nucleic acids
encoding the Fhit proteins, analogs, or derivatives, and FHIT
antisense nucleic acids.
[0027] The invention provides methods for prevention or treatment
of disorders of overproliferation (e.g., cancer and
hyperproliferative disorders) by administering compounds that
promote Fhit activity (e.g., Fhit, an agonist of Fhit; nucleic
acids that encode Fhit).
[0028] The invention also provides methods of prevention and
treatment of disorders of overproliferation, wherein the patient is
hemizygous for a dominant-negative FHIT mutation, by administering
compounds that specifically antagonize the FHIT mutant nucleic acid
or protein (e.g., antibodies or antisense nucleic acids specific to
the mutant).
[0029] Animal models, diagnostic methods and screening methods for
predisposition to disorders, and methods to identify Fhit agonists
and antagonists, are also provided by the invention.
[0030] The present invention also relates to methods of production
of the Fhit proteins, derivatives and analogs, such as, for
example, by recombinant means.
[0031] In a particular embodiment of the invention described by way
of example in Section 6, a human FHIT sequence is disclosed and
shown to be mutated in various cancers.
4. DESCRIPTION OF THE FIGURES
[0032] FIGS. 1A-1B. Organization of the FHIT gene relative to the
3p14.2 FRA3B and translocation sites. A scheme of the normal 3p14.2
region is shown (A) with the chromosomal region (not to scale)
represented by the top line with positions of STS markers (position
of D3S1234 relative to the gene is not known), the FRA3B
represented by the hybrid c13 break and the t(3;8) translocation
break point shown. The dashed portion represents the region
involved in the homozygous deletions in tumor cell lines. Three of
the YAC clones used in developing the above markers, map and cosmid
contig are shown with the cosmid contig below and the distribution
of exons in the FHIT transcript shown below the contig. Black and
dotted boxes represent coding and noncoding exons, respectively;
asterisks indicate exons with start and stop codons. One exon (E5)
falls within the defined homozygously deleted region. Exons 1 (E1),
2 (E2) and 3 (E3) fall centromeric to the t(3;8) translocation
break and exon 4 (E4) and 6-10 E6-E10) flank the homozygously
deleted region on the centromeric and telomeric sides,
respectively. Organization of types of aberrant transcripts from
tumor cell lines are illustrated in part B, with zigzag regions
representing insertions of new sequence, usually repetitive, into
the aberrant transcripts. CCL234 and 235 are colon
carcinoma-derived cell lines in which homozygous deletion in the
fragile region was not detected. In CCL234 RNA, only an
abnormal-sized FHIT transcript was detected by RT-PCR amplification
and sequencing; the shorter transcript was shown to result from
splicing of exon 3 to exon 5, with omission of the noncoding exon
4, leaving the coding region intact. With CCL235 RNA as template,
apparently normal and aberrant RT-PCR products were amplified, with
the aberrant product resulting from splicing of exon 4 to exon 8
with a repetitive insert of 140 bp (contributing an in frame Met
codon) between E4 and E8. RT-PCR amplification of RNA from HeLa
cells, a cervical carcinoma-derived cell line which exhibited a
deletion or a rearrangement of DNA near the t(3;8) translocation,
revealed normal and aberrant-sized products, the smallest product
resulting from splicing of exon 4 to exon 9. RT-PCR amplification
of RNA from KatoIII, a gastric carcinoma-derived cell line with
discontinuous deletions involving the D3S1481 locus and an
.about.50 kbp region between exons 5 and 6, apparently leaving all
FHIT exons intact, resulted in only an aberrant-sized product which
is missing exons 4 through 7, with an 86 bp repeat, inserted
downstream of exon 3, contributing an in frame Met codon.
Amplification of the RT product from HT29, a colon
carcinoma-derived cell line with a large deletion (-200 kbp, about
the size of the 648D4 YAC), which included exon 5, gave only an
aberrant-sized product resulting from splicing of exon 3 to exon 7.
Numerous other tumor-derived cell lines from lung carcinoma (1/3
tested), osteosarcoma (1/1), NPC (3/3), ovarian carcinoma (2/2),
and hematopoietic (4/5) tumors, exhibited aberrant FHIT
transcription products. The RF48 cell line, from a stomach
carcinoma without deletion, showed a normal-sized product, as did a
lymphoblastoid line with the t(3;8) translocation, a melanoma
(WM1158) and a kidney carcinoma (RC17)-derived cell line. Other
colon and stomach carcinoma-derived lines with deletion (AGS,
LS180, LoVo), or without deletion (Colo320), showed aberrant
reverse transcriptase-polymerase chain reaction (RT-PCR) products
(not shown).
[0033] FIGS. 2A-2B. Structure of normal and aberrant FHIT cDNAs.
The nucleotide (SEQ ID NO:1) and predicted amino acid (SEQ ID NO:2)
sequences of the FHIT gene are shown (A) with positions of exons
indicated by arrowheads above the sequence and positions of primers
used in nested PCR and RACE reactions indicated by arrows below the
sequence. A schematic presentation of some of the aberrant
transcripts observed in uncultured tumor tissues of digestive
organs is shown in B. Only transcripts which showed deletion of
coding sequence in Table 3 are presented. The top line in B shows
the intact FHIT cDNA map. The thick and thin bars show the coding
and untranslated regions, respectively. The positions of splice
sites are shown by downward arrows, according to the nucleotide
numbers shown above in A. The class I transcripts lack exon 5 while
class II transcripts retain exon 5 but generally lose exon 8. In
the transcripts with asterisks, insertions of various lengths were
observed downstream of exon 4. E1-10 indicate exons 1-10.
[0034] FIGS. 3A-3C. Expression of the FHIT gene in normal tissues
and tumors. Northern blot (A, B) and RT-PCR analysis (C) of normal
and tumor-derived FHIT mRNA. Panel A shows a northern blot of
normal mRNAs (2 .mu.g/lane) from spleen (lane 1), thymus (lane 2),
prostate (lane 3), testis (lane 4), ovary (lane 5), small intestine
(lane 6), colon (mucosal lining) (lane 7), and peripheral blood
leukocytes (lane 8), hybridized with the FHIT cDNA probe. Panel B
shows a northern blot of mRNAs (2 .mu.g/lane) from normal small
intestine (lane 1) and mRNAs from tumor-derived cell lines: KatoIII
(lane 2), HK1 (lane 3), LoVo (lane 4), CNE2 (lane 5), CNE1 (lane
6), Colo320 (lane 7), LS180 (lane 8), hybridized with the FHIT cDNA
probe (panel B, upper). The same blot was hybridized with a
.beta.-actin cDNA probe (panel B, lower). Panel C shows amplified
products observed after nested RT-PCR amplification of mRNAs from
matched uncultured tumor (T) and normal (N) tissues of the same
patients (J4, 9625, 5586, E37, E32, E3), or mRNAs from tumor
tissues only (J9, J7, J3, J1, E3). Arrowheads show the positions of
amplified products with abnormal DNA sequence. The details of the
DNA sequences of corresponding transcripts are shown in Table 2,
and FIG. 2B. White dots in the tumor lanes show the position of
transcripts with normal DNA sequence.
[0035] FIGS. 4A-4B. (A) Alignment of amino acid sequences of HIT
family proteins and translations of FHIT cDNAs. Alignment was
performed using BOXSHADE version 3.0. Outlining in thick lines
indicates two or more identical residues at a position; outlining
in thin lines indicates similarity. The PAPH1 (SEQ ID NO:3)
(accession #U32615) and CAPH1 (SEQ ID NO:4) (accession #U28375)
designate the S. pombe and S. cerevisiae diadenosine 5',5'''-P1, P4
tetraphosphate asymmetric hydrolases (aph1). PHIT (SEQ ID NO:6)
indicates the HIT family member from cyanabacterian Synechococcus
Sp. (accession #P32084), BHIT (SEQ ID NO:5), the protein kinase C
inhibitor from B. Taurus (bovine; accession #P16436)), MHIT (SEQ ID
NO:7) from M. hyorhinis (mycoplasma, accession #M37339), YHIT (SEQ
ID NO:8) from S. cerevisiae (accession #Q04344); the Fhit protein
is 69% similar to the S. pombe (PAPH1) gene over a length of 109
amino acids. (B) In vitro translation products from recombinant
plasmids containing different alleles of the FHIT gene: pFHIT1 with
a deletion of noncoding exon 4 (lane 1); pFHIT2 with an insertion
of 72 bp between exons 4 and 5 (lane 2); pFHIT3 with a wildtype
FHIT lacking exon 1 (lane 3); the pFHIT full-length wildtype gene
in Bluescript (lane 4); control reaction, in vitro translation from
the pBCAH vector, carrying a portion of the extracellular region of
the PTPRG gene (predicted molecular weight 40 kDa) (lane 5).
[0036] FIG. 5. Organization of the FHIT gene relative to documented
chromosome breaks in the 3p14.2 fragile region. One FHIT allele is
disrupted in all the translocation carriers of the t(3;8) family,
with exons 1, 2 and 3 remaining on the derivative 3 chromosome and
exons-4-10, including the entire coding region, being translocated
to the derivative 8 chromosome, as illustrated above. The hybrid
cell line, c13, with a de novo FRA3B break just telomeric to exon
5, has lost most of the FHIT coding region. The KatoIII cells
apparently retain all FHIT exons but encode only an abnormal
transcript which lacks exons 4-7 and thus cannot produce Fhit
protein. The MB436 and HT29 cells have both lost exon 5 through
deletion of different segments of the fragile region.
[0037] FIG. 6. Hydrophilicity plot of the Fhit deduced protein
sequence (SEQ-ID NO:2), plotted using the PEPPLOT program of the
Wisconsin GCG software for DNA and protein analysis.
[0038] FIGS. 7A-7C. Printout of R50713 nucleotide sequence (SEQ ID
NO:9) aligned with the FHIT cDNA sequence (cDNA 7F1) (SEQ ID NO:1),
and the R11128 nucleotide sequence (SEQ ID NO:77). The FHIT coding
region starts at nucleotide 363 and ends at nucleotide 812.
[0039] FIG. 8. Translation in all three reading frames, both 5' and
3' directions, of the R50713 EST sequence. 5'3' Frame 1: SEQ ID
NOS:10-15 and 76; 5'3' Frame 2: SEQ ID NOS:16-19; 5'3' Frame 3: SEQ
ID NOS:20-25; 3'5' Frame 1: SEQ ID NOS:26-31; 3'5' Frame 2: SEQ ID
NOS:32-36; 3'5' Frame 3: SEQ ID NOS:37-40.
[0040] FIG. 9. Translation in all three reading frames, both 5' and
3' directions, of the R11128 EST sequence. 5'3' Frame 1: SEQ ID
NOS:41-44; 5'3' Frame 2: SEQ ID NOS:45-48; 5'3' Frame 3: SEQ ID
NOS:49-56; 3'5' Frame 1: SEQ ID NOS:57-58; 3'5' Frame 2: SEQ ID
NOS:59-64; 3'5' Frame 3: SEQ ID NOS:65-68.
[0041] FIGS. 10A-10B. (A) Alignment of yeast (S. pombe) Ap4A
hydrolase sequence (U32615) (SEQ ID NO:69) with FHIT cDNA (cDNA
7F1) sequence (SEQ ID NO:1). (B) Result of search for homology
stretches between U32615 and cDNA 7F1.
[0042] FIGS. 11A-11B. Expression of the FHIT gene in small cell
lung cancer (SCLC). (A) Expression of the FHIT gene by nested
RT-PCR analysis in SCLC tumors (T) and matched normal (N) tissues.
Case 83L indicates a cell line established from the tumor 83T.
Sizes of the amplified products are shown at the right. (B) A
schematic presentation of the aberrant transcripts of types I and
II observed in tumor tissue of SCLCs. The top line shows the intact
FHIT cDNA sequence. The thick and thin bars show the coding and
untranslated regions, respectively. The positions of splice sites
are shown by downward arrows, according to the nucleotide numbers.
Type I transcripts lack exons 4 to 6, while type II transcripts
lack exons 4 to 8.
[0043] FIG. 12A-12D. Expression of the FHIT gene in small cell lung
cancer and sequences of FHIT transcripts. (A) FHIT amplified
products observed after nested RT-PCR of mRNA from tumor (T) and
normal (N) tissues of case 45 and from tumor (T), normal (N) and
cell line (L) samples of case 83. Arrowheads show the sizes of the
amplified products. (B-D) Sequences of the type I and II abnormal
transcripts observed in SCLCs. Arrows indicate junctions between
exons 3 and 4 in the wild-type transcript (WT), between exons 3 and
7 in the abnormal transcripts of type I and between exons 3 and 9
in the abnormal transcripts of type II. WT sequence: SEQ ID NO:78.
Type I sequence: SEQ ID NO:79. Type II sequence: SEQ ID NO:80.
[0044] FIGS. 13A-13G. Expression of the PHIT gene in non small cell
lung cancer (NSCLC) and sequences of FHIT transcripts. (A)
Expression of the FHIT gene by nested RT-PCR analysis in NSCLC
tumors (T) and paired normal (N) tissues. Arrowheads indicate the
amplified abnormal products. (B-G) Sequences of the abnormal
transcripts observed in NSCLC cases 2, 3 and 17. Arrows indicate
the junctions of exons 4 to 5 in the wild-type products of cases 2
and 17 (2WT, 17WT) and of exon 3 to 4 in the wild type product of
case 3 (3WT). 2A shows the junction between exons 4 and 9 in the
abnormal product of case 2, 3A shows the junction between exons 3
and 8 in the abnormal product of case 3, and 17A shows the junction
between exons 4 and 8 in the abnormal product of case 17. WT
sequence: SEQ ID NO:81. 3WT sequence: SEQ ID NO:82. 17WT sequence:
SEQ ID NO:83. 2A sequence: SEQ ID NO:84. 3A sequence: SEQ ID NO:85.
17A sequence: SEQ ID NO:86.
5. DETAILED DESCRIPTION OF THE INVENTION
[0045] The present invention relates to nucleotide sequences of
FHIT genes and amino acid sequences of their encoded Fhit proteins,
as well as derivatives and analogs thereof, and antibodies
thereto.
[0046] As described by way of example infra, the present inventors
have isolated and characterized a human FHIT gene, that is involved
in esophageal, gastric, colon, kidney, and other cancers. Mutations
in FHIT gene sequences leading to loss of FHIT gene function are
associated with cancer.
[0047] The present invention further relates to the use of FHIT
genes and related nucleic acids and their encoded-proteins or
derivatives or analogs thereof, and antibodies thereto, in assays
for the detection and in treatment/prevention of disease states
associated with chromosomal or molecular abnormalities and/or
increased expression of FHIT, such as cancer. The present invention
also relates to therapeutic compositions comprising Fhit proteins,
derivatives or analogs thereof, antibodies thereto, nucleic acids
encoding the Fhit proteins, derivatives or analogs, and FHIT
antisense nucleic acids.
[0048] The FHIT gene sequence can be from one of many different
species, including but not limited to, vertebrate, mammalian,
bovine, ovine, porcine, equine, rodent and human, in naturally
occurring-sequence or in variant form, or from any source, whether
natural, synthetic, or recombinant. In a specific embodiment
described herein, the FHIT gene sequence is a human sequence. The
Fhit protein can be that present in one of many different species,
including but not limited to, mammalian, bovine, ovine, porcine,
equine, rodent and human, in naturally occurring or variant form,
or from any source, whether natural, synthetic, or recombinant. In
specific embodiment described herein, the Fhit protein is a human
protein.
[0049] As defined herein, a Fhit derivative may be a fragment or
amino acid variant (e.g., an insertion, substitution and/or
deletion derivative) of the Fhit sequence shown in FIG. 2A as long
as the fragment or amino acid variant is capable of displaying one
or more functional activities associated with a full-length Fhit
protein. Such functional activities include but are not limited to
antigenicity, i.e., the ability to bind to an anti-Fhit antibody,
immunogenicity, i.e., the ability to generate an antibody which is
capable of binding a Fhit protein; the ability to inhibit cell
proliferation or inhibit tumor growth; the ability to bind (or
compete with Fhit for binding) to a substrate for Fhit; ability to
multimerize with Fhit; and, possibly, Ap4A or other diadenosine
hydrolase activity. The invention provides fragments of a Fhit
protein consisting of at least 10 amino acids, or of at least 25
amino acids, or of at least 50 amino acids, or of at least 100
amino acids. Nucleic acids-encoding such derivatives or analogs are
also within the scope of the invention. A preferred Fhit protein
variant is one sharing at least 70% amino acid sequence homology, a
particularly preferred Fhit protein variant is one sharing at least
80% amino acid sequence homology and another particularly preferred
Fhit protein variant is one sharing at least 90% amino acid
sequence homology to the naturally occurring Fhit protein over at
least 25, at least 50, at least 75, at least 100, or at least 147
(full-length) contiguous amino acids of the FHIT amino acid
sequence. As used herein, amino acid sequence homology refers to
amino acid sequences having identical amino acid residues or amino
acid sequences containing conservative changes in amino acid
residues. In another embodiment, a FHIT homologous protein is one
that shares the foregoing percentages of sequences identical with
the naturally occurring FHIT protein over the recited lengths of
amino acids. Proteins encoded by nucleic acids hybridizable to a
FHIT gene under non-stringent, moderately stringent, or stringent
conditions are also provided.
[0050] The present invention also relates to therapeutic and
diagnostic methods and compositions based on Fhit proteins and
nucleic acids and anti-Fhit antibodies. The invention provides for
treatment or prevention of disorders of overproliferation. (e.g.,
cancer and hyperproliferative disorders) by administering compounds
that promote Fhit activity (e.g., Fhit proteins and functionally
active analogs and derivatives (including fragments) thereof;
nucleic acids encoding the Fhit proteins, analogs, or derivatives,
agonists of Fhit).
[0051] The invention also provides methods of treatment or
prevention of disorders of overproliferation in which the subject
is hemizygous for a Fhit dominant-negative mutation by
administering compounds to the subject that specifically
antagonize, or inhibit, the dominant-negative function of the Fhit
mutant gene or protein (e.g., antibodies or Fhit antisense nucleic
acids specific to the mutant).
[0052] Animal models, diagnostic methods and screening methods for
predisposition to disorders are also provided by the invention.
5.1. The FHIT Coding Sequences
[0053] FHIT cDNA, genomic sequences and sequences complementary
thereto are FHIT nucleic acids provided by the present invention.
In a specific embodiment herein, a FHIT cDNA sequence is provided,
thus lacking any introns. Sequences hybridizable thereto,
preferably lacking introns, are also provided. Nucleic acids
comprising FHIT DNA or RNA exon sequences are also provided; in
various embodiments, at least 15, 25 or 50 contiguous nucleotides
of PHIT exon sequences are in the nucleic acid. Also included
within the scope of the present invention are nucleic acids
comprising FHIT cDNA or RNA consisting of at least 8 nucleotides,
at least 15 nucleotides, at least 25 nucleotides, at least 50
nucleotides, at least 100 nucleotides, at least 200 nucleotides, or
at least 350 nucleotides. In various embodiments, nucleic acids are
provided that are less than 2,000, less than 500, less than 275,
less than 200, less than 100, or less than 50 bases (or bp, if
double-stranded). In various embodiments, the nucleic acids are
less than 300 kb, 200 kb, 100 kb, 50 kb, or 10 kb. Nucleic acids
can be single-stranded or double-stranded. In specific embodiments,
isolated nucleic acids are provided that comprise at least 15
contiguous nucleotides of FHIT coding sequences but which do not
comprise all or a portion of any FHIT intron. In a specific
embodiment, the nucleic acid comprises at least one FHIT coding
exon (exon 5, 6, 7, 8 or 9). In another embodiment, the nucleic
acid substantially lacks the FHIT intron between exon 5 and 6, yet
contains exon 5 and at least one other FHIT coding exon selected
from among exon 6, exon 7, exon 8, and exon 9. In yet another
embodiment, the nucleic acid comprises at least one FHIT exon
selected from among exon 1, 2, 3, 4 and 5, and contains at least
one FHIT exon selected from among exon 6, 7, 8, 9 and 10, and is
preferably less than 10 kb in size. In a preferred embodiment the
FHIT exon sequences appear in the nucleic acid in the order in
which they appear in the genome; in an alternative embodiment, the
exon sequences do not appear in the same order. In another
embodiment, the nucleic acid comprises all the FHIT exons (exons
1-10) or all the FHIT coding exons (exons 5-9) in contiguous
fashion, and thus lacks introns. In yet another specific
embodiment, the nucleic acid comprising FHIT gene exon sequences
does not contain sequences of a genomic flanking gene (i.e., 5' or
3' to the PHIT gene in the genome). In a specific embodiment
herein, a FHIT genomic sequence is provided, thus containing
introns.
[0054] The invention also provides single-stranded oligonucleotides
for use as primers in PCR that amplify a FHIT sequence-containing
fragment, e.g., an oligonucleotide having the sequence of a
hybridizable portion (at least .about.8 nucleotides) of a FHIT
gene, and another oligonucleotide having the reverse complement of
a downstream sequence in the same strand of the FHIT gene, such
that each oligonucleotide primes synthesis in a direction toward
the other. The oligonucleotides are preferably in the range of
10-35 nucleotides in length.
[0055] The full length cDNA sequence for human FHIT is depicted in
FIG. 2A (SEQ ID NO: 1), with the coding region thereof spanning
nucleotide numbers 1-441 of FIG. 2A. Sequence analysis of the FHIT
cDNA of FIG. 2A reveals an open reading frame of 441 nucleotides,
encoding a protein of 147 amino acids (SEQ ID NO:2).
[0056] In accordance with the present invention, any polynucleotide
sequence which encodes the amino acid sequence of a FHIT gene
product can be used to generate recombinant molecules which direct
the expression of Fhit. Included within the scope of the present
invention are nucleic acids consisting of at least 8 nucleotides
that are useful as probes or primers (i.e., a hybridizable portion)
in the detection or amplification of FHIT.
[0057] In a specific embodiment disclosed herein, the invention
relates to the nucleic acid sequence of the human FHIT gene. In a
preferred, but not limiting, aspect of the invention, a human FHIT
cDNA sequence is that present in plasmid p7F1 as deposited with the
ATCC and assigned ATCC Accession Number 69977. Such a sequence can
be cloned and sequenced, for example, as described in Section 6,
infra. The invention also relates to nucleic acid sequences
hybridizable or complementary to the foregoing sequences or
equivalent to the foregoing-sequences in that the equivalent
nucleic acid sequences also encode a protein product displaying
Fhit functional activity.
[0058] Nucleic acids encoding fragments and derivatives of FHIT are
additionally described infra.
[0059] The invention also relates to nucleic acids hybridizable to
or complementary to the above-described nucleic acids comprising
FHIT sequences. In specific aspects, nucleic acids are provided
which comprise a sequence complementary to at least 10, 25, 50,
100, or 200 nucleotides or the entire coding region of a FHIT gene.
In a specific embodiment, a nucleic acid which is hybridizable to a
FHIT nucleic acid, or to a nucleic acid encoding a Fhit derivative,
under conditions of low stringency is provided. By way of example
and not limitation, procedures using such conditions of low
stringency are as follows (see also Shilo and Weinberg, 1981, Proc.
Natl. Acad. Sci. USA 78:6789-6792): Filters containing DNA are
pretreated for 6 h at 40.degree. C. in a solution containing 35%
formamide, 5.times.SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1%
PVP, 0.1% Ficoll, 1% BSA, and 500 .mu.g/ml denatured salmon sperm
DNA. Hybridizations are carried out in the same solution with the
following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100
.mu.g/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and
5-20.times.10.sup.6 cpm .sup.32P-labeled probe is used. Filters are
incubated in hybridization mixture for 18-20 h at 40.degree. C.,
and then washed for 1.5 h at 55.degree. C. in a solution containing
2.times.SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The
wash solution is replaced with fresh solution and incubated an
additional 1.5 h at 60.degree. C. Filters are blotted dry and
exposed for autoradiography. If necessary, filters are washed for a
third time at 65-68.degree. C. and reexposed to film other
conditions of low stringency which may be used are well known in
the art (e.g., as employed for cross-species hybridizations).
[0060] In another specific embodiment, a nucleic acid which is
hybridizable to a FHIT nucleic acid under conditions of high
stringency is provided (see infra).
[0061] In a preferred aspect, polymerase chain reaction (PCR) is
used to amplify a desired nucleic acid sequence in a library or
from a tissue source by using oligonucleotide primers representing
known FHIT sequences. Such primers may be used to amplify sequences
of interest from an RNA or DNA source, preferably a cDNA library.
PCR can be carried out, e.g., by use of a Perkin-Elmer Cetus
thermal cycler and Taq polymerase (Gene Amp.TM.). The DNA being
amplified can include mRNA or cDNA or genomic DNA from any
eukaryotic species. One can choose to synthesize several different
degenerate primers, for use in the PCR reactions. It is also
possible to vary the stringency of hybridization conditions used in
priming the PCR reactions, to allow for greater or lesser degrees
of nucleotide sequence homology between the FHIT gene being cloned
and the known FHIT gene. Other means for primer dependent
amplification of nucleic acids are known to those of skill in the
art and can be used.
[0062] After successful amplification of a segment of a FHIT gene
(e.g., an allelic or polymorphic variant or species homolog of a
known FHIT gene) that segment may be molecularly cloned and
sequenced, and utilized as a probe to isolate a complete cDNA or
genomic clone. This, in turn, will permit the determination of the
gene's complete nucleotide sequence, the analysis of its
expression, and the production of its protein product for
functional analysis, as described infra. In this fashion,
additional genes encoding Fhit proteins may be identified.
Alternatively, the FHIT gene of the present invention may be
isolated through an exon trapping system, using genomic DNA (Nehls
et al., 1994, Oncogene 9(8):2169-2175; Verna et al., 1993, Nucleic
Acids Res. 21(22):5198:5202; Auch et al., 1990, Nucleic Acids Res.
18(22):6743-6744).
[0063] Potentially, any eukaryotic cell can serve as the nucleic
acid source for the molecular cloning of the FHIT gene. The nucleic
acid sequences encoding FHIT can be isolated from, for example,
human, porcine, bovine, feline, avian, equine, canine, rodent, as
well as additional primate sources. The DNA may be obtained by
standard procedures known in the art from, for example, cloned DNA
(e.g., a DNA "library"), by chemical synthesis, by cDNA cloning, or
by the cloning of genomic DNA, or fragments thereof, purified from
a desired cell. (See, for example, Sambrook et al., 1989, Molecular
Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.; Glover, D. M. (ed.), 1985, DNA
Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol.
I, II.) Clones derived from genomic DNA may contain regulatory and
intron DNA regions in addition to coding regions while clones
derived from cDNA will contain only FHIT exon sequences. Whatever
the source, the gene should be molecularly cloned into a suitable
vector for propagation of the gene. In a particular embodiment, a
preferred source of nucleic acid for the isolation of FHIT gene
sequences is from kidney or stomach or lung cells.
[0064] In the molecular cloning of the gene from genomic DNA, DNA
fragments are generated, some of which will encode the desired
gene. The DNA may be cleaved at specific sites using various
restriction enzymes. Alternatively, one may use DNAse in the
presence of manganese to fragment the DNA, or the DNA can be
physically sheared, as for example, by sonication. The linear DNA
fragments can then be separated according to size by standard
techniques, including but not limited to, agarose and
polyacrylamide gel electrophoresis and column chromatography.
[0065] Once the DNA fragments are generated, identification of the
specific DNA fragment containing the desired gene may be
accomplished in a number of ways. For example, a FHIT gene of the
present invention or its specific RNA, or a fragment thereof, such
as a probe or primer, may be isolated and labeled and then used in
hybridization assays to detect a generated FHIT gene (Benton, W.
and Davis, R., 1977, Science 196:180; Grunstein, M. And Hogness,
D., 1975, Proc. Natl. Acad. Sci. USA 72:3961). Those DNA fragments
sharing substantial sequence homology to the probe will hybridize
under high stringency conditions. The phrase "high stringency
conditions" as used herein refers to those hybridizing conditions
that (1) employ low ionic strength and high temperature for
washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS
at 50.degree. C.; (2) employ during hybridization a denaturing
agent such as formamide, for example, 50% (vol/vol) formamide with
0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50
mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium
citrate at 42.degree. C.; or (3) employ 50% formamide, 5.times.SSC
(0.75 M NaCl, 0.075 M sodium pyrophosphate, 5.times. Denhardt's
solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10%
dextran sulfate at 42.degree. C., with washes at 42.degree. C. in
0.2.times.SSC and 0.1% SDS.
[0066] It is also possible to identify the appropriate fragment by
restriction enzyme digestion(s) and comparison of fragment sizes
with those expected according to a known restriction map. Further
selection can be carried out on the basis of the properties of the
gene. Alternatively, the presence of the gene may be detected by
assays based on the physical, chemical, or immunological properties
of its expressed product. For example, cDNA clones, or genomic DNA
clones which hybrid-select the proper mRNAs, can be selected which
produce a protein that has similar or identical electrophoretic
migration, isolectric focusing behavior, proteolytic digestion
maps, binding activity or antigenic properties as known for FHIT.
Alternatively, the FHIT protein may be identified by binding of
labeled antibody to the putatively FHIT expressing clones, e.g., in
an ELISA (enzyme-linked immunosorbent assay)-type procedure.
[0067] The FHIT gene can also be identified by mRNA selection by
nucleic acid hybridization followed by in vitro translation. In
this procedure, fragments are used to isolate complementary mRNAs
by hybridization. Such DNA fragments may represent available,
purified FHIT DNA of another FHIT gene.
[0068] Immunoprecipitation analysis or functional assays of the in
vitro translation products of the isolated products of the isolated
mRNAs identifies the mRNA and, therefore, the complementary DNA
fragments that contain the desired sequences. In addition, specific
mRNAs may be selected by adsorption of polysomes isolated from
cells to immobilized antibodies specifically directed against FHIT
protein. A radiolabelled FHIT cDNA can be synthesized using the
selected mRNA (from the adsorbed polysomes) as a template. The
radiolabelled mRNA or cDNA may then be used as a probe to identify
the FHIT DNA fragments from among other genomic DNA fragments.
[0069] Alternatives to isolating the FHIT genomic DNA include, but
are not limited to, chemically synthesizing the gene sequence
itself from a known sequence or making cDNA to the mRNA which
encodes the FHIT protein. For example, RNA useful in cDNA cloning
of the FHIT gene can be isolated from cells which express FHIT,
e.g., kidney or stomach or lung cells. Other methods are known to
those of skill in the art and are within the scope of the
invention.
[0070] The identified and isolated gene can then be inserted into
an appropriate cloning vector. A large number of vector-host
systems known in the art may be used. Possible vectors include, but
are not limited to, plasmids or modified viruses, but the vector
system must be compatible with the host cell used. Such vectors
include, but are not limited to, bacteriophages such as lambda
derivatives, or plasmids such as PBR322 or pUC plasmid derivatives
or the Bluescript vector (Stratagene). The insertion into a cloning
vector can, for example, be accomplished by ligating the DNA
fragment into a cloning vector which has complementary cohesive
termini. However, if the complementary restriction sites used to
fragment the DNA are not present in the cloning vector, the ends of
the DNA molecules may be enzymatically modified. Alternatively, any
site desired may be produced by ligating nucleotide sequences
(linkers) onto the DNA termini; these ligated linkers may comprise
specific chemically synthesized oligonucleotides encoding
restriction endonuclease recognition sequences. In an alternative
method, the cleaved vector and FHIT gene may be modified by
homopolymeric tailing. Recombinant molecules can be introduced into
host cells via transformation, transfection, infection,
electroporation, or other methods known to those of skill in the
art, so that many copies of the gene sequence are generated.
[0071] In an alternative method, the desired gene may be identified
and isolated after insertion into a suitable cloning vector in a
"shot gun" approach. Enrichment for the desired gene, for example,
by size fractionization, can be done before insertion into the
cloning vector.
[0072] In specific embodiments, transformation of host cells with
recombinant DNA molecules that incorporate the isolated FHIT gene,
cDNA, or synthesized DNA sequence enables is generation of multiple
copies of the gene. Thus, the gene may be obtained in large
quantities by growing transformants, isolating the recombinant DNA
molecules from the transformants and, when necessary, retrieving
the inserted gene from the isolated recombinant DNA.
[0073] Oligonucleotides containing a portion of the FHIT coding or
non-coding sequences, or which encode a portion of the FHIT protein
(e.g., primers for use in PCR) can be synthesized by standard
methods commonly known in the art. Such oligonucleotides preferably
have a size in the range of 8 to 25 nucleotides. In a specific
embodiment herein, such oligonucleotides have a size in the range
of 15 to 25 nucleotides or 15 to 35 nucleotides.
[0074] The FHIT sequences provided by the instant invention include
those nucleotide sequences encoding substantially the same amino
acid sequences as found in native Fhit proteins, and those encoded
amino acid sequences with functionally equivalent amino acids, as
well as those encoding other Fhit derivatives or analogs, as
described infra for Fhit derivatives and analogs.
5.2. Expression of the FHIT Gene
[0075] In accordance with the present invention, nucleotide
sequences coding for a FHIT protein, derivative, e.g. fragment, or
analog thereof, can be inserted into an appropriate expression
vector, i.e., a vector which contains the necessary elements for
the transcription and translation of the inserted protein-coding
sequence, for the generation of recombinant DNA molecules that
direct the expression of a FHIT protein. Such PHIT polynucleotide
sequences, as well as other polynucleotides or their complements,
may also be used in nucleic acid hybridization assays, Southern and
Northern blot analysis, etc. In a specific embodiment, a human FHIT
gene, or a sequence encoding a functionally active portion of a
human FHIT gene is expressed. In yet another embodiment, a
derivative or fragment of a human FHIT gene is expressed.
[0076] Due to the inherent degeneracy of the genetic code, other
DNA sequences which encode substantially the same or a functionally
equivalent FHIT amino acid sequence, is within the scope of the
invention. Such DNA sequences include those which are capable of
hybridizing to the human FHIT sequence under stringent
conditions.
[0077] Altered DNA sequences which may be used in accordance with
the invention include deletions, additions or substitutions of
different nucleotide residues resulting in a sequence that encodes
the same or a functionally equivalent gene product. The gene
product itself may contain deletions, additions or substitutions of
amino acid residues within an FHIT sequence, which result in a
silent change thus producing a functionally equivalent FHIT
protein. Such amino acid substitutions may be made on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity, and/or the amphipathic nature of the residues
involved. For example, negatively charged amino acids include
aspartic acid and glutamic acid; positively charged amino acids
include lysine and arginine; amino acids with uncharged polar head
groups having similar hydrophilicity values include the following:
leucine, isoleucine, valine; glycine, alanine; asparagine,
glutamine; serine, threonine; phenylalanine, tyrosine.
[0078] The DNA sequences of the invention may be engineered in
order to alter a FHIT coding sequence for a variety of ends
including but not limited to alterations which modify processing
and expression of the gene product. For example, mutations may be
introduced using techniques which are well known in the art, e.g.,
site-directed mutagenesis, to insert new restriction sites,
etc.
[0079] In another embodiment of the invention, a FHIT gene sequence
or a derivative thereof is ligated to a non-FHIT sequence to encode
a chimeric fusion protein. A fusion protein may also be engineered
to contain a cleavage site located between a PHIT sequence and the
non-FHIT protein sequence, so that the FHIT protein may be cleaved
away from the non-FHIT moiety. In a specific embodiment, the FHIT
amino acid sequence present in the fusion protein consists of at
least 10 contiguous amino acids, at least 25 contiguous amino
acids, at least 50 contiguous amino acids, at least 75 contiguous
amino acids, at least 100 contiguous amino acids, or at least 147
amino acids (full-length) of the Fhit protein sequence.
[0080] In an alternate embodiment of the invention, the coding
sequence of a FHIT is synthesized in whole or in part, using
chemical methods well known in the art. See, for example, Caruthers
et al., 1980, Nuc. Acids Res. Symp. Ser. 7:215-233; Crea and Horn,
1980, Nuc. Acids Res. 9(10):2331; Matteucci and Caruthers, 1980,
Tetrahedron Letters 21:719; and Chow and Kempe, 1981, Nuc. Acids
Res. 9(12):2807-2817. Alternatively, the protein itself could be
produced using chemical methods to synthesize an FHIT amino acid
sequence in whole or in part. For example, peptides can be
synthesized by solid phase techniques, cleaved from the resin, and
purified by preparative high performance liquid chromatography.
(e.g., see Creighton, 1983, Proteins Structures And Molecular
Principles, W.H. Freeman and Co., N.Y. pp. 50-60). The composition
of the synthetic peptides may be confirmed by amino acid analysis
or sequencing (e.g., the Edman degradation procedure; see
Creighton, 1983, Proteins, Structures and Molecular Principles,
W.H. Freeman and Co., N.Y., pp. 34-49.
[0081] In order to express a biologically active FHIT protein or
derivative thereof, a polynucleotide sequence encoding a FHIT
protein, or a derivative thereof, is inserted into an
appropriate-expression vector, i.e., a vector which contains the
necessary elements for the transcription and translation of the
inserted coding sequence. The FHIT gene products as well as host
cells or cell lines transfected or transformed with recombinant
FHIT expression vectors can be used for a variety of purposes.
These include but are not limited to generating antibodies (i.e.,
monoclonal or polyclonal) that immunospecifically bind a FHIT
protein. Anti-FHIT antibodies can be used in detecting or measuring
levels of a FHIT protein in patient samples.
5.2.1. Expression Systems
[0082] Methods which are well known to those skilled in the art can
be used to construct expression vectors containing a FHIT coding
sequence and appropriate transcriptional/translational control
signals. These methods include in vitro recombinant DNA techniques,
synthetic techniques and in vivo recombination/genetic
recombination. See, for example, the techniques described in
Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual 2d
ed., Cold Spring Harbor Laboratory, N.Y. and Ausubel et al., 1989,
Current Protocols in Molecular Biology, Greene Publishing
Associates and Wiley Interscience, N.Y.
[0083] A variety of host-expression vector systems may be utilized
to express a FHIT coding sequence. These include but are not
limited to microorganisms such as bacteria transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors containing an PHIT coding sequence; yeast transformed with
recombinant yeast expression vectors containing an FHIT coding
sequence; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing an PHIT coding
sequence; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing an FHIT coding
sequence; or animal cell systems. The expression elements of these
systems vary in their strength and specificities. Depending on the
host/vector system utilized, any of a number of suitable
transcription and translation elements, including constitutive and
inducible promoters, may be used in the expression vector. For
example, when cloning in bacterial systems, inducible promoters
such as pL of bacteriophage .lamda., plac, ptrp, ptac (ptrp-lac
hybrid promoter) and the like may be used; when cloning in insect
cell systems, promoters such as the baculovirus polyhedrin promoter
may be used; when cloning in plant cell systems, promoters derived
from the genome of plant cells (e.g., heat shock promoters; the
promoter for the small subunit of RUBISCO; the promoter for the
chlorophyll a/b binding protein) or from plant viruses (e.g., the
35S RNA promoter of CaMV; the coat protein promoter of TMV) may be
used; when cloning in mammalian cell systems, promoters derived
from the genome of mammalian cells (e.g., metallothionein promoter)
or from mammalian viruses (e.g., the adenovirus late promoter; the
vaccinia virus 7.5 K promoter) may be used; when generating cell
lines that contain multiple copies of an FHIT DNA, SV40-, BPV- and
EBV-based vectors may be used with an appropriate selectable
marker.
[0084] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
FHIT protein expressed. For example, when large quantities of FHIT
protein are to be produced for the generation of antibodies,
vectors which direct the expression of high levels of fusion
protein products that are readily purified may be desirable. Such
vectors include but are not limited to the E. coli expression
vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the
FHIT coding sequence may be ligated into the vector in frame with
the lac Z coding region so that a hybrid AS-lac Z protein is
produced; pIN vectors (Inouye & Inouye, 1985, Nucleic acids
Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem.
264:5503-5509); and the like. pGEX vectors may also be used to
express foreign polypeptides as fusion proteins with glutathione
S-transferase (GST) (Smith and Johnson, 1988, Gene 7:31-40). In
general, such fusion proteins are soluble and can easily be
purified from lysed cells by adsorption to glutathione-agarose
beads followed by elution in the presence of free glutathione. The
pGEX vectors are designed to include thrombin or factor Xa protease
cleavage sites so that the cloned polypeptide of interest can be
released from the GST moiety.
[0085] In yeast, a number of vectors containing constitutive or
inducible promoters may be used. For a review see, Current
Protocols in Molecular Biology, Vol. 2, 1988, Ed. Ausubel et al.,
Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant et
al., 1987, Expression and Secretion Vectors for Yeast, in Methods
in Enzymology, Ed. Wu & Grossman, 1987, Acad. Press, N.Y.
153:516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash.,
D.C., Ch. 3; and Bitter, 1987, Heterologous Gene Expression in
Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad.
Press, N.Y. 152:673-684; and The Molecular Biology of the Yeast
Saccharomyces, 1982, Eds. Strathern et al., Cold Spring. Harbor
Press, Vols. I and II.
[0086] In cases where plant expression vectors are used, the
expression of an FHIT coding sequence may be driven by any of a
number of promoters. For example, viral promoters such as the 35S
RNA and 19S RNA promoters of CaMV (Brisson et al., 1984, Nature
310:511-514), or the coat protein promoter of TMV (Takamatsu et
al., 1987, EMBO J. 6:307-311) may be used; alternatively, plant
promoters such as the small subunit of RUBISCO (Coruzzi et al.,
1984, EMBO J. 3:1671-1680; Broglie et al., 1984, Science
224:838-843); or heat shock promoters, e.g., soybean hsp17.5-E or
hsp17.3-B (Gurley et al., 1986, Mol. Cell. Biol. 6:559-565) may be
used. These constructs can be introduced into plant cells using Ti
plasmids, Ri plasmids, plant virus vectors, direct DNA
transformation, microinjection, electroporation, etc. For reviews
of such techniques see, for example, Weissbach & Weissbach,
1988, Methods for Plant Molecular Biology, Academic Press, NY,
Section VIII, pp. 421-463; and Grierson & Corey, 1988, Plant
Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9.
[0087] An alternative expression system which could be used to
express a FHIT gene is an insect system. In one such system,
Autographa californica nuclear polyhedrosis virus (AcNPV) is used
as a vector to express foreign genes. The virus grows in Spodoptera
frugiperda cells. A FHIT coding sequence may be cloned into
non-essential regions (for example the polyhedrin gene) of the
virus and placed under control of an AcNPV promoter (for example,
the polyhedrin promoter). Successful insertion of a FHIT coding
sequence will result in inactivation of the polyhedrin gene and
production of non-occluded recombinant virus (i.e., virus lacking
the proteinaceous coat coded for by the polyhedrin gene). These
recombinant viruses are then used to infect Spodoptera frugiperda
cells in which the inserted gene is expressed. (e.g., see Smith et
al., 1983, J. Virol. 46:584; Smith, U.S. Pat. No. 4,215,051).
[0088] In mammalian host cells, a number of viral based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, a FHIT coding sequence may be ligated to an
adenovirus transcription/translation control complex, e.g., the
late promoter and tripartite leader sequence. This chimeric gene
may then be inserted in the adenovirus genome by in vitro or in
vivo recombination. Insertion in a non-essential region of the
viral genome (e.g., region E1 or E3) will result in a recombinant
virus that is viable and capable of expressing a FHIT in infected
hosts. (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci.
USA 81:3655-3659). Alternatively, the vaccinia 7.5 K promoter may
be used. (See, e.g., Hackett et al., 1982, Proc. Natl. Acad. Sci.
USA 79:7415-7419; Madkett et al., 1984, J. Virol. 49:857-864;
Panicali et al., 1982, Proc. Natl. Acad. Sci. USA
79:4927-4931).
[0089] Specific initiation signals may also be required for
efficient translation of an inserted FHIT coding sequences. These
signals include the ATG initiation codon and adjacent sequences. In
cases where an entire FRIT gene, including its own initiation codon
and adjacent sequences, is inserted into the appropriate expression
vector, no additional translational control signals may be needed.
However, in cases where only a portion of a FHIT coding sequence is
inserted, lacking the 5' end, exogenous translational control
signals, including the ATG initiation codon, must be provided.
Furthermore, the initiation codon must be in phase with the reading
frame of a FHIT coding sequence to ensure translation of the entire
insert. These exogenous translational control signals and
initiation codons can be of a variety of origins, both natural and
synthetic. The efficiency of expression may be enhanced by the
inclusion of appropriate transcription enhancer elements,
transcription terminators, etc. (see Bittner et al., 1987, Methods
in Enzymol. 153:516-544).
[0090] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., phosphorylation) and processing (e.g.,
cleavage) of protein products may be important for the function of
the protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins. Appropriate cells lines or host systems can be chosen
to ensure the correct modification and processing of the foreign
protein expressed. To this end, eukaryotic host cells which possess
the cellular machinery for proper processing of the primary
transcript, and phosphorylation of the gene product may be used.
Such mammalian host cells include but are not limited to CHO, VERO,
BHK, HeLa, COS, MDCK, 293, WI38, etc.
[0091] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express a FHIT protein may be engineered. Rather than
using expression vectors which contain viral origins of
replication, host cells can be transformed with FHIT DNA controlled
by appropriate expression control elements (e.g., promoter,
enhancer, sequences, transcription terminators, polyadenylation
sites, etc.), and a selectable marker. Following the introduction
of foreign DNA, engineered cells may be allowed to grow for 1-2
days in an enriched media, and then are switched to a selective
media. The selectable marker in the recombinant plasmid confers
resistance to the selection and allows cells to stably integrate
the plasmid into their chromosomes and grow to form foci which in
turn can be cloned and expanded into cell lines. This method may
advantageously be used to engineer cell lines which express a FHIT
protein. The present invention provides a method for producing a
recombinant FHIT protein comprising culturing a host cell
transformed with a recombinant expression vector encoding a FHIT
protein such that the FHIT protein is expressed by the cell and
recovering the expressed FHIT protein.
[0092] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et
al., 1977, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 48:2026), and adenine
phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes
can be employed in tk-, hgprt- or aprt- cells, respectively. Also,
antimetabolite resistance can be used as the basis of selection for
dhfr, which confers resistance to methotrexate (Wigler et al.,
1980, Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc.
Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to
mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad.
Sci. USA 78:2072); neo, which confers resistance to the
aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol.
150:1); and hygro, which confers resistance to hygromycin (Santerre
et al., 1984, Gene 30:147). Recently, additional selectable genes
have been described, namely trpB, which allows cells to utilize
indole in place of tryptophan; hisD, which allows cells to utilize
histinol in place of histidine (Hartman & Mulligan, 1988, Proc.
Natl. Acad. Sci. USA 85:8047); and ODC (ornithine decarboxylase)
which confers resistance to the ornithine decarboxylase inhibitor,
2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, L., 1987, In:
Current Communications in Molecular Biology, Cold Spring Harbor
Laboratory, Ed.).
5.2.2. Identification of Transfectants or Transformants That
Express FHIT
[0093] The host cells which contain the coding sequence and which
express the biologically active gene product may be identified by
at least four general approaches; (a) DNA-DNA or DNA-RNA
hybridization; (b) the presence or absence of "marker" gene
functions; (c) assessing the level of transcription as measured by
the expression of FHIT mRNA transcripts in the host cell; and (d)
detection of the gene product as measured by immunoassay or by its
biological activity.
[0094] In the first approach, the presence of the FHIT coding
sequence inserted in the expression vector can be detected by
DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide
sequences that are homologous to the FHIT coding sequence,
respectively, or portions or derivatives thereof.
[0095] In the second approach, the recombinant expression
vector/host system can be identified and selected based upon the
presence or absence of certain "marker" gene functions (e.g.,
thymidine kinase activity, resistance to antibiotics, resistance to
methotrexate, transformation phenotype, occlusion body formation in
baculovirus, etc.). For example, if the human FHIT coding sequence
is inserted within a marker gene sequence of the vector,
recombinant cells containing the FHIT coding sequence can be
identified by the absence of the marker gene function.
Alternatively, a marker gene can be placed in tandem with a FHIT
sequence under the control of the same or different promoter used
to control the expression of the FHIT coding sequence. Expression
of the marker in response to induction or selection indicates
expression of the FHIT coding sequence.
[0096] In the third approach, transcriptional activity of a FHIT
gene can be assessed by hybridization assays. For example, RNA can
be isolated and analyzed by Northern-blot using a probe having
sequence homology to a FHIT coding sequence or transcribed
noncoding sequence or particular portions thereof. Alternatively,
total nucleic acid of the host cell may be extracted and
quantitatively assayed for hybridization to such probes.
[0097] In the fourth approach, the levels of a FHIT protein product
can be assessed immunologically, for example by Western blots,
immunoassays such as radioimmuno-precipitation, enzyme-linked
immunoassays and the like.
5.3. Purification of the Expressed Gene Product
[0098] Once a recombinant which expresses the FHIT gene sequence is
identified, the gene product can be analyzed. This is achieved by
assays based on the physical or functional properties of the
product, including radioactive labelling of the product followed by
analysis by gel electrophoresis, immunoassay, or other detection
methods known to those of skill in the art.
[0099] Once the FHIT protein is identified, it may be isolated and
purified by standard methods including chromatography (e.g., ion
exchange, affinity, and sizing column chromatography),
centrifugation, differential solubility, or by any other standard
technique for the purification of proteins. The functional
properties may be evaluated using any suitable assay.
[0100] Alternatively, once a FHIT protein produced by a recombinant
is identified, the amino acid sequence of the protein can be
deduced from the nucleotide sequence of the chimeric gene contained
in the recombinant. As a result, the protein can be synthesized by
standard chemical methods known in the art (e.g., see Hunkapiller
et al., 19.84, Nature 310:105-111).
[0101] In a specific embodiment of the present invention, such FHIT
proteins, whether produced by recombinant DNA techniques or by
chemical synthetic methods, include but are not limited to those
containing, as a primary amino acid sequence, all or part of the
amino acid sequence substantially as depicted in FIG. 2A (SEQ ID
NO:2), as well as fragments and other derivatives, and analogs
thereof.
5.4. Generation of Antibodies to Fhit
[0102] According to the invention, Fhit protein, its fragments or
other derivatives, or analogs thereof, may be used as an immunogen
to generate antibodies which recognize such an immunogen. Such
antibodies include but are not limited to polyclonal, monoclonal,
chimeric, single chain, Fab fragments, and an Fab expression
library. In a specific embodiment, antibodies to a human Fhit
protein are produced.
[0103] Various procedures known in the art may be used for the
production of polyclonal antibodies to a Fhit protein or derivative
or analog. For the production of antibody, various host animals can
be immunized by injection with the native Fhit protein, or a
synthetic version, or derivative (e.g., fragment) thereof,
including but not limited to rabbits, mice, rats, etc. Various
adjuvants may be used to increase the immunological response,
depending on the host species, and including but not limited to
Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanins, dinitrophenol, and potentially useful human adjuvants
such as BCG (bacille Calmette-Guerin) and corynebacterium
parvum.
[0104] For preparation of monoclonal antibodies directed toward a
Fhit protein sequence or analog thereof, any technique which
provides for the production of antibody molecules by continuous
cell lines in culture may be used. For example, the hybridoma
technique originally developed by Kohler and Milstein (1975, Nature
256:495-497), as well as the trioma technique, the human B-cell
hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72),
and the EBV-hybridoma technique to produce human monoclonal
antibodies (Cole et al., 1985, in Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc., pp. 77-96). In an additional
embodiment of the invention, monoclonal antibodies can be produced
in germ-free animals utilizing recent technology (PCT/US90/02545).
According to the invention, human antibodies may be used and can be
obtained by using human hybridomas (Cote et al., 1983, Proc. Natl.
Acad. Sci. USA 80:2026-2030) or by transforming human B cells with
EBV virus in vitro (Cole et al., 1985, in Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, pp. 77-96). In fact, according to the
invention, techniques developed for the production of "chimeric
antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA
81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda et
al., 1985, Nature 314:452-454) by splicing the genes from a mouse
antibody molecule specific for FHIT together with genes from a
human antibody molecule of appropriate biological activity can be
used; such antibodies are within the scope of this invention.
[0105] According to the invention, techniques described for the
production of single chain antibodies (U.S. Pat. No. 4,946,778) can
be adapted to produce Fhit-specific single chain antibodies. An
additional embodiment of the invention utilizes the techniques
described for the construction of Fab expression libraries (Huse et
al., 1989, Science 246:1275-1281) to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity for Fhit proteins, derivatives, or analogs.
[0106] In a specific embodiment, a molecule comprising a fragment
of the Fhit protein is used as an immunogen. For example, since
hydrophilic regions are believed most likely to contain antigenic
determinants, a peptide corresponding to or containing a
hydrophilic portion of a Fhit protein is preferably used as
immunogen.
[0107] Antibody fragments which contain the idiotype of the
molecule can be generated by known techniques. For example, such
fragments include but are not limited to: the F(ab').sub.2 fragment
which can be produced by pepsin digestion of the antibody molecule;
the Fab' fragments which can be generated by reducing the disulfide
bridges of the F(ab').sub.2 fragment, and the Fab fragments which
can be generated by treating the antibody molecule with papain and
a reducing agent.
[0108] In the production of antibodies, screening for the desired
antibody can be accomplished by techniques known in the art, e.g.
ELISA (enzyme-linked immunosorbent assay). For example, to select
antibodies which recognize a specific domain of a Fhit protein, one
may assay generated hybridomas for a product which binds to a Fhit
fragment containing such domain. For selection of an antibody
specific to human Fhit, one can select on the basis of positive
binding to human Fhit and a lack of binding to, for example, mouse
Fhit.
[0109] The foregoing antibodies can be used in methods known in the
art relating to the localization and activity of the protein
sequences of the invention, e.g., for imaging these proteins,
measuring levels thereof in appropriate physiological samples, in
diagnostic methods, etc.
5.5. Structure of the FHIT Gene and Protein
[0110] The structure of the FHIT gene and protein can be analyzed
by various methods known in the art.
5.5.1. Genetic Analysis
[0111] The cloned DNA or cDNA-corresponding to the FHIT gene can be
analyzed by methods including but not limited to Southern
hybridization (Southern, E. M., 1975, J. Mol. Biol. 98:503-517),
Northern hybridization (see, e.g., Freeman et al., 1983, Proc.
Natl. Acad. Sci. USA 80:4094-4098), restriction endonuclease
mapping (Maniatis, T., 1982, Molecular Cloning, A Laboratory, Cold
Spring Harbor, N.Y.), and DNA sequence analysis. Polymerase chain
reaction (PCR; U.S. Pat. Nos. 4,683,202, 4,683,195, and 4,889,818;
Gyllenstein et al., 1988, Proc. Natl. Acad. Sci. USA 85:7652-7656;
Ochman et al., 1988, Genetics 120:621-623; Loh et al., 1989,
Science 243:217-220) followed by Southern hybridization with a
FHIT-specific probe can allow the detection of the FHIT gene in DNA
from various cell types. In one embodiment, Southern hybridization
may be used to determine the genetic linkage of FHIT. PCR followed
by hybridization assay can also be used to detect or measure FHIT
RNA or 3p14.2 chromosomal or molecular abnormalities. Northern
hybridization analysis can be used to determine the expression
levels of the PHIT gene. Other assays are described in Section
5.11. Various cell types, at various states of development or
activity can be tested for FHIT expression. The stringency of the
hybridization conditions for both Southern and Northern
hybridization, or dot blots, can be manipulated to ensure detection
of nucleic acids with the desired degree of relatedness to the
specific FHIT probe used.
[0112] Restriction endonuclease mapping can be used to roughly
determine the genetic structure of the FHIT gene. Restriction maps
derived by restriction endonuclease cleavage can be confirmed by
DNA sequence analysis.
[0113] DNA sequence analysis can be performed by any techniques
known in the art, including but not limited to the method of Maxam
and Gilbert (1980, Meth. Enzymol. 65:499-560), the Sanger dideoxy
method (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74:5463),
the use of T7 DNA polymerase (Tabor and Richardson, U.S. Pat. No.
4,795,699), or use of an automated DNA sequenator (e.g., Applied
Biosystems, Foster City, Calif.). The cDNA sequence of a
representative FHIT gene comprises the sequence substantially as
depicted in FIG. 2A (SEQ ID NO: 1), and described in Section 6,
infra.
5.5.2. Protein Analysis
[0114] The amino acid sequence of the Fhit protein can be derived
by deduction from the DNA sequence, or alternatively, by direct
sequencing of the protein, e.g., with an automated amino acid
sequencer. The amino acid sequence of a representative Fhit protein
comprises the sequence substantially as depicted in FIG. 2A (SEQ ID
NO: 2), and detailed in Section 6, infra, with the representative
mature protein that is shown by amino acid numbers 1-147.
[0115] The Fhit protein sequence can be further characterized by a
hydrophilicity analysis (Hopp, T. and Woods, K., 1981, Proc. Natl.
Acad. Sci. USA 78:3824). A hydrophilicity profile can be used to
identify the hydrophobic and hydrophilic regions of the Fhit
protein and the corresponding regions of the gene sequence which
encode such regions.
[0116] Secondary structural analysis (Chou, P. and Fasman, G.,
1974, Biochemistry 13:222) can also be done, to identify regions of
the Fhit protein that assume specific secondary structures.
[0117] Manipulation, translation, and secondary structure
prediction, as well as open reading frame prediction and plotting,
can also be accomplished using computer software programs available
in the art.
[0118] Other methods of structural analysis can also be employed.
These include but are not limited to X-ray crystallography
(Engstom, A., 1974, Biochem. Exp. Biol. 11:7-13) and computer
modeling (Fletterick, R. and Zoller, M. (eds.), 1986, Computer
Graphics and Molecular Modeling, in Current Communications in
Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y.).
5.6. Fhit Proteins, Derivatives and Analogs
[0119] The invention further relates to Fhit proteins, and
derivatives (including but not limited to fragments) and analogs of
Fhit proteins. Nucleic acids encoding Fhit protein derivatives and
protein analogs are also provided. Molecules comprising Fhit
proteins or derivatives are also provided. In one embodiment, the
Fhit proteins are encoded by the Fhit nucleic acids described in
Section 5.1 supra. In particular aspects, the proteins,
derivatives, or analogs are of Fhit proteins of animals.
[0120] The production and use of derivatives and analogs related to
Fhit are within the scope of the present invention. In a specific
embodiment, the derivative or analog is functionally active, i.e.,
capable of exhibiting one or more functional activities associated
with a full-length, wild-type Fhit protein. As one example, such
derivatives or analogs which have the desired immunogenicity or
antigenicity can be used, for example, in immunoassays, for
immunization, for inhibition of Fhit activity, etc. As another
example, such derivatives or analogs which have hydrolase activity
are provided. Derivatives or analogs that retain, or alternatively
lack or inhibit, a desired Fhit property of interest (e.g.,
inhibition of cell proliferation, tumor inhibition), can be used as
inducers, or inhibitors, respectively, of such property and its
physiological correlates. A specific embodiment relates to a Fhit
fragment that can be bound by an anti-Fhit antibody. Derivatives or
analogs of Fhit can be tested for the desired activity by
procedures known in the art, including but not limited to the
assays described infra.
[0121] In particular, Fhit derivatives can be made by altering FHIT
sequences by substitutions, additions or deletions that provide for
functionally equivalent molecules. Due to the degeneracy of
nucleotide coding sequences, other DNA sequences which encode
substantially the same amino acid sequence as a FHIT gene may be
used in the practice of the present invention. These include but
are not limited to nucleotide sequences comprising all or portions
of FHIT genes which are altered by the substitution of different
codons that encode a functionally equivalent amino acid residue
within the sequence, thus producing a silent change. Likewise, the
Fhit derivatives of the invention include, but are not limited to,
those containing, as a primary amino acid sequence, all or part of
the amino acid sequence of a Fhit protein including altered
sequences in which functionally equivalent amino acid residues are
substituted for residues within the sequence resulting in a silent
change. For example, one or more amino acid residues within the
sequence can be substituted by another amino acid of a similar
polarity which acts as a functional equivalent, resulting in a
silent alteration. Substitutes for an amino acid within the
sequence may be selected from other members of the class to which
the amino acid belongs. For example, the nonpolar (hydrophobic)
amino acids include alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan and methionine. The polar neutral amino
acids include glycine, serine, threonine, cysteine, tyrosine,
asparagine, and glutamine. The positively charged (basic) amino
acids include arginine, lysine and histidine. The negatively
charged (acidic) amino acids include aspartic acid and glutamic
acid.
[0122] In a specific embodiment of the invention, proteins
consisting of or comprising a fragment of a Fhit protein consisting
of at least 10 (continuous) amino acids of the Fhit protein is
provided. In other embodiments, the fragment consists of at least
20 or 50 amino acids of the Fhit protein. In specific embodiments,
such fragments are not larger than 35, 100 or 140 amino acids.
Derivatives or analogs of Fhit include but are not limited to those
molecules comprising regions that are substantially homologous to
Fhit or fragments thereof (e.g., in various embodiments, at least
60% or 70% or 80% or 90% or 95% identity over an amino acid
sequence of identical size or when compared to an aligned sequence
in which the alignment is done by a computer homology program known
in the art) or whose encoding nucleic acid is capable of
hybridizing to a coding FHIT sequence, under stringent, moderately
stringent, or nonstringent conditions.
[0123] The Fhit derivatives and analogs of the invention can be
produced by various methods known in the art. The manipulations
which result in their production can occur at the gene or protein
level. For example, the cloned FHIT gene sequence can be modified
by any of numerous strategies known in the art (Maniatis, T., 1990,
Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.). The sequence can be cleaved
at appropriate sites with restriction endonuclease(s), followed by
further enzymatic modification if desired, isolated, and ligated in
vitro. In the production of the gene encoding a derivative or
analog of Fhit, care should be taken to ensure that the modified
gene remains within the same translational reading frame as Fhit,
uninterrupted by translational stop signals, in the gene region
where the desired Fhit activity is encoded.
[0124] Additionally, the Fhit-encoding nucleic acid sequence can be
mutated in vitro or in vivo, to create and/or destroy translation,
initiation, and/or termination sequences, or to create variations
in coding regions and/or form new restriction endonuclease sites or
destroy preexisting ones, to facilitate further in vitro
modification. Any technique for mutagenesis known in the art can be
used, including but not limited to, chemical mutagenesis, in vitro
site-directed mutagenesis (Hutchinson, C., et al., 1978, J. Biol.
Chem 253:6551), etc.
[0125] Manipulations of the Fhit sequence may also be made at the
protein level. Included within the scope of the invention are Fhit
protein fragments or other derivatives or analogs which are
differentially modified during or after translation, e.g., by
glycosylation, acetylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to an antibody molecule or other cellular ligand,
etc. Any of numerous chemical modifications may be carried out by
known techniques, including but not limited to specific chemical
cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8
protease, NaBH.sub.4; acetylation, formylation, oxidation,
reduction; metabolic synthesis in the presence of tunicamycin;
etc.
[0126] In addition, analogs and derivatives of Fhit can be
chemically synthesized. For example, a peptide corresponding to a
portion of a Fhit protein which comprises the desired domain (see
Section 5.6.1), or which mediates the desired activity in vitro,
can be synthesized by use of a peptide synthesizer. Furthermore, if
desired, nonclassical amino acids or chemical amino acid analogs
can be introduced as a substitution or addition into the Fhit
sequence. Non-classical amino acids include but are not limited to
the D-isomers of the common amino acids, .alpha.-amino isobutyric
acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, .gamma.-Abu,
.epsilon.-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid,
3-amino propionic acid, ornithine, norleucine, norvaline,
hydroxyproline, sarcosine, citrulline, cysteic acid,
t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,
.beta.-alanine, fluoro-amino acids, designer amino acids such as
.beta.-methyl amino acids, C.alpha.-methyl amino acids,
N.alpha.-methyl amino acids, and amino acid analogs in general.
Furthermore, the amino acid can be D (dextrorotary) or L
(levorotary).
[0127] In a specific embodiment, the Fhit derivative is a chimeric,
or fusion, protein comprising a Fhit protein or fragment thereof
(preferably consisting of at least a domain or motif of the Fhit
protein, or at least 10 amino acids of the Fhit protein) joined at
its amino- or carboxy-terminus via a peptide bond to an amino acid
sequence of a different protein. In one embodiment, such a chimeric
protein is produced by recombinant expression of a nucleic acid
encoding the protein (comprising a Fhit-coding sequence joined
in-frame to a coding sequence for a different protein). Such a
chimeric product can be made by ligating the appropriate nucleic
acid sequences encoding the desired amino acid sequences to each
other by methods known in the art, in the proper coding frame, and
expressing the chimeric product by methods commonly known in the
art. Alternatively, such a chimeric product may be made by protein
synthetic techniques, e.g., by use of a peptide synthesizer.
Chimeric genes comprising portions of FHIT fused to any
heterologous protein-encoding sequences may be constructed.
[0128] In another specific embodiment, the Fhit derivative is a
molecule comprising a region of homology with a Fhit protein. By
way of example, in various embodiments, a first protein region can
be considered "homologous" to a second protein region when the
amino acid sequence of the first region is at least 30%, 40%, 50%,
60%, 70%, 75%, 80%, 90%, or 95% identical, when compared to any
sequence in the second region of an equal number of amino acids as
the number contained in the first region or when compared to an
aligned sequence of the second region that has been aligned by a
computer homology program known in the art. For example, a molecule
can comprise one or more regions homologous to a Fhit domain (see
Section 5.6.1) or a portion thereof or a full-length Fhit
protein.
5.7. Assays of Fhit Proteins, Derivatives and Analogs
[0129] The functional activity of Fhit proteins, derivatives and
analogs can be assayed by various methods.
[0130] For example, in one embodiment, where one is assaying for
the ability to bind or compete with wild-type Fhit for binding to
anti-Fhit antibody, various immunoassays known in the art can be
used, including but not limited to competitive and non-competitive
assay systems using techniques such as radioimmunoassays, ELISA
(enzyme linked immunosorbent assay), "sandwich" immunoassays,
immunoradiometric assays, gel diffusion precipitin reactions,
immunodiffusion assays, in situ immunoassays (using colloidal gold,
enzyme or radioisotope labels, for example), western blots,
precipitation reactions, agglutination assays (e.g., gel
agglutination assays, hemagglutination assays), complement fixation
assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc. In one embodiment, antibody
binding is detected by detecting a label on the primary antibody.
In another embodiment, the primary antibody is detected by
detecting binding of a secondary antibody or reagent to the primary
antibody. In a further embodiment, the secondary antibody is
labelled. Many means are known in the art for detecting binding in
an immunoassay and are within the scope of the present
invention.
[0131] In another embodiment, where a Fhit-binding protein is
identified, the binding can be assayed, e.g., by means well-known
in the art.
[0132] In another embodiment, should a Fhit protein have hydrolase
activity, hydrolase assays can be used to measure Fhit hydrolase
activity. Such assays can be carried out by methods well known in
the art.
[0133] In addition, assays known in the art can be used to detect
or measure the ability to inhibit cell proliferation, in vitro or
in vivo.
[0134] Other methods will be known to the skilled artisan and are
within the scope of the invention.
5.8. Therapeutic Uses: Treatment and Prevention of Disorders
Involving Overproliferation of Cells
[0135] The invention provides for treatment or prevention of
various diseases and disorders by administration of a therapeutic
compound (termed herein "Therapeutic"). Such "Therapeutics" include
but are not limited to: Fhit proteins and analogs and derivatives
(including fragments) thereof (e.g., as described hereinabove);
antibodies thereto (as described hereinabove); nucleic acids
encoding the Fhit proteins, analogs, or derivatives (e.g., as
described hereinabove); and Fhit agonists, and antagonists of
mutant FHIT genes or proteins (e.g., antibodies or antisense
nucleic acids). In a preferred embodiment, disorders involving cell
overproliferation are treated or prevented by administration of a
Therapeutic that promotes Fhit function. The above is described in
detail in the subsections below.
[0136] Generally, administration of products of a species origin or
species reactivity (in the case of antibodies) that is the same
species as that of the patient is preferred. Thus, in a preferred
embodiment, a human Fhit protein, derivative, or analog, or nucleic
acid, or an antibody to a human Fhit protein or human FHIT
antisense nucleic acid, is therapeutically or prophylactically
administered to a human patient.
[0137] Additional descriptions and sources of Therapeutics that can
be used according to the invention are found in Sections 5.1
through 5.7 herein.
[0138] A FHIT polynucleotide and its Fhit protein product can be
used for therapeutic/prophylactic purposes for diseases involving
cell overproliferation, as well as other disorders associated with
chromosomal translocations or inversions or molecular abnormalities
associated with the FHIT locus, and/or decreased expression of
wild-type FHIT RNA or protein and/or expression of a mutant FHIT
RNA or protein and/or expression of a mutant FHIT RNA or protein. A
FHIT polynucleotide, and its FHIT protein product, may be used for
therapeutic/prophylactic purposes alone or in combination with
other therapeutics useful in the treatment of cancer and
hyperproliferative or dysproliferative disorders.
[0139] Diseases and disorders involving cell overproliferation are
treated or prevented by administration of a Therapeutic that
promotes (i.e., increases or supplies) Fhit function. Examples of
such a Therapeutic include but are not limited to Fhit proteins,
derivatives, or fragments that are functionally active,
particularly that are active in inhibiting cell proliferation
(e.g., as demonstrated in in vitro assays or in animal models), and
nucleic acids encoding a Fhit protein or functionally active
derivative or fragment thereof (e.g., for use in gene therapy).
Other Therapeutics that can be used, e.g., Fhit agonists, can be
identified using in vitro assays or animal models, examples of
which are described infra.
[0140] In specific embodiments, Therapeutics that promote Fhit
function are administered therapeutically (including
prophylactically): (1) in diseases or disorders involving an
absence or decreased (relative to normal or desired) level of Fhit
functional protein or of Fhit function, for example, in patients
where Fhit protein is lacking, genetically defective, biologically
inactive or underactive, or underexpressed; or (2) in diseases or
disorders wherein in vitro (or in vivo) assays indicate the utility
of Fhit agonist administration. The absence or decreased level in
Fhit protein or function can be readily detected, e.g., by
obtaining a patient tissue sample (e.g., from biopsy tissue) and
assaying it in vitro for RNA or protein levels, structure and/or
activity of the expressed Fhit RNA or protein (see Section 5.11
infra re assays used in diagnosis). Many methods standard in the
art can be thus employed, including but not limited to immunoassays
to detect and/or visualize Fhit protein (e.g., Western blot,
immunoprecipitation followed by sodium dodecyl sulfate
polyacrylamide gel electrophoresis, immunocytochemistry, etc.)
and/or hybridization assays to detect Fhit expression by detecting
and/or visualizing Fhit mRNA or cDNA (e.g., Northern assays, dot
blots, in situ hybridization, and preferably those assays described
in Section 5.11), etc.
[0141] Diseases and disorders involving cell overproliferation that
can be treated or prevented include but are not limited to
malignancies, premalignant conditions (e.g., hyperplasia,
metaplasia, dysplasia), benign tumors, hyperproliferative
disorders, benign dysproliferative disorders, etc. Examples of
these are detailed below.
5.8.1. Malignancies
[0142] Malignancies and related disorders that can be treated or
prevented by administration of a Therapeutic that promotes Fhit
function (e.g., a full-length Fhit protein or functional derivative
thereof or nucleic acid encoding the foregoing) include but are not
limited to those listed in Table 1 (for a review of such disorders,
see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co.,
Philadelphia): TABLE-US-00001 TABLE 1 MALIGNANCIES AND RELATED
DISORDERS Leukemia acute leukemia acute lymphocytic leukemia acute
myelocytic leukemia myeloblastic promyelocytic myelomonocytic
monocytic erythroleukemia chronic leukemia chronic myelocytic
(granulocytic) leukemia chronic lymphocytic leukemia Polycythemia
vera Lymphoma Hodgkin's disease non-Hodgkin's disease Multiple
myeloma Waldenstrom's macroglobulinemia Heavy chain disease Solid
tumors sarcomas and carcinomas fibrosarcoma myxosarcoma liposarcoma
chondrosarcoma osteogenic sarcoma osteosarcoma chordoma
angiosarcoma endotheliosarcoma lymphangiosarcoma
lymphangioendotheliosarcoma synovioma mesothelioma Ewing's tumor
leiomyosarcoma rhabdomyosarcoma colon carcinoma colorectal
carcinoma pancreatic cancer breast cancer ovarian cancer prostate
cancer squamous cell carcinoma basal cell carcinoma adenocarcinoma
sweat gland carcinoma sebaceous gland carcinoma papillary carcinoma
papillary adenocarcinomas cystadenocarcinoma medullary carcinoma
bronchogenic carcinoma renal cell carcinoma hepatoma bile duct
carcinoma choriocarcinoma seminoma embryonal carcinoma Wilms' tumor
cervical cancer uterine cancer testicular tumor lung carcinoma
small cell lung carcinoma non small cell lung carcinoma bladder
carcinoma epithelial carcinoma glioma astrocytoma medulloblastoma
craniopharyngioma ependymoma pinealoma hemangioblastoma acoustic
neuroma oligodendroglioma menangioma melanoma neuroblastoma
retinoblastoma nasopharyngeal carcinoma esophageal carcinoma
[0143] In a specific embodiment, digestive tract tumors are treated
or prevented, including but not limited to esophageal, stomach,
colon, and colorectal cancers. In another specific embodiment,
airway cancers such as lung cancers (e.g., small cell lung
carcinoma) and nasopharyngeal carcinoma are treated or prevented.
In yet other specific embodiments, malignancy or dysproliferative
changes (such as metaplasias and dysplasias), or hyperproliferative
disorders, are treated or prevented in the head, neck, cervix,
kidney, stomach, skin, ovary, bladder, breast, colon, lung, or
uterus. In other specific embodiments, sarcoma, or leukemia is
treated or prevented. In another particular embodiment,
osteosarcoma or renal cell carcinoma is treated or prevented.
5.8.2. Premalignant Conditions
[0144] The Therapeutics of the invention that promote Fhit activity
can also be administered to treat premalignant conditions and to
prevent progression to a neoplastic or malignant state, including
but not limited to those disorders listed in Table 1. Such
prophylactic or therapeutic use is indicated in conditions known or
suspected of preceding progression to neoplasia or cancer, in
particular, where non-neoplastic cell growth consisting of
hyperplasia, metaplasia, or most particularly, dysplasia has
occurred (for review of such abnormal growth conditions, see
Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders
Co., Philadelphia, pp. 68-79.) Hyperplasia is a form of controlled
cell proliferation involving an increase in cell number in a tissue
or organ, without significant alteration in structure or function.
As but one example, endometrial hyperplasia often precedes
endometrial cancer. Metaplasia is a form of controlled cell growth
in which one type of adult or fully differentiated cell substitutes
for another type of adult cell. Metaplasia can occur in epithelial
or connective tissue cells. Atypical metaplasia involves a somewhat
disorderly metaplastic epithelium. Dysplasia is frequently a
forerunner of cancer, and is found mainly in the epithelia; it is
the most disorderly form of non-neoplastic cell growth, involving a
loss in individual cell uniformity and in the architectural
orientation of cells. Dysplastic cells often have abnormally large,
deeply stained nuclei, and exhibit pleomorphism. Dysplasia
characteristically-occurs where there exists chronic irritation or
inflammation, and is often found in the cervix, respiratory
passages, oral cavity, and gall bladder.
[0145] In a preferred embodiment of the invention, a patient in
whose DNA is detected a mutation in the PHIT gene, particularly a
deletion, and most particularly a homozygous mutation, is thereby
determined to have a predisposition to cancer and is treated by
administration of a Fhit protein or functional derivative thereof
or nucleic acid encoding the same (gene therapy).
[0146] Alternatively or in addition to the presence of abnormal
cell growth characterized as hyperplasia, metaplasia, or dysplasia,
the presence of one or more characteristics of a transformed
phenotype, or of a malignant phenotype, displayed in vivo or
displayed in vitro by a cell sample from a patient, can indicate
the desirability of prophylactic/therapeutic administration of a
Therapeutic that promotes Fhit function. As mentioned supra, such
characteristics of a transformed phenotype include morphology
changes, looser substratum attachment, loss of contact inhibition,
loss of anchorage dependence, protease release, increased sugar
transport, decreased serum requirement, expression of fetal
antigens, disappearance of the 250,000 dalton cell surface protein,
etc. (see also id., at pp. 84-90 for characteristics associated
with a transformed or malignant phenotype).
[0147] In a specific embodiment, leukoplakia, a benign-appearing
hyperplastic or dysplastic lesion of the epithelium, or Bowen's
disease, a carcinoma in situ, are pre-neoplastic lesions indicative
of the desirability of prophylactic intervention.
[0148] In another embodiment, fibrocystic disease (cystic
hyperplasia, mammary dysplasia, particularly adenosis (benign
epithelial hyperplasia)) is indicative of the desirability of
prophylactic intervention.
[0149] In other embodiments, a patient which exhibits one or more
of the following predisposing factors for malignancy is treated by
administration of an effective amount of a Therapeutic: a
chromosomal translocation associated with a malignancy (e.g., the
Philadelphia chromosome for chronic myelogenous leukemia, t(14;18)
for follicular lymphoma, etc.), familial polyposis or Gardner's
syndrome (possible forerunners of colon cancer), benign monoclonal
gammopathy (a possible forerunner of multiple myeloma), and a first
degree kinship with persons having a cancer or precancerous disease
showing a Mendelian (genetic) inheritance pattern (e.g., familial
polyposis of the colon, Gardner's syndrome, hereditary exostosis,
polyendocrine adenomatosis, medullary thyroid carcinoma with
amyloid production and pheochromocytoma, Peutz-Jeghers syndrome,
neurofibromatosis of Von Recklinghausen, retinoblastoma, carotid
body tumor, cutaneous melanocarcinoma, intraocular melanocarcinoma,
xeroderma pigmentosum, ataxia telangiectasia, Chediak-Higashi
syndrome, albinism, Fanconi's aplastic anemia, and Bloom's
syndrome; see Robbins and Angell, 1976, Basic Pathology, 2d Ed.,
W.B. Saunders Co., Philadelphia, pp. 112-113) etc.)
[0150] In a specific embodiment, a Therapeutic of the invention is
administered to a human patient to prevent progression to breast,
colon, lung, stomach or uterine cancer, or melanoma or sarcoma.
5.8.3. Hyperproliferative and Dysproliferative Disorders
[0151] In another embodiment of the invention, a Therapeutic that
promotes Fhit activity is used to treat or prevent
hyperproliferative or benign dysproliferative disorders. Specific
embodiments are directed to treatment or prevention of benign
tumors, fibrocystic conditions, and tissue hypertrophy (e.g.,
prostatic hyperplasia). In specific embodiments, a patient having
an intestinal polyp, colon polyp, or esophageal dysplasia is
treated by administration of a Therapeutic.
5.8.4. Gene Therapy
[0152] In a specific embodiment, nucleic acids comprising a
sequence encoding a Fhit protein or functional derivative thereof,
are administered to promote Fhit function, by way of gene therapy.
Gene therapy refers to therapy performed by the administration of a
nucleic acid to a subject.
[0153] A FHIT polynucleotide may be used in the treatment of
various disease states associated with chromosome 3p14.2
abnormalities, such as cancers, and/or decreased expression of
wild-type FHIT RNA or protein. By introducing FHIT gene sequences
into cells, gene therapy can be used to treat conditions associated
with under-expression of functional FHIT RNA or protein.
Accordingly, the present invention provides a method for treating a
disease state associated with a chromosome 3p14.2 abnormality in
mammal suffering from a disease state associated with a chromosome
3p14.2 abnormality comprising administering a therapeutically
effective amount of a nucleic acid encoding a functional Fhit
protein to a mammal suffering from a disease state associated with
a chromosome 3p14.2 abnormality. In this embodiment of the
invention, the nucleic acid produces its encoded protein that
mediates a therapeutic effect by promoting Fhit function, thereby,
e.g., inhibiting tumor or cancer appearance or progression.
[0154] Any of the methods for gene therapy available in the art can
be used according to the present invention. Exemplary methods are
described below.
[0155] For general reviews of the methods of gene therapy, see
Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu,
1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.
Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and
Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May,
1993, TIBTECH 11(5):155-215). Methods commonly known in the art of
recombinant DNA technology which can be used are described in
Ausubel et al. (eds.), 1993, Current Protocols in Molecular
Biology, John Wiley & Sons, NY; and Kriegler, 1990, Gene
Transfer and Expression, A Laboratory Manual, Stockton Press,
NY.
[0156] In a preferred aspect, the Therapeutic comprises a FHIT
nucleic acid that is part of an expression vector that expresses a
Fhit protein or fragment or chimeric protein thereof in a suitable
host. In particular, such a nucleic acid has a promoter operably
linked to the FHIT coding region, said promoter being inducible or
constitutive, and, optionally, tissue-specific. In another
particular embodiment, a nucleic acid molecule is used in which the
FHIT coding sequences and any other desired sequences are flanked
by regions that promote homologous recombination at a desired site
in the genome, thus providing for intrachromosomal expression of
the FHIT nucleic acid (Koller and Smithies, 1989, Proc. Natl. Acad.
Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature
342:435-438).
[0157] Delivery of the nucleic acid into a patient may be either
direct, in which case the patient is directly exposed to the
nucleic acid or nucleic acid-carrying vector, or indirect, in which
case, cells are first transformed with the nucleic acid in vitro,
then transplanted into the patient. These two approaches are known,
respectively, as in vivo or ex vivo gene therapy.
[0158] In a specific embodiment, the nucleic acid is directly
administered in vivo, where it is expressed to produce the encoded
product. This can be accomplished by any of numerous methods known
in the art, e.g., by constructing it as part of an appropriate
nucleic acid-expression vector and administering it so that it
becomes intracellular, e.g., by infection using a defective or
attenuated retroviral or other viral vector (see U.S. Pat. No.
4,980,286), or by direct injection of naked DNA, or by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or
coating with lipids or cell-surface receptors or transfecting
agents, encapsulation in liposomes, microparticles, or
microcapsules, or by administering it in linkage to a peptide which
is known to enter the nucleus, by administering it in linkage to a
ligand subject to receptor-mediated endocytosis (see e.g., Wu and
Wu, 1987, J. Biol. Chem. 262:4429-4432) (which can be used to
target cell types specifically expressing the receptors), etc. In
another embodiment, a nucleic acid-ligand complex can be formed in
which the ligand comprises a fusogenic viral peptide to disrupt
endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be
targeted in vivo for cell specific uptake and expression, by
targeting a specific receptor (see, e.g., PCT Publications WO
92/06180 dated Apr. 16, 1992 (Wu et al.); WO 92/22635 dated Dec.
23, 1992 (Wilson et al.); WO92/20316 dated Nov. 26, 1992 (Findeis
et al.); WO93/14188 dated Jul. 22, 1993 (Clarke et al.), WO
93/20221 dated Oct. 14, 1993 (Young)). Alternatively, the nucleic
acid can be introduced intracellularly and incorporated within host
cell DNA for expression, by homologous recombination (Koller and
Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra
et al., 1989, Nature 342:435-438).
[0159] In a specific embodiment, a viral vector that contains the
FHIT nucleic acid is used. For example, a retroviral vector can be
used (see Miller et al., 1993, Meth. Enzymol. 217:581-599). These
retroviral vectors have been modified to delete retroviral
sequences that are not necessary for packaging of the viral genome
and integration into host cell DNA. The FHIT nucleic acid to be
used in gene therapy is cloned into the vector, which facilitates
delivery of the gene into a patient. More detail about retroviral
vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302,
which describes the use of a retroviral vector to deliver the mdr1
gene to hematopoietic stem cells in order to make the stem cells
more resistant to chemotherapy. Other references illustrating the
use of retroviral vectors in gene therapy are: Clowes et al., 1994,
J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473;
Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and
Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel.
3:110-114.
[0160] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and
Development 3:499-503 present a review of adenovirus-based gene
therapy. Bout et al., 1994, Human Gene Therapy 5:3-10 demonstrated
the use of adenovirus vectors to transfer genes to the respiratory
epithelia of rhesus monkeys. Other instances of the use of
adenoviruses in gene therapy can be found in Rosenfeld et al.,
1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;
and Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234.
[0161] Adeno-associated virus (AAV) has also been proposed for use
in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med.
204:289-300. Herpesviruses can also be used.
[0162] Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or viral
infection. Usually, the method of transfer includes the transfer of
a selectable marker to the cells. The cells are then placed under
selection to isolate those cells that have taken up and are
expressing the transferred gene. Those cells are then delivered to
a patient.
[0163] In this embodiment, the nucleic acid is introduced into a
cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences, cell
fusion, chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells (see e.g.,
Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al.,
1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther.
29:69-92) and may be used in accordance with the present invention,
provided that the necessary developmental and physiological
functions of the recipient cells are not disrupted. The technique
should provide for the stable transfer of the nucleic acid to the
cell, so that the nucleic acid is expressible by the cell and
preferably heritable and expressible by its cell progeny.
[0164] The resulting recombinant cells can be delivered to a
patient by various methods known in the art. In a preferred
embodiment, epithelial cells are injected, e.g., subcutaneously. In
another embodiment, recombinant skin cells may be applied as a skin
graft onto the patient. Recombinant blood cells (e.g.,
hematopoietic stem or progenitor cells) are preferably administered
intravenously. The amount of cells envisioned for use depends on
the desired effect, patient state, etc., and can be determined by
one skilled in the art.
[0165] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include but are not limited to epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes; blood cells such as T lymphocytes, B lymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,
granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells, e.g., as obtained from bone
marrow, umbilical cord blood, peripheral blood, fetal liver,
etc.
[0166] In a preferred embodiment, the cell used for gene therapy is
autologous to the patient.
[0167] In an embodiment in which recombinant cells are used in gene
therapy, a FHIT nucleic acid is introduced into the cells such that
it is expressible by the cells or their progeny, and the
recombinant cells are then administered in vivo for therapeutic
effect. In a specific embodiment, stem or progenitor cells are
used. Any stem and/or progenitor cells which can be isolated and
maintained in vitro can potentially be used in accordance with this
embodiment of the present invention. Such stem cells include but
are not limited to hematopoietic stem cells (HSC), stem cells of
epithelial tissues such as the skin and the lining of the gut,
embryonic heart muscle cells, liver stem cells (PCT Publication WO
94/08598, dated Apr. 28, 1994), and neural stem cells (Stemple and
Anderson, 1992, Cell 71:973-985).
[0168] Epithelial stem cells (ESCs) or keratinocytes can be
obtained from tissues such as the skin and the lining of the gut by
known procedures (Rheinwald, 1980, Meth. Cell Bio. 21A:229). In
stratified epithelial tissue such as the skin, renewal occurs by
mitosis of stem cells within the germinal layer, the layer closest
to the basal lamina. Stem cells within the lining of the gut
provide for a rapid renewal rate of this tissue. ESCs or
keratinocytes obtained from the skin or lining of the gut of a
patient or donor can be grown in tissue culture (Rheinwald, 1980,
Meth. Cell Bio. 21A:229; Pittelkow and Scott, 1986, Mayo Clinic
Proc. 61:771). If the ESCs are provided by a donor, a method for
suppression of host versus graft reactivity (e.g., irradiation,
drug or antibody administration to promote moderate
immunosuppression) can also be used.
[0169] With respect to hematopoietic stem cells (HSC), any
technique which provides for the isolation, propagation, and
maintenance in vitro of HSC can be used in this embodiment of the
invention. Techniques by which this may be accomplished include (a)
the isolation and establishment of HSC cultures from bone marrow
cells isolated from the future host, or a donor, or (b) the use of
previously established long-term HSC cultures, which may be
allogeneic or xenogeneic. Non-autologous HSC are used preferably in
conjunction with a method of suppressing transplantation immune
reactions of the future host/patient. In a particular embodiment of
the present invention, human bone marrow cells can be obtained from
the posterior iliac crest by needle aspiration (see, e.g., Kodo et
al., 1984, J. Clin. Invest. 73:1377-1384). In a preferred
embodiment of the present invention, the HSCs can be made highly
enriched or in substantially pure form. This enrichment can be
accomplished before, during, or after long-term culturing, and can
be done by any techniques known in the art. Long-term cultures of
bone marrow cells can be established and maintained by using, for
example, modified Dexter cell culture techniques (Dexter et al.,
1977, J. Cell Physiol. 91:335) or Witlock-Witte culture techniques
(Witlock and Witte, 1982, Proc. Natl. Acad. Sci. USA
79:3608-3612).
[0170] In a specific embodiment, the nucleic acid to be introduced
for purposes of gene therapy comprises an inducible promoter
operably linked to the coding region, such that expression of the
nucleic acid is controllable by controlling the presence or absence
of the appropriate inducer of transcription.
[0171] Additional methods that can be adapted for use to deliver a
nucleic acid encoding a Fhit protein or functional derivative
thereof are described in Section 5.8.5.
5.8.5. Antagonizing Dominant-Negative FHIT Mutations for Treatment
or Prevention of Disorders of Overproliferation
[0172] The invention also provides methods of treating or
preventing disorders of overproliferation (e.g., cancer,
hyperproliferative disorders) in which the patient has a hemizygous
FHIT mutation (presumably a dominant-negative FHIT mutation) by
specifically antagonizing (administering an antagonist to) the
mutant FHIT gene or protein (and not wild-type FHIT or Fhit).
Hemizygosity for a FHIT mutation can be detected by observing the
presence of both normal and mutant FHIT DNA (e.g., cDNA) or RNA in
a sample from a patient, e.g., by methods as described in Sections
5.11 and 6 hereof.
[0173] For example, in a specific embodiment, an effective amount
of antisense oligonucleotide that inhibits the expression of the
mutant FHIT gene, and not the wild-type FHIT gene, is administered.
For example, if the hemizygous FHIT mutation in the patient is a
deletion of at least a portion of one or more FHIT exons, the
antisense oligonucleotide can comprise a hybridizable sequence
complementary to the junction formed by the deletion, said junction
being present in the mutant FHIT gene but not the wild-type FHIT
gene. Thus, the antisense oligonucleotide comprises a sequence
complementary to contiguous sequences from two exons not naturally
found contiguous in wild-type FHIT cDNA.
[0174] In another specific embodiment, an antibody can be used
therapeutically or prophylactically to specifically antagonize the
hemizygous Fhit mutant protein. For example, such an antibody can
specifically recognize an epitope in a Fhit deletion mutant formed
by the fusion of sequences not naturally contiguous in the
wild-type Fhit protein. For therapeutic purposes, a Fhit mutant
protein can be used as immunogen to make anti-Fhit antibodies that
neutralize the activity of the Fhit mutant protein and not
wild-type Fhit protein. Accordingly, the present invention provides
a method for treating a disease state associated with a FHIT
abnormality in a mammal suffering from a disease state associated
with a FHIT abnormality comprising administering a therapeutically
effective amount of an anti-Fhit antibody specific to the abnormal
FHIT gene or protein to a mammal suffering from a disease state
associated with a FHIT abnormality.
[0175] In another specific embodiment, a recombinant nucleic acid
consisting of non-FHIT sequences flanked by FHIT sequences so as to
promote homologous recombination specifically with a mutant FHIT
gene in a patient, is introduced into the patient, in order to
"knock out" (inhibit the effect of) the mutant, particularly where
such mutant is believed to be a dominant-negative one.
[0176] Antisense oligonucleotides are described in further detail
below.
5.8.5.1. Antisense Regulation of Mutant FHIT Gene Expression
[0177] In a specific embodiment, mutant function of Fhit or FHIT is
specifically inhibited by use of FHIT antisense nucleic acids. The
present invention provides the therapeutic or prophylactic use of
nucleic acids of at least six nucleotides that are antisense to a
gene or cDNA encoding a mutant Fhit. A FHIT "antisense" nucleic
acid as used herein refers to a nucleic acid capable of hybridizing
to a portion of a FHIT RNA (preferably mRNA) or mutant form thereof
by virtue of some sequence complementarity (other than to
nonspecific sequences such as a polyA tail). The antisense nucleic
acid may be complementary to a coding and/or noncoding region of a
FHIT mRNA. Such antisense nucleic acids have utility as
Therapeutics that inhibit dominant-negative mutant Fhit function,
and can be used in the treatment or prevention of disorders as
described supra in Section 5.8 and its subsections.
[0178] The antisense nucleic acids of the invention can be
oligonucleotides that are double-stranded or single-stranded, RNA
or DNA or a modification or derivative thereof, which can be
directly administered to a cell, or which can be produced
intracellularly by transcription of exogenous, introduced
sequences.
[0179] The invention further provides pharmaceutical compositions
comprising an effective amount of the FHIT antisense nucleic acids
of the invention in a pharmaceutically acceptable carrier, as
described in Section 5.1.0.
[0180] In another embodiment, the invention is directed to methods
for inhibiting the expression specifically of a FHIT mutant nucleic
acid sequence in a prokaryotic or eukaryotic cell comprising
providing the cell with an effective amount of a composition
comprising an FHIT antisense nucleic acid of the invention.
[0181] The FHIT antisense nucleic acids are of at least six
nucleotides and are preferably oligonucleotides (ranging from 6 to
about 50 oligonucleotides). In specific aspects, the
oligonucleotide is at least 10 nucleotides, at least 15
nucleotides, at least 100 nucleotides, or at least 200 nucleotides.
The oligonucleotides can be DNA or RNA or chimeric mixtures or
derivatives or modified versions thereof, single-stranded or
double-stranded. The oligonucleotide can be modified at the base
moiety, sugar moiety, or phosphate backbone. The oligonucleotide
may include other appending groups such as peptides, or agents
facilitating transport across the cell membrane (see, e.g.,
Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556;
Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT
Publication No. WO 88/09810, published Dec. 15, 1988) or
blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134,
published Apr. 25, 1988), hybridization-triggered cleavage agents
(see, e.g., Krol et al., 1988, BioTechniques 6:958-976) or
intercalating agents (see, e.g., Zon, 1988, Pharm. Res.
5:539-549).
[0182] In a preferred aspect of the invention, a FHIT antisense
oligonucleotide is provided, preferably of single-stranded DNA. In
a most preferred aspect, such an oligonucleotide comprises a
sequence antisense to a junction of two non-normally contiguous
sequences in a FHIT gene deletion mutant, most preferably, of a
human FHIT gene mutant. The oligonucleotide may be modified at any
position on its structure with substituents generally known in the
art.
[0183] The FHIT antisense oligonucleotide may comprise at least one
modified base moiety which is selected from the group including but
not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthihe, xantine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0184] In another embodiment, the oligonucleotide comprises at
least one modified sugar moiety selected from the group including
but not limited to arabinose, 2-fluoroarabinose, xylulose, and
hexose.
[0185] In yet another embodiment, the oligonucleotide comprises at
least one modified phosphate backbone selected from the group
consisting of a phosphorothioate, a phosphorodithioate, a
phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a
methylphosphonate, an alkyl phosphotriester, and a formacetal or
analog thereof.
[0186] In yet another embodiment, the oligonucleotide is an
.alpha.-anomeric oligonucleotide. An .alpha.-anomeric
oligonucleotide forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .beta.-units, the
strands run parallel to each other (Gautier et al., 1987, Nucl.
Acids Res. 15:6625-6641).
[0187] The oligonucleotide may be conjugated to another molecule,
e.g., a peptide, hybridization triggered cross-linking agent,
transport agent, hybridization-triggered cleavage agent, etc.
[0188] Oligonucleotides of the invention may be synthesized by
standard methods known in the art, e.g. by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). These include techniques for chemically
synthesizing oligodeoxyribonucleotides well known in the art such
as for example solid phase phosphoramidite chemical synthesis. As
examples, phosphorothioate oligonucleotides may be synthesized by
the method of Stein et al. (1988, Nucl. Acids Res. 16:3209),
methylphosphonate oligonucleotides can be prepared by use of
controlled pore glass polymer supports (Sarin et al., 1988, Proc.
Natl. Acad. Sci. U.S.A. 85:7448-7451), etc. Alternatively, RNA
molecules may be generated by in vitro and in vivo transcription of
DNA sequences encoding the antisense RNA molecule. Such DNA
sequences may be incorporated into a wide variety of vectors which
incorporate suitable RNA polymerase promoters such as the T7 or SP6
polymerase promoters. Alternatively, antisense cDNA constructs that
synthesize antisense RNA constitutively or inducibly, depending on
the promoter used, can be introduced stably into cell lines.
[0189] In a specific embodiment, the PHIT antisense oligonucleotide
comprises catalytic RNA, or a ribozyme (see, e.g., PCT
International Publication WO 90/11364, published Oct. 4, 1990;
Sarver et al., 1990, Science 247:1222-1225). Ribozymes are
enzymatic RNA molecules capable of catalyzing the specific cleavage
of RNA. The mechanism of ribozyme action involves sequence specific
hybridization of the ribozyme molecule to complementary target RNA,
followed by a endonucleolytic cleavage. Within the scope of the
invention are engineered hammerhead motif ribozyme molecules that
specifically and efficiently catalyze endonucleolytic cleavage of
mutant FHIT RNA sequences.
[0190] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage-sites which include the following sequences, GUA,
GUU and GUC. Once identified, short RNA sequences of between 15 and
20 ribonucleotides corresponding to the region of the target gene
containing the cleavage site may be evaluated for predicted
structural features such as secondary structure that may render the
oligonucleotide sequence unsuitable. The suitability of candidate
targets may also be evaluated by testing their accessibility to
hybridization with complementary oligonucleotides, using
ribonuclease protection assays.
[0191] In another embodiment, the oligonucleotide is a
2'-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.
15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987,
FEBS Lett. 215:327-330).
[0192] In an alternative embodiment, the FHIT antisense nucleic
acid of the invention is produced intracellularly by transcription
from an exogenous sequence. For example, a vector can be introduced
in vivo such that it is taken up by a cell, within which cell the
vector or a portion thereof is transcribed, producing an antisense
nucleic acid (RNA) of the invention. Such a vector would contain a
sequence encoding the FHIT antisense nucleic acid. Such a vector
can remain episomal or become chromosomally integrated, as long as
it can be transcribed to produce the desired antisense RNA. Such
vectors can be constructed by recombinant DNA technology methods
standard in the art. Vectors can be plasmid, viral, or others known
in the art, used for replication and expression in mammalian cells.
Expression of the sequence encoding the FHIT antisense RNA can be
by any promoter known in the art to act in mammalian, preferably
human, cells. Such promoters can be inducible or constitutive. Such
promoters include but are not limited to: the SV40 early promoter
region (Bernoist and Chambon, 1981, Nature 290:304-310), the
promoter contained in the 3' long terminal repeat of Rous sarcoma
virus (Yamamoto et al., 1980, Cell 22:787-797), the herpes
thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad.
Sci. U.S.A. 78:1441-1445), the regulatory sequences of the
metallothionein gene (Brinster et al., 1982, Nature 296:39-42),
etc.
[0193] The antisense nucleic acids of the invention comprise a
sequence complementary to at least a portion of an RNA transcript
of a FHIT gene, preferably a human mutant FHIT gene. However,
absolute complementarity, although preferred, is not required. A
sequence "complementary to at least a portion of an RNA," as
referred to herein, means a sequence having sufficient
complementarity to be able to hybridize with the RNA, forming a
stable duplex; in the case of double-stranded FRIT antisense
nucleic acids, a single strand of the duplex DNA may thus be
tested, or triplex formation may be assayed. The ability to
hybridize will depend on both the degree of complementarity and the
length of the antisense nucleic acid. Generally, the longer the
hybridizing nucleic acid, the more base mismatches with a FHIT RNA
it may contain and still form a stable duplex (or triplex, as the
case may be). One skilled in the art can ascertain a tolerable
degree of mismatch by use of standard procedures to determine the
melting point of the hybridized complex.
[0194] The FHIT antisense nucleic acids can be used to treat (or
prevent) malignancies or hyperproliferative disorders, of a cell
type which has been shown to express mutant FHIT RNA. Malignant,
neoplastic, and pre-neoplastic cells which can be tested for such
expression include but are not limited to those described supra in
Sections 5.8. In a preferred embodiment, a single-stranded DNA
antisense FHIT oligonucleotide is used.
[0195] Malignant (particularly, tumor) cell types which express
FHIT RNA can be identified by various methods known in the art.
Such methods include but are not limited to hybridization with a
FHIT-specific nucleic acid (e.g., by Northern hybridization, dot
blot hybridization, in situ hybridization), observing the ability
of RNA from the cell type to be translated in vitro into Fhit
protein, etc. (see the assays described for diagnosis in Section
5.11). In a preferred aspect, primary tumor tissue from a patient
can be assayed for FHIT expression prior to treatment.
[0196] Pharmaceutical compositions of the invention, comprising an
effective amount of a FHIT antisense nucleic acid in a
pharmaceutically acceptable carrier, can be administered to a
patient having a malignancy which is of a type that expresses
mutant FHIT RNA that is specifically antagonized by the antisense
nucleic acid.
[0197] The amount of FHIT antisense nucleic acid which will be
effective in the treatment of a particular disease state or
condition will depend on the nature of the disease state or
condition, and can be determined by standard clinical techniques.
Where possible, it is desirable to determine the antisense
cytotoxicity of the tumor type to be treated in vitro, and then in
useful animal model systems prior to testing and use in humans.
[0198] In a specific embodiment, pharmaceutical compositions
comprising FHIT antisense nucleic acids are administered via
liposomes, microparticles, or microcapsules. In various embodiments
of the invention, it may be useful to use such compositions to
achieve sustained release of the FHIT antisense nucleic acids. In a
specific embodiment, it may be desirable to utilize liposomes
targeted via antibodies to specific identifiable tumor antigens
(Leonetti et al., 1990, Proc. Natl. Acad. Sci. USA 87:2448-2451;
Renneisen et al., 1990, J. Biol. Chem. 265:16337-16342).
[0199] In a particular embodiment of the invention, antisense FHIT
oligonucleotides or anti-Fhit antibodies that specifically
antagonize a mutant PHIT gene or protein present in a patient, are
administered to the patient in combination with administration to
the patient of FHIT gene therapy (administration of wild-type Fhit
function) or functional Fhit protein or agonists.
5.9. Demonstration of Therapeutic or Prophylactic Utility
[0200] The FHIT polynucleotides, FHIT protein products, derivatives
and analogs thereof, and antibodies thereto, and antisense nucleic
acids of the invention can be tested in vivo for the desired
therapeutic or prophylactic activity. For example, such compounds
can be tested in suitable animal model systems prior to testing in
humans, including but not limited to rats, mice, chicken, cows,
monkeys, rabbits, etc. For in vivo testing, prior to administration
to humans, any animal model system known in the art may be
used.
5.10. Therapeutic/Prophylactic Methods and Compositions
[0201] The invention provides methods of treatment and prophylaxis
by administration to a subject of an effective amount of a
Therapeutic, i.e., a FHIT nucleic acid, FHIT protein, derivative or
analog thereof, or antibody thereto of the present invention. In a
preferred aspect, the Therapeutic is substantially purified. The
subject is preferably an animal, including but not limited to
animals such as cows, pigs, chickens, etc., and is preferably a
mammal, and most preferably human. The subject can be a fetus,
child, or adult.
[0202] In a specific embodiment, a non-human mammal is the
subject.
[0203] Formulations and methods of administration that can be
employed when the Therapeutic comprises a nucleic acid are
described in Sections 5.8.4 and 5.8.5.1 above; additional
appropriate formulations and routes of administration can be
selected from among those described hereinbelow.
[0204] Various delivery systems are known and can be used to
administer a Therapeutic of the invention, e.g., encapsulation in
liposomes, microparticles, microcapsules, expression by recombinant
cells, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987,
J. Biol. Chem. 262:4429-4432), construction of a therapeutic
nucleic acid as part of a retroviral or other vector, etc. Methods
of introduction include but are not limited to intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, and oral routes. The compounds may be administered by
any convenient route, for example by infusion or bolus injection,
by absorption through epithelial or mucocutaneous linings (e.g.,
oral mucosa, rectal and intestinal mucosa, etc.) and may be
administered together with other biologically active agents.
Administration can be systemic or local. In addition, it may be
desirable to introduce the pharmaceutical compositions of the
invention into the central nervous system by any suitable route,
including intraventricular and intrathecal injection;
intraventricular injection may be facilitated by an
intraventricular catheter, for example, attached to a reservoir,
such as an Ommaya reservoir.
[0205] In a specific embodiment, it may be desirable to administer
the pharmaceutical compositions of the invention locally to the
area in need of treatment; this may be achieved by, for example,
and not by way of limitation, local infusion during surgery,
topical application, e.g., in conjunction with a wound dressing
after surgery, by injection, by means of a catheter, by means of a
suppository, or by means of an implant, said implant being of a
porous, non-porous, or gelatinous material, including membranes,
such as sialastic membranes, or fibers. In one embodiment,
administration can be by direct injection at the site (or former
site) of a malignant tumor or neoplastic or pre-neoplastic
tissue.
[0206] In a specific embodiment where the Therapeutic is a nucleic
acid encoding a protein therapeutic, the nucleic acid can be
administered in vivo to promote expression of its encoded protein,
by constructing it as part of an appropriate nucleic acid
expression vector and administering it so that it becomes
intracellular, e.g., by use of a retroviral vector (see U.S. Pat.
No. 4,980,286), or by direct injection, or by use of microparticle
bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with
lipids or cell-surface receptors or transfecting agents, or by
administering it in linkage to a homeobox-like peptide which is
known to enter the nucleus (see e.g., Joliot et al., 1991, Proc.
Natl. Acad. Sci. USA 88:1864-1868), etc. Alternatively, a nucleic
acid therapeutic can be introduced intracellularly and incorporated
within host cell DNA for expression, by homologous
recombination.
[0207] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of a therapeutic, and a pharmaceutically
acceptable carrier or excipient. Such a carrier includes but is not
limited to saline, buffered saline, dextrose, water, glycerol,
ethanol, and combinations thereof. The carrier and composition can
be sterile. The formulation should suit the mode of
administration.
[0208] The composition, if desired, can also contain minor amounts
of wetting or emulsifying agents, or pH buffering agents. The
composition can be a liquid solution, suspension, emulsion, tablet,
pill, capsule, sustained release formulation, or powder. The
composition can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. Oral formulation can
include standard carriers such as pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, etc.
[0209] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0210] The Therapeutics of the invention can be formulated as
neutral or salt forms. Pharmaceutically acceptable salts include
those formed with free amino groups such as those derived from
hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and
those formed with free carboxyl groups such as those derived from
sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0211] The amount of the Therapeutic of the invention which will be
effective in the treatment of a particular disorder or condition
will depend on the nature of the disorder or condition, and can be
determined by standard clinical techniques. In addition, in vitro
assays may optionally be employed to help identify optimal dosage
ranges. The precise dose to be employed in the formulation will
also depend on the route of administration, and the seriousness of
the disease or disorder, and should be decided according to the
judgment of the practitioner and each patient's circumstances.
However, suitable dosage ranges for intravenous administration are
generally about 20-500 micrograms of active compound per kilogram
body weight. Suitable dosage ranges for intranasal administration
are generally about 0.01 pg/kg body weight to 1 mg/kg body weight.
Effective doses may be extrapolated from dose-response curves
derived from in vitro or animal model test systems.
[0212] Suppositories generally contain active ingredient in the
range of 0.5% to 10% by weight; oral formulations preferably
contain 10% to 95% active ingredient.
[0213] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
5.11. Diagnostic Uses
[0214] A FHIT polynucleotide and nucleic acids complementary
thereto, its Fhit protein product, fragments thereof, and
antibodies thereto can be used for diagnostic purposes for
disorders involving overproliferation of cells, as well as other
disorders associated with chromosomal translocations and inversions
or molecular abnormalities associated with the FHIT gene, and/or
decreased expression of wild-type FHIT RNA or protein.
[0215] Such molecules can also be used in diagnostic assays, such
as immunoassays, to detect, prognose, diagnose, or monitor various
conditions, diseases, and disorders associated with expression of
mutant FHIT transcripts or monitor the treatment thereof.
Accordingly, in specific embodiments, cancer or premalignant
changes or hyperproliferative or benign dysproliferative disorders
in tissue is diagnosed by detecting the presence of one or more
mutant FHIT transcripts, alone or in combination with a decrease in
expression of wild-type FHIT transcript, in patient samples
relative to FHIT expression in an analogous non-diseased sample
(from the patient or another person, as determined experimentally
or as is known as a standard level in such samples). For diagnostic
purposes, a FHIT polynucleotide may be used to detect mutant FHIT
gene expression in disease states.
[0216] The subject, or patient, is an animal, e.g., a mammal, and
is preferably human, and can be a fetus, child, or adult.
[0217] As illustrated infra, the FHIT gene sequence is associated
with cancers, particularly associated with translocations and
deletions within the FHIT gene. In specific embodiments, diseases
and disorders involving overproliferation of cells can be
diagnosed, or their suspected presence can be screened for, or a
predisposition to develop such disorders can be detected, by
detecting decreased levels of wild-type Fhit protein, wild-type
FHIT RNA, or Fhit functional activity, or by detecting mutations in
FHIT RNA, DNA, cDNA, or protein (e.g., translocations or deletions
in FHIT nucleic acids, truncations in the FHIT gene or protein,
changes in nucleotide or amino acid sequence relative to wild-type
Fhit) that cause decreased expression or activity of Fhit or a
dominant-negative effect. Such diseases and disorders include but
are not limited to those described in Section 5.8 and its
subsections. By way of example, levels of Fhit protein can be
detected by immunoassay, levels of FHIT RNA can be detected by
hybridization assays (e.g., Northern blots, dot blots) or RT-PCR,
translocations, deletions, and point mutations in FHIT nucleic
acids can be detected by Southern blotting, RFLP analysis, PCR of
cDNA using primers that preferably generate a fragment spanning at
least most of the FHIT gene, sequencing of the FHIT genomic DNA or
cDNA obtained from the patient, etc.
[0218] In a preferred embodiment, levels of FHIT mRNA (or cDNA) or
protein in a patient sample are detected or measured or analyzed by
size and/or sequence, in which aberrant levels, size or sequence
indicate that the subject has, or has a predisposition to
developing, a malignancy or hyperproliferative disorder; in which
the decreased levels are relative to the levels present in an
analogous sample from a portion of the body or from a subject not
having the malignancy or hyperproliferative disorder, as the case
may be.
[0219] FHIT gene sequences may be used diagnostically for the
detection of diseases states resulting from chromosomal or
molecular abnormalities, e.g., translocations, inversions and
deletions, involving the FHIT gene. Nucleic acids comprising FHIT
nucleotide sequences of at least 8 nucleotides, at least 15
nucleotides, at least 25 nucleotides, at least 50 nucleotides, at
least 100 nucleotides, at least 200 nucleotides, at least 300
nucleotides, and preferably less than 500 nucleotides, and the
nucleic acids described in Section 5.1, may be used as probes in
hybridization assays for the detection and measurement of FHIT gene
sequences. Nucleic acids of not more than 5 kilobases, of not more
than 10 kilobases, not more than 25 kilobases, not more than 50
kilobases or not more than 70 kilobases which are hybridizable to a
FHIT gene, cDNA, or complementary strand can be used as probes in
hybridization assays for the detection and measurement of FHIT
nucleotide sequences. As an example, the FHIT DNA sequence may be
used in hybridization assays, e.g., Southern or Northern analysis,
including in situ hybridization assays, of patient's samples to
diagnose abnormalities of FHIT expression. Hybridization assays can
be used to detect, prognose, diagnose, or monitor malignancies,
associated with aberrant changes in FHIT expression and/or activity
as described supra. In particular, such a hybridization assay is
carried out by a method comprising contacting a sample containing
nucleic acid with a nucleic acid probe capable of hybridizing to
FHIT DNA (e.g., cDNA) or RNA, under conditions such that
hybridization can occur, and detecting or measuring any resulting
hybridization. In particular, hybridization assays can be used to
detect the presence of abnormalities associated with expression of
mutant FHIT mRNA, by hybridizing mRNA or cDNA from a patient sample
to a FHIT probe, and analyzing by size and/or sequence the
resulting hybridized nucleic acids. For example, assays which can
be used include, but are not limited to Northern blots, dot blots,
etc. A particular hybridization assay is Northern blot analysis of
a patient sample using FHIT gene probes of at least 15 nucleotides
up to the full length cDNA sequence shown in FIG. 2A. Another
hybridization assay is in situ hybridization analysis of a patient
sample using anti-FHIT antibodies or FHIT nucleotide hybridization
probes. Such techniques are well known in the art, and are in fact
the basis of many commercially available diagnostic kits.
[0220] In a specific embodiment, cancer or other disorder of cell
overproliferation (e.g., those described in Sections 5.8.1-5.8.3
above), is diagnosed or prognosed by detecting a mutation in the
FHIT gene or its produced RNA in a sample derived from a patient.
The mutation can be a translocation, deletion, insertion or
substitution/point mutation. In a preferred embodiment, the
mutation is a deletion of all or a portion of at least one coding
exon (i.e., exon 5, 6, 7, 8 or 9), preferably exon 5 or exon 8. In
a preferred embodiment, the deletion is a homozygous deletion. In
another embodiment, the mutation is a mutation that causes a
frameshift upstream of exon 8, or otherwise causes a lack of the
wild-type open reading frame (ORF) of exon 8 in the patient's FHIT
RNA.
[0221] In other specific embodiments, the mutation is a deletion of
FHIT exons 4-6 resulting in a fusion of exon 3 sequences to exon 7
sequences in a FHIT RNA or cDNA, or the mutation is a deletion of
FHIT exons 4-8 resulting in a fusion of exon 3 sequences to exon 9
sequences in a FHIT RNA or cDNA.
[0222] In another particular embodiment, the mutation that is
detected is an insertion into a coding region of the FHIT gene or
an insertion downstream of exon 4, or an insertion in the 5'
noncoding region between exon 4 and 5. In a specific embodiment,
the mutation in the FHIT gene coding sequence is detected by
detecting an aberrant sized FHIT cDNA or mRNA from the subject
(i.e., FHIT RNA or cDNA that has a different size than the
wild-type FHIT RNA (that is present or expected to be present in
normal individuals not having or pre-disposed to a cancer
associated with a FHIT mutation, e.g., the .about.1.1 kb
transcript)).
[0223] In another embodiment, diagnosis or prognosis is carried out
by detecting an aberrant sized FHIT cDNA or mRNA from the subject
as well as the loss of one FHIT allele in the subject.
[0224] Polynucleotide sequences of FHIT consisting of at least 8 to
25 nucleotides that are useful as primers in primer dependent
nucleic acid amplification methods may be used for the detection of
mutant FHIT genomic or RNA sequences in patient samples. Primer
dependent nucleic acid amplification methods useful in the present
invention include, but are not limited to, polymerase chain
reaction (PCR), competitive PCR, cyclic probe reaction, and ligase
chain reaction. Such techniques are well known by those of skill in
the art. A preferred nucleic acid amplification method of the
present invention is reverse transcriptase PCR (RT-PCR) (Siebert et
al., 1992, Nature 359:557-558).
[0225] In a particular embodiment of the present invention, each
primer of a pair of primers for use in a primer dependent nucleic
acid amplification method is selected from a different exon of the
genomic FHIT nucleotide sequences. For example, if one primer of a
pair or primers is selected from exon 1 of the FHIT genomic
sequence, the second primer will be selected from exon 2, 3, 4, 5,
6, 7, 8, 9 or 10 of the FHIT genomic sequence. As another example,
if one primer of a pair of primers is selected from exon 2 of the
FHIT genomic sequence, the second primer will be selected from exon
1, 3, 4, 5, 6, 7, 8, 9 or 10 of the FHIT genomic sequence.
Resulting amplified genomic nucleotide sequences will contain
amplified intron sequences and will be of a larger size than
amplified cDNA nucleotide sequences that will not contain amplified
intron sequences. Similarly, amplified cDNA sequences having a
deletion mutation can be distinguished from amplified wild-type
sequences due to the size difference of the resulting amplified
sequences (the deletion mutant will generate a shorter amplified
fragment). For amplification of cDNA nucleotide sequences, the
primer sequences should be selected from exons sequences that are
sufficiently far enough apart to provide a detectable amplified
nucleotide sequence.
[0226] In a specific embodiment, cancer or other disorder of cell
proliferation or a predisposition thereto is detected or diagnosed
in a subject by detecting mutation(s) within the FHIT gene as
follows: A sample containing RNA of tissue or cells of a patient is
obtained, and the RNA is reverse-transcribed into cDNA by methods
commonly known in the art; preferably this step is followed by
amplifying fragments comprising FHIT coding sequences within the
cDNA, and detecting one or more mutation(s) within the FHIT coding
sequences within the amplified fragment. The amplification can be
by any suitable methods known in the art, and is preferably done by
polymerase chain reaction (PCR). RT-PCR is preferred due to the
great size (>500 kb) of the FHIT gene in the genome, which
renders one unable to amplify a single fragment containing most of
the FHIT exons from a genomic sample, whereas amplification of such
a fragment is readily accomplished from a cDNA sample. The primers
for use in PCR are upstream and downstream primers that prime
synthesis by a polymerase toward each other, and are preferably in
the range of 8-35 nucleotides, preferably separated by in the range
of 10-2,000 nucleotides in the FHIT mRNA. In a preferred
embodiment, each primer comprises a hybridizable sequence contained
within an exon of the FHIT gene or within 200 nucleotides flanking
(5' or 3' to) an exon of the FHIT gene. In a specific embodiment,
the first primer hybridizes 5' to exon 5 (preferably containing
sequences of exon 4 or 5' thereto) and the second primer hybridizes
on the other strand 5' to the intron between exons 5 and 6 (such
that an amplified fragment from wild-type FHIT cDNA would contain
exon 5). In another specific embodiment, the second primer
hybridizes on the other strand 5' to exon 6. In other specific
embodiments, the first and second primers respectively hybridize on
opposite strands 5' to the 3' terminus of exon 4 and 5' to exon 8;
5' to the 3' terminus of exon 4 and 5' to exon 9; and 5' to exon 1
and 5' to exon 10, such that the resulting amplified fragment would
contain the exon sequences normally present between where the
primers hybridize should they be present in the cDNA. Thus, for
example, in the foregoing examples, the PCR primer pairs are
adapted to amplify a fragment of wild-type FHIT cDNA comprising
FHIT exon 5, exon 5 plus exon 6, sequences between the 3' terminus
of exon 4 and exon 8, sequences between the 3' terminus of exon 4
and exon 9, and exons 1 through 10, respectively. The presence of
one or more mutations in the cDNA can be detected by detecting an
aberrantly sized (preferably amplified) fragment (compared to those
fragment(s) produced by a wild-type FHIT transcript), e.g., by
subjecting the cDNA to size separation such as by agarose gel
electrophoresis or column chromatography. In a preferred
embodiment, the presence of one or more mutations in the cDNA is
detected by sequencing of the cDNA, or more preferably, of the
isolated fragments amplified from the cDNA. The amplified fragments
can be isolated by methods known in the art, e.g., agarose gel
electrophoresis and recovery from the gel band and/or column
chromatography. Such sequencing can be carried out by standard
methods commonly known in the art, and can be automated or
manual.
[0227] In yet another specific embodiment, mutation(s) in the FHIT
gene or mRNA from a patient can be detected by other methods
commonly known in the art, e.g., Northern hybridization. By way of
example but not limitation, RNA from a patient's tissue is
separated by gel electrophoresis, transferred to a filter or other
solid phase, and hybridized to labelled DNA probes. The hybridized
RNAs are then visualized by detecting the label. Preferably,
numerous DNA probes are used, from different portions of the FHIT
cDNA.
[0228] In another embodiment, Southern hybridization can be used to
detect gross mutations in FHIT DNA. For example, genomic DNA
isolated from a patient, separated by gel electrophoresis,
transferred to a filter or other solid phase, and hybridized with a
FHIT probe (e.g., an oligonucleotide containing a FHIT gene
sequence, affixed to a detectable label). Preferably, a
multiplicity of FHIT probes are used, hybridizable to sequences
within each of the coding exons, and particularly preferably,
including probe(s) hybridizable to sequences within exon 5.
[0229] In another embodiment, a translocation within the FHIT gene
is detected by methods commonly known in the art. For example, in a
preferred embodiment, a sample comprising PHIT genomic DNA, or,
preferably FHIT cDNA (e.g., cDNA of total polyA mRNA) from a
patient is subjected to PCR by use of primers that prime synthesis
across the suspected translocation junction. For example, one
primer can have a sequence hybridizable to chromosome 3 (preferably
within the FHIT gene upstream of exon 4, e.g., a sequence within
exon 1, 2 or 3) and one primer can have a sequence hybridizable to
chromosome 8 (downstream of the translocation event); amplification
of a fragment indicates the presence of a translocation between
chromosomes 3 and 8. Additionally or alternatively performing PCR
by priming with primers, each having a sequence within the FHIT
gene (see e.g., description supra regarding primers for RT-PCR)
will result in an amplified fragment only if at least one FHIT
allele contains the primer sequences undisrupted by a translocation
event in between them.
[0230] Detection of homozygous mutations (mutations in both
alleles) in FHIT genes are deemed more severe indicators of the
presence of, or a predisposition to, cancer than hemizygous
mutations (of one allele) in FHIT genes.
[0231] As used herein, patient samples which can be used include,
but are not limited to, fresh or frozen tissue samples, which can
be used in in situ hybridization assays; cell or tissue from
biopsies and, in general, patient samples containing nucleic acid,
which can be used in assays that measure or quantitate or analyze
FHIT nucleic acid.
[0232] The FHIT gene sequences of the present invention may be used
diagnostically for the detection of chromosome 3p14.2
abnormalities, in particular, translocations with chromosome 8, and
deletions. Accordingly, the present invention provides a process
for detecting a target sequence indicative of or including a
chromosome 3p14.2 abnormality in a sample, comprising the steps of
amplifying the target sequence in the sample using a first primer
of 8 to 25 nucleotides, preferably 18-25 nucleotides, complementary
to the nucleotide sequence of SEQ ID NO: 1, and a second primer
complementary to a region telomeric or centromeric to the FHIT gene
and detecting any resulting amplified target sequence in which the
presence of the amplified target sequence is indicative of the
abnormality. The present invention also provides a method of
diagnosing a malignancy associated with chromosome 3p14.2
abnormalities in a patient comprising, detecting said chromosome
3p14.2 abnormality according to the method above in which the
presence of an amplified target sequence indicative of a mutant
FHIT transcript indicates the presence of a cancer or precancerous
condition in the patient. The resultant amplified target sequence
can be detected on gel electrophoresis and compared with a normal
sample or standard that does not contain a chromosome 3p14.2
abnormality. The amplification of genomic DNA target sequences may
require generating long PCR products. PCR techniques for generating
long PCR products are described in Science (1994) 263:1564-1565;
PCR kits for generating long PCR products are available from Perkin
Elmer and Takara Shuzo Co., Ltd. The present invention also
provides a method for detecting a target nucleotide sequence
indicative of or including at least a portion of a chromosome
3p14.2 abnormality (thereby indicative of the presence of or a
predisposition to a disorder of cell overproliferation) in a
nucleic acid sample, comprising the steps of hybridizing the sample
with a nucleic acid probe of not more than 10 kilobases, comprising
FHIT cDNA sequences selected from among at least exon 1, 2, 3 or 4
and selected from among at least exon 7, 8 or 9, or a sequence
absolutely complementary thereto, and detecting or measuring the
amount of any resulting hybridization between the probe and the
target sequence within the sample. Alternatively, the probe
comprises at least 310 contiguous nucleotides of a FHIT cDNA, or at
least 266 contiguous nucleotides of FHIT cDNA coding sequences. The
resultant hybridization between the probe and the target sequence
within the sample can be detected using gel electrophoresis and can
be compared to a target sequence from a normal sample or standard
that does not contain the abnormality. The present invention also
provides a method of diagnosing a malignancy associated with a FHIT
abnormality in a patient comprising detecting said FHIT abnormality
according to the method above in which the presence of the
amplified target sequence indicates the presence of a malignancy in
the patient. Absolute complementarity between a hybridization probe
and a target sequence, although preferred, is not required. A
sequence "complementary to at least a portion of", as referred to
herein, means a sequence having sufficient complementarity to be
able to hybridize with the nucleic acid, forming a stable
hybridization complex. The ability to hybridize will depend on both
the degree of complementarity and the length of the nucleic acid.
Generally, the longer the hybridizing nucleic acid, the more base
mismatches with a FHIT RNA it may contain and still form a stable
duplex (or triplex, as the case may be). One skilled in the art can
ascertain a tolerable degree of mismatch by use of standard
procedures to determine the melting point of the hybridized
complex.
[0233] An additional aspect of the present invention relates to
diagnostic kits for the detection or measurement of FHIT gene
sequences and FHIT protein. Kits for diagnostic use are provided,
that comprise in one or more containers an anti-Fhit antibody, and,
optionally, a labeled binding partner to the antibody.
Alternatively, the anti-Fhit antibody can be labeled (with a
detectable marker, e.g., a chemiluminescent, enzymatic,
fluorescent, or radioactive moiety). A kit is also provided that
comprises in one or more containers a nucleic acid probe capable of
hybridizing to FHIT RNA. Accordingly, the present invention
provides a diagnostic kit comprising, in a container a compound
comprising a probe of not more than 10 kilobases and comprising
FHIT cDNA sequences comprising at least one of exon 1, 2, 3 or 4
and at least one of exon 7, 8 or 9; or its complement.
Alternatively, the probe comprises at least 310 contiguous
nucleotides of a FHIT cDNA, or at least 266 contiguous nucleotides
of FHIT cDNA coding sequences. Alternatively, the present invention
provides a diagnostic kit comprising, in one or more containers, a
pair of primers of at least 8-35, preferably 8-25, nucleotides in
which at least one of said primers is hybridizable to SEQ ID NO: 1
or its complement and wherein said primers are capable of priming
cDNA synthesis in an amplification reaction. In a specific
embodiment, a kit can comprise in one or more containers a pair of
primers (e.g., each in the size range of 8-35 nucleotides) that are
capable of priming amplification [e.g., by polymerase chain
reaction (see e.g., Innis et al., 1990, PCR Protocols, Academic
Press, Inc., San Diego, Calif.), ligase chain reaction (see EP
320,308) use of Q.beta. replicase, cyclic probe reaction, or other
methods known in the art] under appropriate reaction conditions of
at least a portion of a FHIT nucleic acid. The present invention
also provides a diagnostic kit in which at least one of the primers
is hybridizable to SEQ ID NO: 1 or its complement and in which one
of the primers is hybridizable to a DNA sequence located telomeric
or centromeric to the FHIT gene. In another embodiment, the kit
comprises a primer pair such as described supra for use in
diagnostic assays. In a specific embodiment, one of the foregoing
compounds of the container can be detectably labeled. A kit can
optionally further comprise in a container a predetermined amount
of a purified Fhit protein or nucleic acid, e.g., for use as a
standard or control.
[0234] The amplification reaction of the present invention may be a
polymerase chain reaction, competitive PCR and competitive
reverse-transcriptase PCR (Clementi et al., 1994, Genet Anal Tech
Appl 11(1):1-6 and Siebert et al., 1992, Nature 359:557-558);
cyclic probe reaction, which allows for amplification of a target
sequence using a hybrid RNA/DNA probe and RNase (ID Biomedical);
ligase chain reaction (Wu et al., 1989, Genomics 4:560-569). In a
particular embodiment, the chromosomal abnormality associated with
a FHIT locus can be detected as described in PCT Publication No.
WO92/19775, dated Nov. 12, 1992. In a specific embodiment, the FHIT
probe used in a hybridization assay is detectably labeled. Such a
label can be any known in the art including, but not limited to,
radioactive labels, fluorescent labels, biotin, chemiluminescent
labels, etc.
[0235] In a specific embodiment in which the assay used employs
primers, at least one primer can be detectably labeled. In another
embodiment, one of a primer pair is attached to a moiety providing
for capture, e.g., a magnetic bead.
[0236] Anti-FHIT antibodies may be generated and used
diagnostically to detect the presence of mutant Fhit protein in
patient samples, and/or the absence of wild-type Fhit protein,
thereby identifying disease states associated with chromosome
3p14.2 abnormalities such as disorders of cell
overproliferation.
[0237] For example, in one embodiment, where one is detecting or
measuring mutant Fhit protein by assaying for binding to anti-Fhit
antibody, various immunoassays known in the art can be used,
including but not limited to competitive and non-competitive assay
systems using techniques such as radioimmunoassays, ELISA (enzyme
linked immunosorbent assay), "sandwich" immunoassays,
immunoradiometric assays, gel diffusion precipitin reactions,
immunodiffusion assays, in situ immunoassays (using colloidal gold,
enzyme or radioisotope labels, for example), western blots, in situ
hybridizations, precipitation reactions, agglutination assays
(e.g., gel agglutination assays, hemagglutination assays),
complement fixation assays, immunofluorescence assays, protein A
assays, and immunoelectrophoresis assays, etc. In one embodiment,
antibody binding is detected by detecting a label on the primary
antibody. In another embodiment, the primary antibody is detected
by detecting binding of a secondary antibody or reagent to the
primary antibody. In a further embodiment, the secondary antibody
is labelled. Many means are known in the art for detecting binding
in an immunoassay and are within the scope of the present
invention. In particular, such an immunoassay is carried out by a
method comprising contacting a sample derived from a patient with
an anti-Fhit antibody under conditions such that immunospecific
binding can occur, and detecting or measuring the amount of any
immunospecific binding by the antibody. In a specific embodiment,
antibody to a Fhit protein can be used to assay a patient tissue or
serum sample for the presence of a FHIT protein where an increased
level of FHIT protein is an indication of a diseased condition. In
one embodiment of the present invention, the FHIT protein is
detected or measured by immunocytochemistry of a patient sample. In
another embodiment, assays to measure the levels of FHIT protein or
RNA can be used to monitor therapy of disease associated with
increased expression of FHIT. For example, a decrease in levels of
FHIT RNA or protein after therapy, relative to the level found
before therapy, may be indicative of a favorable response to
therapy. An increase in such levels after therapy may be indicative
of a poor response to therapy.
[0238] For detection of Fhit protein sequences, a diagnostic kit of
the present invention comprises, in one or more containers, an
anti-Fhit antibody which optionally can be detectably labeled. In a
different embodiment, the kit can comprise in a container, a
labeled specific binding portion of an antibody. As used herein,
the term detectable label refers to any label which provides
directly or indirectly a detectable signal and includes, for
example, enzymes, radiolabelled molecules, fluorescent molecules,
particles, chemiluminesors, enzyme substrates or cofactors, enzyme
inhibitors, or magnetic particles. Examples of enzymes useful as
detectable labels in the present invention include alkaline
phosphatase and horse radish peroxidase. A variety of methods are
available for linking the detectable labels to proteins of interest
and include for example the use of a bifunctional agent, such as,
4,4'-difluoro-3,3'-dinitro-phenylsulfone, for attaching an enzyme,
for example, horse radish peroxidase, to a protein of interest. The
attached enzyme is then allowed to react with a substrate yielding
a reaction product which is detectable. The present invention
provides a method for detecting a Fhit protein in a patient sample,
comprising, contacting the patient sample with an anti-Fhit
antibody under conditions such that immunospecific binding can
occur, and detecting or measuring the amount of any immunospecific
binding by the antibody. The method preferably also comprises
subjecting the protein to size fractionation and/or sequence
determination.
[0239] Samples can be any sample from a patient containing FHIT
protein, e.g., tissue sections.
[0240] In diagnosing disease states, the functional activity of
Fhit proteins, derivatives and analogs may be assayed by various
methods. Accordingly, the present invention also provides a method
of diagnosing a malignancy or other disorder associated with
chromosome 3p14.2 (FHIT) abnormalities in a patient comprising,
detecting expression of a mutant Fhit protein in a sample from the
patient, in which the presence of a mutant Fhit protein indicates
the presence of a malignancy or other disorder associated with FHIT
abnormalities in the patient.
[0241] In a specific embodiment of the invention, prenatal
diagnosis of a disorder of cell overproliferation or a
predisposition thereto, is carried out. For example, one can first
obtain tissue (e.g., blood cells) from an expectant parent. If one
or more of the expectant parents have a FHIT mutation, thus
indicating possible inheritance of this mutation by the offspring,
amniocentesis or some other method of fetal tissue sampling can
then be carried out to obtain fetal cells which can then be tested
for the presence of FHIT mutant DNA or RNA or protein by methods as
described above (e.g., RT-PCR to detect mutant FHIT RNA).
[0242] In another embodiment, the levels of FHIT protein or RNA
expression may be used to stage or monitor disease, with the
appearance of or an increase in mutant Fhit protein or RNA
expression, and/or a decrease of or loss in wild-type Fhit protein
or RNA expression, relative to that present in a sample derived
from the subject at an earlier time, indicates disease
progression.
[0243] The ability of antibodies, peptides or other molecules to
modulate the effect of Fhit protein on disease states may be
monitored. For example, the expression of FHIT gene sequences or
Fhit protein sequences may be detected as described, supra, both
before and after administration of a therapeutic composition, e.g.,
comprising a FHIT nucleotide sequence, Fhit protein sequence,
derivative or analog thereof, or antibody thereto, or antisense
nucleic acid of the present invention.
[0244] In another embodiment, presence of FHIT mutation(s),
particularly homozygous ones, can be used as indicators of adverse
outcome to therapy or recurrence of the disorder in patients with
disorders of cell overproliferation.
[0245] Other methods will be known to the skilled artisan and are
within the scope of the invention.
5.12. Screening for Fhit Agonists and Antagonists
[0246] FHIT nucleic acids, proteins, and derivatives also have uses
in screening assays to detect molecules that specifically bind to
FHIT nucleic acids, proteins, or derivatives and thus have
potential use as agonists or antagonists of Fhit, in particular,
molecules that thus affect cell proliferation. In a preferred
embodiment, such assays are performed to screen for molecules with
potential utility as anti-cancer drugs or lead compounds for drug
development. The invention thus provides assays to detect molecules
that specifically bind to FHIT nucleic acids, proteins, or
derivatives. For example, recombinant cells expressing FHIT nucleic
acids can be used to recombinantly produce Fhit proteins in these
assays, to screen for molecules that bind to a Fhit protein.
Molecules (e.g., putative binding partners of Fhit) are contacted
with the Fhit protein (or fragment thereof) under conditions
conducive to binding, and then molecules that specifically bind to
the Fhit protein are identified. Similar methods can be used to
screen for molecules that bind to Fhit derivatives or nucleic
acids. Methods that can be used to carry out the foregoing are
commonly known in the art.
[0247] In a specific embodiment of the present invention, a Fhit
protein and/or cell line that expresses a Fhit protein can be used
to screen for antibodies, peptides, or other molecules that bind to
the FHIT protein and thus may act as agonists or antagonists of
FHIT protein. For example, anti-Fhit antibodies capable of
neutralizing the activity of a dominant-negative mutant Fhit
protein may be used to inhibit or prevent a disease state
associated with cell overproliferation such as cancer.
[0248] Screening of organic or peptide libraries with recombinantly
expressed mutant Fhit protein may be useful for identification of
therapeutic molecules that function to inhibit the activity of
mutant Fhit protein. Screening against wild-type Fhit protein can
then be carried out to select for antagonists specific to the
mutant Fhit protein, i.e., that do not inhibit (or bind) the
wild-type Fhit protein. Synthetic and naturally occurring products
can be screened in a number of ways deemed routine to those of
skill in the art.
[0249] By way of example, diversity libraries, such as random or
combinatorial peptide or nonpeptide libraries can be screened for
molecules that specifically bind to Fhit. Many libraries are known
in the art that can be used, e.g., chemically synthesized
libraries, recombinant (e.g., phage display libraries), and in
vitro translation-based libraries.
[0250] Examples of chemically synthesized libraries are described
in Fodor et al., 1991, Science 251:767-773; Houghten et al., 1991,
Nature 354:84-86; Lam et al., 1991, Nature 354:82-84; Medynski,
1994, Bio/Technology 12:709-710; Gallop et al., 1994, J. Medicinal
Chemistry 37(9):1233-1251; Ohlmeyer et al., 1993, Proc. Natl. Acad.
Sci. USA 90:10922-10926; Erb et al., 1994, Proc. Natl. Acad. Sci.
USA 91:11422-11426; Houghten et al., 1992, Biotechniques 13:412;
Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA 91:1614-1618;
Salmon et al., 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712; PCT
Publication No. WO 93/20242; and Brenner and Lerner, 1992, Proc.
Natl. Acad. Sci. USA 89:5381-5383.
[0251] Examples of phage display libraries are described in Scott
and Smith, 1990, Science 249:386-390; Devlin et al., 1990, Science,
249:404-406; Christian, R. B., et al., 1992, J. Mol. Biol.
227:711-718); Lenstra, 1992, J. Immunol. Meth. 152:149-157; Kay et
al., 1993, Gene 128:59-65; and PCT Publication No. WO 94/18318
dated Aug. 18, 1994.
[0252] In vitro translation-based libraries include but are not
limited to those described in PCT Publication No. WO 91/05058 dated
Apr. 18, 1991; and Mattheakis et al., 1994, Proc. Natl. Acad. Sci.
USA 91:9022-9026.
[0253] By way of examples of nonpeptide libraries, a benzodiazepine
library (see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA
91:4708-4712) can be adapted for use. Peptoid libraries (Simon et
al., 1992, Proc. Natl. Acad. Sci. USA 89:9367-9371) can also be
used. Another example of a library that can be used, in which the
amide functionalities in peptides have been permethylated to
generate a chemically transformed combinatorial library, is
described by Ostresh et al. (1994, Proc. Natl. Acad. Sci. USA
91:11138-11142).
[0254] Screening the libraries can be accomplished by any of a
variety of commonly known methods. See, e.g., the following
references, which disclose screening of peptide libraries: Parmley
and Smith, 1989, Adv. Exp. Med. Biol. 251:215-218; Scott and Smith,
1990, Science 249:386-390; Fowlkes et al., 1992; BioTechniques
13:422-427; Oldenburg et al., 1992, Proc. Natl. Acad. Sci. USA
89:5393-5397; Yu et al., 1994, Cell 76:933-945; Staudt et al.,
1988, Science 241:577-580; Bock et al., 1992, Nature 355:564-566;
Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA 89:6988-6992;
Ellington et al., 1992, Nature 355:850-852; U.S. Pat. No.
5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346,
all to Ladner et al.; Rebar and Pabo, 1993, Science 263:671-673;
and PCT Publication No. WO 94/18318.
[0255] In a specific embodiment, screening can be carried out by
contacting the library members with a Fhit protein (or nucleic acid
or derivative) immobilized on a solid phase and harvesting those
library members that bind to the protein (or nucleic acid or
derivative). Examples of such screening methods, termed "panning"
techniques are described by way of example in Parmley and Smith,
1988, Gene 73:305-318; Fowlkes et al., 1992, BioTechniques
13:422-427; PCT Publication No. WO 94/18318; and in references
cited hereinabove.
[0256] In another embodiment, the two-hybrid system for selecting
interacting proteins in yeast (Fields and Song, 1989, Nature
340:245-246; Chien et al., 1991, Proc. Natl. Acad. Sci. USA
88:9578-9582) can be used to identify molecules that specifically
bind to a Fhit protein or derivative.
5.13. Animal Models
[0257] The invention also provides animal models.
[0258] In one embodiment, animal models for diseases and disorders
involving cell overproliferation (e.g., as described in Section
5.8.1) are provided. Such an animal can be initially produced by
promoting homologous recombination between a FHIT gene in its
chromosome and an exogenous FHIT gene that has been rendered
biologically inactive (preferably by insertion of a heterologous
sequence, e.g., an antibiotic resistance gene). In a preferred
aspect, this homologous recombination is carried out by
transforming embryo-derived stem (ES) cells with a vector
containing the insertionally inactivated FHIT gene, such that
homologous recombination occurs, followed by injecting the ES cells
into a blastocyst, and implanting the blastocyst into a foster
mother, followed by the birth of the chimeric animal ("knockout
animal") in which a FHIT gene has been inactivated (see Capecchi,
1989, Science 244:1288-1292). The chimeric animal can be bred to
produce additional knockout animals. Such animals can be mice,
hamsters, sheep, pigs, cattle, etc., and are preferably non-human
mammals. In a specific embodiment, a knockout mouse is
produced.
[0259] Such knockout animals are expected to develop or be
predisposed to developing diseases or disorders involving cell
overproliferation (e.g., malignancy) and thus can have use as
animal models of such diseases and disorders, e.g., to screen for
or test molecules (e.g., potential anti-cancer therapeutics) for
the ability to inhibit overproliferation (e.g., tumor formation)
and thus treat or prevent such diseases or disorders.
[0260] In a different embodiment of the invention, transgenic
animals that have incorporated and express a dominant-negative
mutant FHIT gene have use as animal models of diseases and
disorders involving cell overproliferation. Such animals can be
used to screen for or test molecules for the ability to
specifically inhibit the dominant-negative mutant and thus treat or
prevent such diseases and disorders.
6. THE HUMAN FHIT GENE, SPANNING THE CHROMOSOME 3p14.2 FRAGILE SITE
AND RENAL CARCINOMA ASSOCIATED TRANSLOCATION BREAKPOINT, IS
ABNORMAL IN DIGESTIVE TRACT CANCERS
[0261] As described herein, we have isolated and characterized a
human gene involved in esophageal, gastric, colon, kidney, and
other cancers. A 200-300 kilobase (kb) region of chromosome 3p14.2,
including the fragile site locus, FRA3B, is involved in homozygous
deletions in multiple tumor-derived cell lines and in hemizygous
deletions in esophageal, gastric, colon, kidney and other cancers.
Exon amplification from a cosmid contig covering this 200-300
kilobase region allowed identification of the human FHIT gene, a
member of the zinc-binding histidine triad gene family, which
encodes a ubiquitous 1.1 kilobase transcript and a 16.8 kDa protein
with homology to a protein kinase C inhibitor gene, another member
of the HIT family.
[0262] The FHIT locus is composed of 10 small exons distributed
over at least 500 kilobases, with the three 5' most untranslated
exons mapping centromeric to the clear cell renal carcinoma
associated 3p14.2 translocation breakpoint; the remaining exons map
telomeric to this translocation breakpoint with exon 5, the first
amino acid coding exon, falling within the homozygously deleted
fragile region, FRA3B, and exons 6-10 mapping telomeric to the
tumor cell common deleted region and the FRA3B region. Aberrant
transcripts of the PHIT locus were found in approximately 50% of
esophageal, stomach and colon carcinomas, and the familial t(3;8)
renal carcinomas have lost one FHIT allele due to disruption by the
translocation.
[0263] The aberrant FHIT transcripts usually resulted from abnormal
splicing, which often deleted exon 5 or 8, resulting in transcripts
which could not encode Fhit protein. Thus, chromosome abnormalities
at 3p14.2 and FRA3B, resulting in loss of the Fhit protein, are
involved in initiation and/or progression of several important
types of human cancer.
6.1. Results
The Cosmid Contig
[0264] From the 648D4 cosmid library, clones were selected
initially using the BE758-6, A6URA, A3, and 1300E3, probes, which
were distributed across the homozygously deleted region as shown in
FIG. 1A. Cosmid end-clones were then isolated and used for the next
round of cosmid screening. The cosmid map was assembled by
PCR-amplification of the starting STSs (DNA sequence tags) and new
ones developed from cosmid ends, using cosmid DNA templates.
Additionally, each new STS was tested against the YAC contig (also
shown in FIG. 1A), against cell lines with homozygous deletions and
rodent-human hybrids retaining portions of chromosome 3 (LaForgia
et al., 1993, Cancer Res. 53:3118-3124; Druck et al., 1995, Cancer
Res. 55:5348-5355; Bullrich et al., 1995, Cytogenet. Cell Genet.
70:250-254). Six cosmids were assembled into a contig which covered
the homozygously deleted region.
[0265] To define more precisely the homozygously deleted region,
which we will refer to as the fragile region, 42 STS markers,
spanning the chromosomal region from the PTPRG locus to D3S1234,
derived from cosmid walking and exon trapping, were tested by
PCR-amplification for presence in eleven cancer-derived cell lines
which had been tested previously with a subset of markers (data not
shown; and Lisitsyn et al., 1995, Proc. Natl. Acad. Sci. USA
92:151-155).
[0266] Colon carcinoma-derived LoVo, HT29 and SW480 and gastric
carcinoma-derived AGS cell lines showed similar large deletions
such as depicted by the dotted portion of the top line in FIG. 1A.
Colon carcinoma-derived LS180 and breast carcinoma-derived
MDA-MB436 cells exhibited discontinuous deletions, covering this
same region, with most markers lost but some retained. The gastric
carcinoma-derived KatoIII cells appeared to have lost the D3S1481
marker and the telomeric portion of the fragile region, from AP4/5
to D3S2977 (see FIG. 1A). The HKI cells, derived from a
nasopharyngeal carcinoma (NPC), had lost the region between D3S1481
and the AP4/5 marker, while CNE2, another NPC-derived cell line had
a discontinuous deletion which included a region near the t(3;8)
and the region between D3S1481 and D3S2977. HeLa cells also
exhibited discontinuous deletions with one deleted region near the
t(3;8) and between D3S1481 and AP4/5. The NPC-derived CNE1 cells
were tested with most markers without detection of a deletion.
Thus, there are many different tumor associated 3p14.2 chromosome
breakpoints surrounding the t(3;8), the FRA3B locus and the
homozygously deleted region covered by the cosmid contig.
Isolation of cDNAs
[0267] The six cosmids covering the homozygous deletion, shown in
FIG. 1A, were used in exon trapping experiments aimed at
identifying genes within the deleted region. Putative trapped exons
were sequenced and sequences analyzed using GRAIL 2 of the ORNL
GRAIL server. Several trapped exons were recognized as exons by
Grail 2 and were used as probes on northern blots of poly A.sup.+
RNA from a spectrum of human tissues. Additionally, sequences of
trapped exons were compared against nucleotide sequence databases.
One exon, trapped from a cosmid 76 subclone (c76, FIG. 1A) matched
a number of cDNA sequences from breast (Genbank accession #R53187
and #R86313) and fetal liver and spleen (#R1.1128) libraries
submitted by the Washington University-Merck EST Project. A 23
basepair (bp) oligonucleotide primer designed from this sequence
(FIG. 2A, primer X8) was used in primer extension to obtain a 5'
extended product of the cDNA by a RACE (Rapid amplification of cDNA
ends) reaction (Marathon.TM. cDNA amplification kit, Clontech). The
longest product (370 bp) from the RACE reaction detected a
ubiquitously expressed 1.1-kb mRNA by northern blot analysis of
mRNAs from various normal tissues. The size was similar to the
length of the largest cDNA clone isolated from a normal colon cDNA
library using the same DNA fragment as a probe. The DNA sequence
analysis of this full length clone (FIG. 2A) revealed a long 5'
untranslated region of more than 350 bp followed by an initial
methionine codon and surrounding sequence which fitted Kozak's
rule, an open reading frame (ORF) of 147 amino acids, a 31
untranslated region, a polyadenylation consensus sequence and a
poly A tail. Exon sizes varied widely, e.g., exon 5 having 120
nucleotides, exon 6 having 146 nucleotides, and exon 7 having 30
nucleotides. With reference to FIG. 2A, exon 1 consists of
nucleotide numbers -362 to -213; exon 2 consists of nucleotide
numbers -212 to -164; exon 3 consists of nucleotide numbers -163 to
-111; exon 4 consists of nucleotide numbers -110 to -18; exon 5
consists of nucleotide numbers -17 to 103; exon 6 consists of
nucleotide numbers 104 to 249; exon 7 consists of nucleotide
numbers 250 to 279; exon 8 consists of nucleotide numbers 280 to
348; exon 9 consists of nucleotide numbers 349 to 449; and exon 10
consists of nucleotide numbers 450 to 733.
[0268] A hydrophilicity plot for the Fhit protein was carried out
and is shown in FIG. 6.
[0269] This FHIT cDNA, as well as the matching sequences from the
EST database, were translated and open reading frame (ORF) amino
acid sequences (FIG. 2A) compared to the protein databases. The
longest EST in the 5' direction was R50713 (which contained
sequence found in the 3' end of FHIT exon 7, exon 8, and exon 9).
The longest EST in the 3' direction was R11128 (which contained
sequence found in half of exon 2, and in exons 3-6). EST R53187 had
the longest span of sequences corresponding to the FHIT cDNA,
including 297 nucleotides identical to the FHIT cDNA sequence from
exon 2 through a portion of exon 5. Among the best matches in the
database retrieved by computer searches, this 297 nucleotide
sequence was the longest stretch of identity with the FHIT cDNA
sequence. The next longest stretch of identity was found in EST
11128, with 287 nucleotides identical to the PHIT cDNA sequence
starting within exon 2 until 3 bases before the end of exon 6. A
printout of the R50713 nucleotide sequence aligned with the FHIT
cDNA sequence (cDNA 7F1) and the R11128 nucleotide sequence is
shown in FIGS. 7A-7C. As will be noted, neither of the R53187 nor
R11128 nucleotide sequences, or any of numerous other EST
sequences, span the full FHIT protein coding region. Also, the
translations of the R50713, and R11128 sequences in all three
reading frames, in both orientations, are shown in FIGS. 8 and 9,
respectively, and from none of the translated sequences shown in
FIG. 8 or 9 can the Fhit protein sequence be deduced.
[0270] The full length FHIT cDNA probe was then hybridized to
northern blots carrying mRNA from a spectrum of tissues. As shown
in FIG. 3A, the cDNA detected the ubiquitously expressed 1.1-kb
transcript.
Relationship of the cDNA to the Genomic Map of the Region
[0271] Oligonucleotide primers from the initially trapped exon were
used to generate intron sequences from cosmid 76; these sequences
were used in turn to prepare primers and probes to map the exon (E5
in FIG. 1A) on the cosmids, YACs and DNA from cancer cell lines
with deletions, as illustrated in FIG. 1A. Using cDNA as template,
oligonucleotide primer pairs bracketing the exons upstream and
downstream of exon 5 were then used to amplify cDNA fragments to
serve as probes for mapping the 5' and 3' flanking exons on the
cosmid contig; these probes demonstrated that the cDNA sequences 5'
and 3' of exon 5 were not within the 648D4 cosmid contig covering
the homozygous deletions. Thus, cosmid libraries from YACs 850A6
and 750F1, which extend centromeric and telomeric to the fragile
region deletions, respectively, as shown in FIG. 1A, were prepared
and screened with the 5' and 3' cDNA probes flanking exon 5.
Cosmids containing the remaining exons were then used to derive
intron sequences using cDNA primers, and the structure of the gene
determined as shown in FIG. 1A. The cDNA consisted of 10 exons
which were distributed among 3 YAC clones (FIG. 1A); exons 1
through 4 mapped to YAC clone 850A6, exon 5 was present in all
three YAC clones, and exons 6 through 10 mapped to YAC clone 750F1.
Only exon 5 fell within the region of homozygous deletion in
tumor-derived cell lines, i.e. within YAC clone 648D4. The coding
region of the ORF began in exon 5 and ended in exon 9, as shown in
detail in FIGS. 2A and 2B.
[0272] Most interestingly, the first three exons (E1, E2 and E3) of
the gene mapped centromeric to the t(3;8) break, between the t(3;8)
break and the 5' end of the PTPRG gene, as determined by
amplification of these exons from the YAC DNAs and DNAs derived
from hybrids carrying portions of chromosome 3, derived from the
t(3;8) break and a FRA3B break (data not shown). Thus, this gene
became a strong candidate for involvement in initiation of the
familial RCCs, because one copy of the gene is disrupted by the
translocation.
[0273] The homology search in amino acid sequence databases showed
a significant homology to a group of proteins which have a
histidine triad motif, designated HIT proteins (Seraphin, 1992, J.
DNA Sequencing & Mapping 3:177-179). The predicted amino acid
sequence of the cDNA for the human gene, designated the Fragile
Histidine Triad gene or the FHIT gene, is shown in FIG. 4A compared
to the other members of the HIT family. The highest homology of the
FHIT protein (.about.50% identity) is to the yeast diadenosine
hydrolases (aph1s), shown in FIG. 4A as PAPH1 and CAPH1, identified
in S. pombe and S. cerevisiae, respectively (Huang et al., 1995,
Biochem. J. 312:925-932). An alignment of the yeast (S. pombe) Ap4A
hydrolase (PAPH1) sequence (U32615) with FHIT (cDNA 7F1) is shown
in FIGS. 10A-10C. There is not extensive homology. When we did a
computer search for homology stretches between the yeast hydrolase
and the FHIT nucleotide sequences, the result was the small region
of nucleotide homology shown in FIG. 10B The consensus sequence for
the HIT family proteins is shown below the amino acid sequences in
FIG. 4A.
[0274] To recapitulate, the PHIT gene, which may be the human
cognate gene for the yeast Ap.sub.4A hydrolase gene, spans a
>500 kbp region which includes the t(3;8), the FRA3B and a tumor
cell-specific commonly deleted region.
Expression of the FHIT Gene
[0275] We had placed the BE758-6 locus and microsatellite marker,
D3S1300, within the region of common loss in a variety of
tumor-derived cell lines and our LOH study of gastric and colon
tumors detected a high frequency of allelic deletion, often
involving D3S1300, in the region between the t(3;8) and the D3S1234
locus (see FIG. 1A). Now, the localization of both the BE758-6 and
D3S1300 loci within the FHIT gene locus, close to the first coding
exon, exon 5, suggested that the FHIT gene was the target of
deletion in uncultured tumors, as well as tumor-derived cell lines.
To begin an analysis of FHIT transcripts in tumor-derived cells,
mRNAs from tumor-derived cell lines and normal tissues was studied
by northern analysis.
[0276] Poly A.sup.+ RNA from normal tissues and a number of NPC,
colon and gastric tumor-derived cell lines, with and without
apparent deletions in the fragile region, was tested for
hybridization to the FHIT cDNA on northern blots (FIGS. 3A and
B).
[0277] A low level of expression of the FHIT gene occurred in all
human tissues tested, as shown in FIG. 3A for spleen, thymus,
prostate, testis, ovary, small intestine, colon and peripheral
blood lymphocytes. The major transcript was .about.1.1 kb with a
longer transcript at .about.5 kb, which was barely detectable or
undetectable on some blots. Since the 1.1 kb transcript matches the
size of the full-length cDNA, the longer transcript may represent a
precursor RNA which is not fully processed. Similar transcripts
were seen in mRNA from brain, heart, lung, liver, skeletal muscle,
kidney and pancreas, with the putative unprocessed RNA appearing to
be more abundant in lung, small intestine and colon on some
northern blots.
[0278] mRNAs from tumor-derived cell lines with known homozygous
deletions in the fragile region exhibited varying levels of FHIT
transcripts (FIG. 3B), from barely detectable (FIG. 3B, lanes 2-4,
KatoIII, HKI and LoVo mRNA, respectively) to almost a normal level
(lane 8, LS180), relative to normal small intestine mRNA (lane
1).
[0279] Note that the NPC cell lines with (CNE2, HK1; FIG. 3B, lanes
5, 3) and without (CNE1; FIG. 3B, lane 6) homozygous deletions we
had documented expressed barely detectable FHIT mRNA. The
NPC-derived cell line, CNE2, exhibited a possible smaller
transcript (FIG. 3B, lane 5), while Colo320, a colorectal
carcinoma-derived cell line without a deletion, exhibited an
apparently normal-sized FHIT transcript (FIG. 3B, lane 7), although
it should be noted that size alone does not imply presence of a
wildtype transcript. The -1.1 kb bands could harbor transcripts
with one or more small exons missing, since several exons are very
small, e.g. exon 7, 30 nucleotides, exon 2, 49 nucleotides, exon 3,
53 nucleotides. One conclusion of the northern analysis is that
there was no direct relationship between size or abundance of
transcript and detection of homozygous deletions in specific
tumor-derived cell lines, suggesting that there may be small
deletions in some tumor cell lines which have not been detected
with the available markers.
RT-PCR and cDNA Sequence Analysis of Tumor-Derived mRNA
[0280] In order to look for abnormalities in FHIT transcripts from
deleted and nondeleted tumor cell lines, we reverse-transcribed
mRNAs with (dT).sub.17 primer, amplified the cDNA with 5' and 3'
primers and then reamplified using primers inside the original
primers (nested PCR), as described in methods. Positions of the
primers are shown in FIG. 2A. The amplified products were separated
on agarose gels and normal-sized and aberrant fragments were cut
from the gels and sequenced (examples of aberrant bands are shown
for mRNAs of uncultured tumors in FIG. 3C; RT-PCR products from the
tumor cell lines were very similar). The tumor-derived cell lines
exhibited a pattern of products ranging from only one apparently
normal-sized amplified transcript to numerous aberrant bands
without a normal-sized band. Some tumor-derived cell lines
exhibited both an apparently normal-sized and one or more aberrant
bands. The sequencing of the aberrant bands revealed numerous
abnormal products, some examples of which are illustrated in FIG.
1B. Colon tumor-derived CCL235 and CCL234 cell lines did not show
deletion of the STS markers tested, but both showed aberrant
transcripts, as illustrated, with CCL235 exhibiting a normal-sized
product in addition. HT29 and KatoIII cell lines both showed
homozygous deletion, but the KatoIII cell line exhibited a deletion
of the telomeric portion of the homozygously deleted region and not
the region containing exons 4 and 5, nor the region of exon 6,
exons which are all missing in the aberrant RT-PCR product, as
illustrated in FIG. 1B. Numerous other tumor-derived cell lines
also exhibited aberrant RT-PCR products similar to those shown
schematically in FIG. 1B (data not shown). Detailed descriptions of
similar aberrant products from uncultured tumors (FIG. 3C) are
given in Table 2 and FIG. 2B.
[0281] Ten cases of uncultured esophagus, nine of stomach and eight
of colon tumors were analyzed, and aberrant transcripts were
observed in 5, 5 and 3 cases, respectively (summarized in Tables 2
and 3 and illustrated in FIG. 2B. TABLE-US-00002 TABLE 2 Derivation
of FHIT RT-PCR Amplified Products and cDNA Sequences From
Uncultured Tumors of Gastric Organs Cases with aberrant transcripts
No. of cases Number Origin of Tumors analyzed of cases Codes.sup.a
Esophagus 10 5 E3*, E12*, E13* E32*, E37* Stomach 9 5 J1*, J3, J4*,
J7, J9* Colon 8 3 9625*, 5586*, 9575* .sup.aIn cases with asterisks
(*), normal tissues from the same organs were analyzed and did not
exhibit alterations in the coding region sequences.
[0282] TABLE-US-00003 TABLE 3 Aberrant Transcripts Observed in
Uncultured Tumors.sup.1 Tumor-derived Deletion Insertion.sup.4
Putative protein.sup.5 transcript.sup.2 (position.sup.3) Size (bp)
Homology Effect coded in frame *E3 a 280-348 72 NS Ex 8 loss HIT(-)
*E12 a 280-348 -- -- Ex 8 loss HIT(-) -- b 122-516 -- -- FS after
Ex 6 HIT(-) *E13 a -17-249 -- -- Ex 5 & 6 loss -- -- b -17-348
-- -- Ex 5-8 loss -- E32 -- 280-449 -- -- Ex 8 & 9 loss HIT(-)
*E37 a -- 72 NS none intact -- b -73-173 -- -- Ex 5 loss HIT(+)
*9625 a 280-348 -- -- Ex 8 loss HIT(-) -- b -17-279 87 Alu Ex 5-7
loss -- -- c -110-204 -- -- Ex 4 & 5 loss HIT(+) *5586 a
-17-349 135 Alu Ex 5-8 loss -- -- b -17-279 37 NS Ex 5-7 loss --
*9575 a 280-348 -- -- Ex 8 loss HIT(-) -- b 60-181 -- -- FS after
Ex 5 HIT(-) -- c -110-348 -- -- Ex 4-8 loss -- J1 a -110-(-17) --
-- none intact -- b -17-279 -- -- Ex 5-7 loss -- J3 -- -17-279 173
Alu Ex 5-7 loss HIT(+) J4 -- -17-457 305 Alu Ex 5-9 loss -- *J7 a
-110-249 -- -- Ex 4-6 loss -- *J9 a 280-348 -- -- Ex 8 loss HIT(-)
.sup.1All the aberrant transcripts which involve the coding
sequence of the FHIT gene are shown in FIG. 3B. Alu, Alu repeat,
FS, frameshift; NS, no significant homology; Ex, exon. .sup.2In
tumors with asterisks (*), normal transcripts without alteration of
coding region sequence were also observed. .sup.3The positions of
the first and last nucleotides of the deletions are shown according
to the nucleotide numbers in FIG. 2A. .sup.4The position of all
insertions was downstream of exon 4. .sup.5Putative protein coded
in frame with the Fhit protein is shown: HIT(+), protein with HIT
motif; HIT(-), protein without HIT motif; --, no protein in
frame.
The sequence analyses of the aberrant cDNAs revealed absence of
various regions between exons 4 and 9 (Table 3 and FIG. 2B), while
the RT-PCR and cDNA sequence analyses of normal tissue mRNAs from
the same organs did not exhibit any alterations of the coding
region sequence (Table 2, E3, E12, E113, E37, J1, J4, J9, 9625,
5586, 9575). In 8 of 13 cases with aberrant transcripts,
normal-sized transcripts were also observed (FIG. 3C; E3, E12, E13,
E37, 9625, 9575, J7 and J9; E12 and 9575, not shown), while in 5 of
13 cases normal-sized transcripts were not detected (FIG. 3C, J3,
J4), or were barely detected (FIG. 3C, E32, 5586, J1). In most of
the aberrant transcripts, the beginning and the end of the deleted
portions of the transcripts coincided with splice sites (FIG. 2B),
suggesting that the cDNA deletions resulted from the loss of
genomic regions containing or surrounding the relevant lost FHIT
exons. The aberrant transcripts can be classified into two groups
(class I and II, FIG. 2B): class I transcripts lack exon 5, which
has the initial methionine codon of the FHIT ORF, resulting in the
loss of the ORF; class II transcripts have an intact initial
methionine codon but do not include exon 8, except for 9575b, which
exhibited a frameshift after exon 6. Thus, in all the class II
transcripts, the wildtype ORF of exon 8, the histidine triad
containing domain, is not present. Moreover, some of the class II
transcripts exhibited loss only of exon 8 (FIG. 2B; E3, E12a,
9625a, 9575a, J9a), suggesting that exon 8 was the target of
deletion. Since exon 8 encodes the histidine triad motif, it is
likely that neither class I nor class II transcripts, constituting
the major fraction of aberrant transcripts, can encode a fully
functional protein. However, there is an in frame methionine (Met)
codon in exon 6 (see FIG. 2B), and in some cases insertions
contribute an in frame Met (not shown); thus, the majority of
aberrant transcripts could encode partial proteins with or without
the HIT domain as indicated in Table 3. Insertions of various
lengths, of DNA not derived from the FHIT gene, were observed in
some transcripts; insertions were found only downstream of exon 4
(Table 3, 5586a, 5586b, 9625, J3, J4). A minor group of aberrant
transcripts retained intact full length ORFs, but were missing exon
4 (Table 3, J1a), or had insertion of 72 bp of DNA sequence in the
5' noncoding region between exon 4 and 5 (Table 3, E37a, and FIG.
1B). It is possible that such insertions affect translation of the
ORF.
[0283] In order to determine if the wildtype FHIT cDNA and various
cDNAs derived from tumor specific transcripts, which retained the
entire coding region, could be translated in vitro, several
recombinant plasmids were constructed, each containing a FHIT gene
downstream from the T7 promoter and lacking the first noncoding
exon. The pFHIT1 plasmid carried an aberrant cDNA, missing exon 4,
from the CCL234 colon cancer cell line. Plasmid pFHIT2 carried a
cDNA from esophageal tumor E37 with an insertion of 72 bp between
exon 4 and exon 5. The pFHIT3 plasmid contained the wildtype FHIT
gene lacking exon 1. The constructs were used for in vitro
translation by rabbit reticulocyte lysate. Analysis of translation
products (FIG. 4B) showed the predicted 16.8 kDa protein translated
from each cDNA construct.
The FHIT Protein
[0284] The protein sequence predicted by the FHIT cDNA is very
similar (57/109 amino acid identities; 76/109 or 69%, similarities,
as calculated by the NCBI BLAST server) to the S. pombe diadenosine
5',5'''P.sup.1, P.sup.4 tetraphosphate hydrolase, aph1 (Huang et
al., 1995, Biochem. J. 312:925-932), as shown in the amino acid
alignment in FIG. 4A, where PAPH1 represents the S. pombe
sequence.
[0285] The S. pombe aph1 enzyme was cloned by purification of the
enzyme, amino acid sequencing of the N-terminus and design of
primers to amplify a partial cDNA; the full length genomic and a
cDNA of 1.2 kbp were then cloned, sequenced and translated (Huang
et al., 1995, Biochem. J. 312:925-932). By similar methods, a human
hydrolase (APH1) has been cloned, sequenced and translated (Thorne
et al., 1995, Biochem. J. 311:717-721) and, surprisingly, does not
resemble the S. pombe aph1 gene nor the FHIT gene. Since higher
eukaryotes appear to possess a single 16-21 kDa Ap.sub.4A
asymmetrical pyrophosphohydrolase (cited in Thorne et al., 1995,
Biochem. J. 311:717-721), it is thus not clear if the FHIT gene is
a human APH1 enzyme, although it may be a human cognate of the S.
pombe aph1 enzyme.
[0286] The FHIT gene is also very similar to the S. cerevisiae aph1
gene product (CAPH1 in FIG. 4A) with 40% identity and 62%
similarity in the 50 amino acids between 49 and 102 of the FHIT
amino acid sequence, and higher similarity in the HIT domain. The
other proteins or hypothetical proteins in FIG. 4A are all members
of this HIT gene family, a family of proteins present in
prokaryotes, yeast and mammals, described by Seraphin, 1992, DNA
Sequencing & Mapping 3:177-179. The signature feature of the
family is the histidine triad (most commonly HVHVH, amino acids
94-98 of the FHIT protein, FIG. 4A), which for the case of BHIT
(FIG. 4A), the bovine inhibitor of protein kinase C (PKCI1) has
been shown to be a zinc-binding site (Pearson et al., 1990, J.
Biol. Chem. 265:4583-4591; Mozier et al., 1991, FEBS 279:14-18).
The Fhit protein product is only 39% similar to the bovine PKCI1
protein over Fhit amino acids 12-100, as calculated by NCBI BLAST.
Thus, the FHIT gene is not likely to be the human PKCI1 gene.
Functions of the other HIT genes are thus far not known.
Furthermore, structural features of family members have not been
studied extensively. The PKCI1 protein has a predicted content of
23% .alpha. helix and 42% .beta. conformation (31% .beta. sheet and
11% .beta. turn) (Pearson et al., 1990, J. Biol. Chem.
265:4583-4591); the conserved region, including the histidine triad
and upstream region were predicted to be mostly random coil
alternating with .beta. sheet conformation, with the HIT domain
.beta. sheet. This conformation may be preserved in the Fhit
protein. Also, the HIT domain consists of basic and hydrophobic
amino acids and might be expected to be buried inside the protein,
as suggested for the PKCI1 protein (Pearson et al., 1990, J. Biol.
Chem. 265:4583-4591).
6.2. Discussion
[0287] The meaning of fragile sites for cancer has been a subject
of speculation for years and the near coincidence of the
chromosomal position of the FRA3B and the t(3;8) translocation at
3p14.2 has been especially intriguing. The FRA3B is constitutive;
that is, after treatment of peripheral blood lymphocytes with
.about.0.4 .mu.M aphidicolin, which interferes with the action of
DNA polymerase .alpha., the characteristic gaps in chromosome
region 3p14.2 are observed in .about.70% of metaphases from all
individuals. So the structural basis for the induction of gaps is
present in all individuals. It is also known that within the 3p14.2
band, some of the induced gaps represent chromosome breaks, which
occur possibly at several sites in the chromatin of an
.about.200-300 kilobase region (Paradee et al., 1995, Genomics
27:358-361). Thus, the sequences involved in gaps and breaks may
occur in more than one site within the fragile region. At other
fragile sites such as the folate-sensitive fragile sites on X,
FRAXA, FRAXE, FRAXF, the structural basis for the gaps seems to be
variable lengths of CCG or CGG triplet repeats and imperfect
repeats are more stable than perfect repeats (Chung et al., 1993,
Nature Genet. 5:254-258); these fragile sites seem to be single
sites of fragility. Perhaps the FRA3B appears to be the most common
fragile site because it actually represents a collection of
different fragile sites in a small chromosomal region. The specific
sequences responsible for the breaks at FRA3B in hybrid cells have
not been described but we have observed that many tumor-derived
cell lines exhibit apparent discontinuous homozygous deletions.
FIG. 5 diagrams the relationship between the various types of
chromosome breaks in 3p14.2 and the organization of the FHIT gene
relative to the breaks. Note that in FIG. 5, the chromosome breaks
and deletions in the KatoIII gastric carcinoma-derived cells leave
the coding region intact, but we have observed only aberrant FHIT
transcript in this cell line. Thus, inapparent chromosomal
abnormalities must account for the lack of normal transcription in
KatoIII and other tumor cells; one possibility is that two FHIT
alleles are present in KatoIII with hemizygous alterations in the
portions of the FHIT genes not homozygously deleted. Another
possibility is that alteration near an exon affects splicing.
Additionally, some cancer-derived cell lines and uncultured tumors
showed transcripts with alterations to noncoding regions of the
FHIT transcript. These transcripts were transcribed and translated
into full length protein in a coupled system using a reticulocyte
lysate for translation (FIG. 4B), but perhaps in the tumor cells
from which they were derived, the lack of exon 4 or insertion of
new sequences would affect expression of the Fhit protein. Another
puzzle, if the FHIT gene acts as a classical suppressor gene with
inactivation of both alleles, is the presence of normal-sized
transcripts along with aberrant products in the RT-PCR
amplification products of tumor-derived cell lines such as CCL235
(colon), A549 (lung) and HeLa (cervical). It is possible that the
aberrant transcripts, which in most cases might encode partial Fhit
proteins, could interfere with the function of a normal Fhit
protein. The normal-sized products from these cell lines have not
yet been fully sequenced so it is possible that they do not, in
fact, represent normal transcripts. A number of the uncultured
tumors also exhibited aberrant and normal-sized products, and
sequencing showed that some of these normal-sized products were
indeed wildtype products. In these cases, the normal transcripts
could have derived from admixed normal cells.
[0288] We have not yet observed point mutations within the coding
region of any FHIT transcripts, perhaps suggesting that aberrant.
FHIT genes usually are the result of deletions.
[0289] Aphidicolin, which inhibits the action of DNA polymerase
.alpha., induces the gaps and breaks observed in the FRA3B region
in normal metaphases; thus in the digestive tract tumors and tumor
cell lines we have studied, the genomic deletions resulting in
aberrant transcription and loss of functional Fhit protein, could
have been induced by exposure of these organs to other agents which
interfere with DNA replication, such as nicotine, caffeine,
possibly alcohol and other known carcinogens. Interestingly, zinc
deficiency is associated with a high frequency of esophageal tumors
in man (Yang, 1980, Cancer Res. 40:2633-2644) and rat (Fong et al.,
1978, J. Natl. Cancer Inst. 61:145-150); zinc deficiency may cause
proliferation of the epithelial cells lining the esophagus (Yang et
al., 1987, J. Natl. Cancer Inst. 79:1241-1246), so perhaps zinc
deficiency mimics loss of the Fhit protein, which may require bound
zinc for its function. It is, therefore, interesting that FHIT gene
exon 8, carrying the HIT motif, the presumptive zinc binding site,
is a target of deletion in numerous digestive tract tumors.
[0290] Whether or not this region of 3p14.2 contains repeated CCG
or CGG triplets is not yet known, but because there are differences
between the rare, inherited folate-sensitive fragile sites which
have been characterized, and the common, constitutive, aphidicolin
fragile sites, perhaps a different basis for the fragility should
be expected. Thus far, we have noted that there are many Alu
repeats in the telomeric portion of the fragile region (not shown)
and there is a (TAA).sub.15 repeat in this same commonly deleted
region for which the number of repeats is highly variable. Perhaps
other triplet repeats of this type exist in the region. Also in
.about.9 kilobase pairs of sequenced portions of the cosmid S8
(telomeric portion of the fragile region, see FIG. 1A), several Alu
repeats and a LINE element were encountered; the nucleotide content
of the sequenced region was 57.4% A and T residues, while the FHIT
cDNA nucleotide content was 48% A and T. A high A and T content is
characteristic of some characterized origins of DNA replication,
especially in yeast and, in fact, although higher eukaryotic
origins of replication have not been identified, it has been
speculated that Alu repeats may be connected with replication.
Another notable feature of the FHIT gene itself is that nearly all
the exons end with the sequence AG, the usual sequence for splice
acceptor sites. Based on our observation of frequent aberrant
splicing in this fragile region, it is tempting to speculate that
the region is especially rich in sequences resembling splice
acceptor sites.
[0291] Interestingly, we have previously observed a homozygous
deletion in mouse L cells, which involves several N-terminal exons
of the murine Ptprg gene (Wary et al., 1993, Cancer Res.
53:1498-1502), and Pathak et al. (1995, Cancer Genet. Cytogenet.
83:172-173) have shown that mouse-colon and mammary tumors as well
as melanomas have abnormalities in, the proximal region of mouse
chromosome 14 where Ptprg (Wary et al., 1993, Cancer Res.
53:1498-1502) and probably Fhit loci map.
[0292] Studies of FHIT gene RT-PCR products from RNA of numerous
cell lines suggested that PHIT gene abnormalities could be
important not only in airway and digestive tract tumors such as
nasopharyngeal, esophageal, stomach and colorectal carcinomas, but
possibly also in ovarian, cervical and lung tumors, osteosarcoma,
and some leukemias; also a bladder and breast carcinoma cell line
exhibited homozygous deletions in the fragile region (Lisitsyn et
al., 1995, Proc. Natl. Acad. Sci. USA 92:151-155; and out data).
Thus, uncultured tumors of these types should be tested for FHIT
gene abnormalities.
[0293] Clear cell RCCs might also be expected to involve FHIT gene
aberrations because the FHIT gene is disrupted by the familial RCC
translocation break in 3p14.2 and the translocation/FRA3B region is
the target of allelic loss in most sporadic clear cell RCCs (Druck
et al., 1995, Cancer Res. 55:5348-5355). Since the FHIT ORF is
contained in exons 5 through 9, translocated to chromosome 8 in the
t(3;8) family, it is possible that both alleles could still be
expressed in some or all tissues; we have found a few polymorphisms
within the ORF but none yet which distinguishes the two allelic
FHIT transcripts in the t(3;8) lymphoblastoid cell lines (data not
shown). If the FHIT gene disruption is the first "hit" to a
suppressor gene in this family, then the second FHIT allele should
be altered in the t(3;8) tumors. Since we have not yet detected
point mutations in the FHIT gene, the best way to look for
alterations of the FHIT gene in t(3;8) RCCs would be to amplify the
FHIT reverse transcript, as done for uncultured tumors in this
study. We have done this experiment for RNA from two RCC cell lines
and two uncultured RCCs, all from sporadic tumors, and have
observed normal-sized products, which have not yet been cloned and
sequenced. Nor have we yet observed homozygous deletions in RCCs
using a subset of STS markers in the fragile region. Nevertheless,
it would be surprising if the FHIT gene is not involved in some
sporadic RCCs.
[0294] Since the FHIT gene is probably ubiquitously expressed, it
may not be surprising if it can serve as a tumor suppressor gene
for specific tissues of many different organs, apparently
predominantly of the digestive tract, or maybe predominantly organs
with epithelial cell linings. Another common denominator of the
types of tumor exhibiting aberrant FHIT alleles might be that they
are predominantly organs directly exposed to environmental
carcinogens; some of the types of tumors exhibiting FHIT gene
aberrations occur very frequently in restricted regions of the
globe, NPC in China, gastric cancer in southeast Asia, and often
there are environmental factors at play. A possible role for EBV in
promotion of Chinese NPCs might be through-viral DNA integration
into the FRA3B region, suggested by the previous experiments of
Rassool et al. (1992, Am. J. Hum. Genet. 50:1243-1251), showing
apparent preferential integration of exogenous DNA into induced
fragile sites in cultured cells. Similarly human papillomaviruses
associated with cervical carcinomas might promote induction of the
FRA3B, contributing to the loss of heterozygosity on 3p in uterine
cancers (Yokota et al., 1989, Cancer Res. 49:3598-3601), and
possibly to inactivation of the FHIT gene. Perhaps the t(3;8)
family members, carrying one disrupted FHIT gene succumb to kidney
tumors rather than colon or esophageal tumors due to specific types
of environmental agents to which they are exposed.
[0295] We observed strong similarity of the FHIT gene to S. pombe
and S. cerevisiae Ap.sub.4A hydrolases. Specific roles for the
diadenosine, Ap.sub.4A, have not been defined (Huang et al., 1995,
Biochem. J. 312:925-932) and it is not clear that the Ap.sub.4A
hydrolase activity is the only or even the major in vivo function
of these proteins. Expression of the S. pombe aph1 in S. cerevisiae
did not inhibit growth, but for unknown reasons the S. pombe enzyme
was not expressed at a high level (Huang et al., 1995, Biochem. J.
312:925-932). Very little is known of the function of the other
members of the HIT family. If indeed the FHIT gene is the cognate
of the S. pombe aph1 gene identified as an Ap.sub.4A hydrolase,
then the strong conservation (69% similarity) between the yeast and
human gene suggests important functions. Whether the FHIT gene does
or does not encode an Ap.sub.4A hydrolase, it is likely that the
study of the S. pombe and S. cerevisiae hydrolase knockouts and
other types of mutations will be useful in understanding the
functions of the Fhit protein.
[0296] There is some suggestion that as an intracellular regulatory
molecule, the Ap.sub.4A diadenosine may regulate ability of cells
to adapt to metabolic stress such as heat, oxidation, and DNA
damage; thus deviation from abnormal level of Ap.sub.4A may result
in inability of cells to adapt to environmental stresses imposed by
carcinogens or viruses which cause genetic damage.
6.3. Material and Methods
Tissues and Cell Lines
[0297] Matched normal and cancerous tissues from patients with
primary esophageal, colon and stomach carcinomas were obtained
immediately after surgery. Tumors were dissected to eliminate
normal tissue before preparation of DNA. Many cell lines were
obtained from the ATCC. The RC kidney cell lines were kindly
provided by E. Lattime.
RNA Extraction and Reverse Transcription
[0298] Total and poly A.sup.+ mRNA was extracted from cell lines
and tissues using the RNAzol kit (TelTest, Inc., Texas) or the
FastTrack Kit (Invitrogen), respectively. To obtain mRNA from
tissues, fresh specimens were frozen immediately after excision,
and stored at -85.degree. C. or in liquid nitrogen until extraction
of mRNA. RNA was stored as a pellet under ethanol or solubilized in
RNAse-free water and kept at -70.degree. C. Reverse transcription
was performed in 30 .mu.l final volume of 50 mM tris-HCl pH 8.3, 75
mM KCl, 3 mM MgCl.sub.2, 10 mM DTT, 2 .mu.M dNTPs, 500 ng oligo-dT,
600 units MMLV-RT (BRL), 40 units RNasin (Promega), and 2 .mu.g
RNA. This reaction was incubated at 37.degree. C. for 90 min and
boiled for 5 min.
DNA Sequence Analysis
[0299] cDNA, genomic clones and putative exons were sequenced using
primers specific for vector flanking sequences (T3, T7 etc.) and
various synthetic oligonucleotides. RT-PCR products were directly
sequenced after isolation of bands from low melt agarose and
purification by column chromatography (Qiagen, Chatsworth, Calif.).
Sequencing of double-stranded plasmids, PCR products and phage or
cosmid genomic clones was performed using Taq DyeDeoxy Terminator
Cycle Sequencing Kits (Applied Biosystems, Inc. (ABI)); reaction
products were electrophoresed and recorded on the 373 or 377 DNA
sequencer (ABI). Sequences were analyzed using GCG, BLAST, and
GRAIL software.
PCR Amplification
[0300] The oligonucleotides for generating probes, PCR products and
RT-PCR products were designed using the computer program Oligo 4.0
(National Biosciences). For Southern blots, probes were produced by
PCR amplification using various FHIT specific primers, as indicated
in results. Sequences and positions of some primers are shown in
FIG. 2A.
[0301] PCR reactions were carried out in 12.5 or 25 .mu.l final
volume with 1-100 ng of template, 20-40 ng primers, 10 mM tris-HCl
pH 8.3, 50 mM KCl, 0.1 mg/ml gelatin, 15 mM MgCl.sub.2, 200-600 mM
dNTPs and 0.5-2.5 units Taq polymerase (ABI). The amplifications
were performed in a Perkin-Elmer Cetus thermal cycler for 30 cycles
of 94.degree. C. for 30 s (for denaturation), 60.degree. C. (varied
for specific primer pairs) for 30 s (for annealing), and extending
at 72.degree. C. for 30-45 s. The PCR products were visualized in
ethidium bromide stained low melting agarose gels. The bands of
amplified DNA were excised from gels and purified for labeling or
sequencing.
DNA Preparation and Southern Blot Hybridization
[0302] Cellular DNAs were isolated and Southern blots prepared
using conventional methods. Probes were labeled by random-priming
with [.sup.32P]-dCTP (NEN) and hybridized to membranes in 0.75 M
NaCl, 50% formamide at 42.degree. C. overnight. Final washes of
membranes were in 0.1.times.SSC and 0.1% SDS at 65.degree. C. for
30 min. Hybridized filters were exposed to XAR-2 X-ray film (Kodak)
with intensifying screens for times varying from 1 to 72 h.
Identification of YACs
[0303] We and others (Boldog et al., 1993, Proc. Natl. Acad. Sci.
USA 90:8509-8513; Boldog et al., 1994, Genes Chrom. Cancer
11:216-221; Wilke et al., 1994, Genomics 22:319-326; Michaelis et
al., 1995, Cancer Genet. Cytogenet. 81:1-12; Kastury et al., 1996,
Genomics, in press) had previously identified the 850A6 clone from
the Genethon mega YAC library as containing the D3S1300 and D3S1481
markers (Roche et al., 1994, Hum. Mol. Genet. 3:215). Overlapping
YACs were identified by analysis of the Genethon database
information.
Identification of Region Specific STSs
[0304] A number of STSs were available from our work with the 850A6
YAC. The A6URA marker was from the 850A6 URA end, A3 was from an
Alu-vectorette amplified fragment of 850A6; BE758-6 and D3S1480
amplified fragments were used as probes to select phage genomic
clones from which end sequences were obtained and sequence tagged.
A phage genomic clone for D3S1300 was selected from the 850A6 phage
library and end clone D1300E3 isolated. Other D3S and WI marker
primer pairs were obtained from Research Genetics or were
synthesized from sequences provided in the WI database. From
sequencing of a phage genomic subclone from the 648D4 YAC, a
(TAA).sub.15 trinucleotide repeat was found and designated locus
ph13; the AP4/5 STS was derived from partial sequencing of a cosmid
subclone of the 648D4 YAC.
Cosmid Mapping
[0305] High molecular weight YAC containing yeast DNA in agarose
plugs was partially restricted with the Sau3AI enzyme, and
subcloned into a cosmid vector. This cosmid library was initially
screened with DNA probes derived from STSs previously mapped to
this region. The ends of the insert DNAs flanking the cosmid vector
were sequenced to find new STSs, which were used as probes to
rescreen the cosmid libraries.
Exon Trapping and cDNA Cloning
[0306] The cosmid DNAs were partially restricted with Sau3AI
enzyme, run on a 1.0% agarose gel, and fragments larger than 2 kbp
cut out and subcloned into the pSPL vector and transfected into
COS-7 cells, according to the manufacturer's instructions. The DNA
inserts trapped between the splice sites of the vector were
sequenced by a primer supplied with the vector (GIBCO/BRL). The
cDNA was extended in the 5' direction by PCR-amplification of a
total human fetal brain cDNA using an exon-specific primer (X8,
nucleotide numbers -19 to -41) and a RACE reaction kit (Clontech).
The normal colon cDNA library was purchased from Clontech.
FHIT Exon Mapping
[0307] The genomic sequences of exon-intron junctions of the FHIT
gene were determined by sequencing the positive cosmids with
primers derived from the cDNA. Localization of each exon of the
FHIT gene was determined by PCR amplification using primers derived
from each exon with YAC and chromosome 3 hybrid DNAs as templates.
The primer sequences used to obtain cDNA probes flanking exon 5
were: 5'TCTGCTCTGTCCGGTCACA3' (SEQ ID NO:70) (nuc. #-355 to -337)
with primer X8 (shown in FIG. 2A) for 5' flanking exons;
5'ATGTCCTTGTGTGCCCGCT3' (SEQ ID NO:71) (nuc. #105 to 123) with 3D2
(see FIG. 2A) for 3' flanking exons.
Northern Blot and Hybridization
[0308] Two .mu.g of mRNAs were electrophoresed through 1.5% agarose
gel in 2.2 M formaldehyde and 1.times.MOPS buffer and blotted to a
positively charged membrane by standard procedures. Northern blot
filters of multiple normal tissue mRNAs were purchased (Clontech,
Palo Alto, Calif.). The FHIT cDNA probe for hybridization was
obtained using the FHIT cDNA as template for PCR-amplification with
the following primer pair: 5'TGAGGACATGTCGTTCAGATTTGG3' (SEQ ID
NO:72), nuc. #-7 to 17; and 5'CTGTGTCACTGAAAGTAGACC3' (SEQ ID
NO:73), nuc. #449 to 429. Probes were labeled by random-priming
with [.sup.32P]-dCTP and 2.times.10.sup.6 cpm/ml was hybridized to
each filter. Hybridizations were at 42.degree. C. for 16 hours in
SSPE buffer (5.times.SSPE, 10.times. Denhardt's solution, 0.1 mg/ml
carrier DNA, 50% formamide, 2% SDS). Final washes were in
0.1.times.SSC, 0.1% SDS at 50.degree. C. for 30 min before exposure
at -80.degree. C. to XAR-2 films (Kodak) with intensifying screens
on both sides.
Nested RT-PCR and Sequencing of cDNAs
[0309] First strand cDNAs were synthesized and 1 .mu.l of each
product was subjected to a first round of PCR amplification with 30
cycles of 95.degree. C. for 20 sec, 60.degree. C. for 30 sec, and
72.degree. C. for 1 min with 5% dimethylsulfoxide and 0.5 mM
spermidine in 10 .mu.l reaction volume under standard conditions,
using primers 5U2 and 3D2, indicated in FIG. 2A. One .mu.l of the
reaction products, after 20-fold dilution, was subjected to a
second round of PCR amplification using nested primers 5U1 and 3D1
(shown in FIG. 2A), under the conditions noted above, except the
reaction volume was 30 .mu.l. The PCR products were run, on 1.5%
agarose gels, stained with ethidium bromide, purified and 2.5 ng
sequenced using the 5U1 primer.
In Vitro Transcription and Translation
[0310] Three different fragments of DNA, containing the FHIT gene
were obtained by PCR, using oligonucleotides UR5
(5'CTGTAAAGGTCCGTAGTG3' (SEQ ID NO:74), nuc. #-171 to -154 in FIG.
2A) and O6 (5'CTGTGTCACTGAAAGTAGACC3' (SEQ ID NO:75), the reverse
complement of nuc. #429-449). Amplifications were performed in 100
.mu.l final volume of 10 mM Tris-HCI (pH 8.9), 50 mM KCl, 1.5 mM
MgCl.sub.2, 200 .mu.M deoxynucleotide triphosphates, 10 ng RT-PCR
products and 2.5 U Taq polymerase using an Omni Gene Thermal
Cycler. 25 PCR cycles consisted of 94.degree. C. 1 min, 52.degree.
C. 1 min and an extension step at 72.degree. C. 45 sec. PCR
products were separately inserted in a PCRII plasmid using the TA
cloning system (Invitrogen). Recombinant vectors, containing the
normal FHIT and aberrant genes under the control of the T7
promoter, were sequenced and used for in vitro transcription and
translation.
[0311] The in vitro transcription and translation reactions were
performed by TnT Coupled reticulocyte systems (Promega) in a final
volume of 50 .mu.l containing 1/2 volume rabbit reticulocyte
lysate, 1 .mu.g recombinant plasmid DNA, 10 U T7 polymerase, 20
.mu.M amino acid mixture without methionine, 40 .mu.M
.sup.35S-methionine (Amersham) and 40 U RNasin ribonuclease
inhibitor. Reactions were carried out for 90 min at 30.degree. C.
The synthesized proteins were analyzed by SDS polyacrylamide gel
electrophoresis (SDS-PAGE) and autoradiography.
7. THE FHIT GENE AT 3p14.2 IS ABNORMAL IN LUNG CANCER
[0312] The FHIT gene is disrupted by the t(3;8) chromosomal
translocation observed in a family with renal cell carcinoma and
contains the FRA3B fragile site and the target of homozygous
deletions in various human cancer derived cell lines. The study in
Section 6 hereof indicates that FHIT gene abnormalities often occur
in primary digestive tract cancers.
[0313] Deletions of the short arm of chromosome 3 occur at a very
high frequency and in early phases of lung carcinogenesis
suggesting that this chromosomal region contains crucial genes for
lung cancer development. We isolated the FHIT gene, located at the
chromosomal band 3p14.2, and found that it contains the FRA3B
fragile site. The gene is disrupted in the t(3;8) translocation
observed in a family with renal cell carcinoma and resides in a
region which shows allelic losses in various human malignancies. In
this study in Section 7 hereof, we have analyzed the role of the
FHIT gene in cancers associated with carcinogenic exposure, that is
lung cancer of the small-cell and non-small-cell type. Analysis of
59 tumors and paired normal lung tissues was performed by reverse
transcription of FHIT transcripts followed by PCR amplification and
sequencing of products; allelic losses affecting the gene were
evaluated by microsatellite polymorphisms analysis. About 80% of
small-cell lung tumors and 40% of non small-cell lung cancers
showed abnormalities in RNA transcripts of FHIT and 88% of the
tumors exhibited loss of FHIT alleles. Abnormal lung tumor
transcripts lack two or more exons of the FHIT gene. All the cases
showing abnormal transcripts also had loss of one allele of the
FHIT gene. The results indicate abnormalities of this gene in
nearly all SCLC and in a high proportion of NSCLC, suggesting a
critical role for the FHIT gene in lung carcinogenesis.
7.1. Results
RT-PCR and cDNA Sequence Analysis
Small Cell Lung Cancer (SCLC)
[0314] In order to study abnormalities in FHIT transcripts from
tumors and normal tissues, we reverse-transcribed mRNAs and
amplified the cDNAs by nested-PCR as described in methods. Fourteen
primary tumor samples and one cell line (83L) were studied. In two
cases matched normal lung parenchyma was also analyzed. Eleven of
the 14 cases (79%) analyzed by RT-PCR showed the presence of
abnormal transcripts (FIG. 11). The analysis of the amplified
transcripts from the primary tumors consistently revealed the
presence of two abnormal bands of .about.360 bp (type I) and
.about.250 bp (type II). Seven cases displayed both type I and II
abnormal transcripts whereas four cases showed only the type I
band. In two samples (FIG. 11A, case 107; FIG. 12A, case 45) as
well as in the tumor-derived cell line (FIG. 12A, case 83L) the
normal sized transcript was undetectable while in the other nine
cases a normal-sized band of varying intensity was observed.
[0315] In one patient (FIG. 12A, case 83) we examined the primary
tumor, a tumor derived-cell line and a normal lung specimen.
Whereas in the normal lung only the normal transcript was detected,
the primary tumor exhibited the type I and II abnormal transcripts
together with a normal sized transcript and the tumor-derived cell
line displayed the type I abnormal transcript and a novel band of
-420 bp (FIG. 12), probably generated following in vitro
subculturing. Accordingly, cytogenetic analysis of this cell line
revealed extensive chromosomal instability resulting in the
presence of dicentric and tricentric chromosomes, telomeric
associations and double minutes. FISH analysis with a painting
probe of chromosome 3 showed the occurrence of several structural
rearrangements of this chromosome including a translocation of the
3p arm with a breakpoint in 3p14-21 (data not shown).
Interestingly, in the cell line, the normal-sized transcript was
undetectable suggesting that the normal-sized product observed in
the primary tumor reflected the presence of normal cells
infiltrating the tumor specimen.
[0316] Abnormal and normal-sized bands were separated on agarose
gel, cut and sequenced (FIG. 12B). Sequence analysis of the
aberrant bands revealed that the type I transcript corresponded to
absence of exons 4 to 6 (nt -111 to 249) of the FHIT cDNA sequence
(to be accorded GenBank accession # u46922), resulting in a
junction between exons 3 and 7 of the FHIT cDNA. A loss of exons 4
to 8 (nt -111 to 348), resulting in fusion of exons 3 and 9 was
found in the type II transcript. In each aberrant transcript the
fusion junctions coincided with splice sites.
[0317] Sequence analysis of the normal-sized bands revealed that
they contained the normal FHIT cDNA, possibly reflecting the
contribution of normal cells infiltrating the tumor specimens.
[0318] Since both types of aberrant transcripts lacked exon 5,
containing the initial methionine codon of the FHIT open reading
frame (see Section 6), and type II transcripts also lacked nearly
the entire coding region, including exon 8, containing the highly
conserved HIT motif (see Section 6), it is likely that the results
of these aberrant fusion transcripts would lead to loss of FHIT
function.
[0319] Sequencing of the normal-sized RT-PCR product amplified from
normal tissues (cases 45 and 83) revealed presence of the wildtype
FHIT transcript.
Non Small Cell Lung Cancer (NSCLC)
[0320] RNA from 45 primary NSCLC tumors and matching normal lung
parenchyma samples were similarly studied; 18 of 45 tumors (40%)
displayed aberrant RT-PCR products. A detailed description of these
results is summarized in Table 4. TABLE-US-00004 TABLE 4 RT-PCR,
SEQUENCING AND LOH RESULTS IN NSCLC CASE/TYPE RT-PCR* SEQUENCE
LOH.degree. 1.sub.ADC N Normal YES A del ex3.fwdarw.7 (nt
-111.fwdarw.4249) 2.sub.SQC N Normal NE A del ex4.fwdarw.8 + (nt
-17.fwdarw.279) A del ex4.fwdarw.9 (nt -17.fwdarw.348)
3.sup..DELTA..sub.MUCOEp N Normal NE A del ex3.fwdarw.8 (nt
-111.fwdarw.279) 4.sub.SQC N Normal YES A del ex4.fwdarw.8 (nt
-17.fwdarw.279) 5.sub.ADC N Normal YES A del ex3.fwdarw.9 (nt
-111.fwdarw.348) 6.sub.ADC N Normal YES A del ex3.fwdarw.8 (nt
-111.fwdarw.279) 7.sub.ADC N Normal YES A del ex3.fwdarw.9 (nt
-111.fwdarw.348) 8.sub.SQC N Normal YES A del ex3.fwdarw.7 (nt
-111-249) 9.sub.SQC N Normal NE A del ex3.fwdarw.9 (nt
-111.fwdarw.348) 10.sub.SQC N NE YES A 11.sub.ADC N NE YES A
12.sub.ADC N NE YES A 13.sub.SQC N NE YES A 14.sub.ADC N Normal YES
A del ex3.fwdarw.9 (nt -111.fwdarw.348) 15.sub.ADC N NE NI A del
ex3.fwdarw.9 (nt -111.fwdarw.348) 16.sub.ADC N NE NE A A 17.sub.SQC
N NE A del ex4.fwdarw.8 (nt -17.fwdarw.279) 18.sub.ADC N NE NE A N
= Normal Transcript A = Abnormal Transcript .sup..DELTA.In Normal
Lung Parenchyma: del ex3.fwdarw.9 .degree.Loci analyzed: D3S4103,
(ph13), D3S1234, D3S1313, D3S1312 NE: Not Evaluated NI: Non
Informative
[0321] The RT-PCR amplified products from the transcripts present
in these tumors consisted of one or two abnormal bands, always
accompanied by a normal-sized transcript (FIG. 13A). All the paired
normal lung RNAs from the same lung cancer cases showed the
presence of the normal FHIT product only, except one case (case 3
in Table 4) which displayed an aberrant product differing in size
from the aberrant product observed in the corresponding tumor.
Sequence analysis of the aberrant fragments revealed a range of
RT-PCR products with losses of various exons from 4 to 9 (Table 4
and FIG. 13B), including an RT-PCR product-missing exons 4 to 8,
resulting in a junction of exons 3 and 9 (nt -111 to 348), products
missing exons 4 to 6 or 7, fusing exon 3 to exon 7 or 8,
respectively, and products missing exon 5 to exon 7 or 8, resulting
in junctions between exon 4 and 8 or exons 4 and 9, respectively.
All the types of abnormal transcripts observed lacked exon 5, the
first coding exon, and half (6/12) of the abnormal transcripts
characterized by sequence analysis also showed loss of exon 8
containing the HIT domain. Sequence analysis of the normal-sized
transcript amplified from RNA of the normal tissue of these
patients revealed a normal FHIT cDNA sequence. A small
alternatively spliced region at the beginning of exon 10 from
nucleotides 450 to 460, outside the open reading frame of the FHIT
gene, was observed in the normal-sized transcript present in the
tumor and in the corresponding normal tissue of several
patients.
Loss of Heterozygosity (LOH) Analysis
[0322] To look for allelic losses in tumor samples, a PCR-based
approach was used using primers which amplify polymorphic
microsatellite markers internal and flanking the FHIT gene. DNA
from tumor and corresponding normal tissues from 28 NSCLC and 7
SCLC cases were analyzed for allelic losses at locus D3S4103 (ph
13) (see Section 6), internal to the FHIT gene, and at loci located
in flanking regions, centromeric (D3S1312 at 3p14.2) (Druck et al.,
1995, Cancer Res. 55:5348-5353) and telomeric (D3S1234 at 3p14.2
and D3S1313 at 3p14.3) to the FHIT gene. (See Table 5).
TABLE-US-00005 TABLE 5 LOH Frequency at 3p14.2 Loci in SCLC and
NSCLC D3S1234 D3S4103 D3S1212 20/25 (80%) 20/25 (80%) 12/15
(80%)
[0323] In NSCLC samples LOH at loci D3S4103 and D3S1234 was found
in 17 of the 21 informative cases (81%). The combined frequency of
losses affecting these two loci was 88% (23/26 of the informative
cases). Ten of 13 (76%) and 9 of 11 (81%) tumors also showed LOH at
D3S1312 and at D3S1313 loci, respectively, indicating a large
deletion in this genomic region. In SCLC cases, 3 of 4 informative
patients showed LOH at D3S4103 and D3S1234, and 4 of 5 informative
cases had lost at least one of these loci. Overall 12 of 12 (100%)
of the informative tumors which exhibited abnormal FHIT
transcripts, showed allelic losses at one or more of the loci
tested.
7.2. Discussion
[0324] Three distinct chromosomal regions of 3p, which include
3p25, 3p21.3-p21.2 and 3p12-p14.1, are believed to harbour gene(s)
involved in lung cancer on the basis of the high frequency of
allelic loss in primary tumors and defined homozygous deletions in
lung cancer derived cell lines; extensive efforts have been made to
define small common region of loss in order to isolate tumor
suppressor genes in these chromosomal regions.
[0325] Deletions of 3p constitute particularly useful genetic
markers since several studies have reported that they occur in the
early stages of lung carcinogenesis, such as bronchial dysplasia
and metaplasia (Sundaresan et al., 1992, Oncogene 7:1989-1997;
Sozzi et al., 1991, Cancer Res. 51:400-404; Hung et al., 1995, JAMA
273:558-563). Moreover allelic loss on chromosome 3p in primary
SCLC has been suggested to represent an unfavorable prognostic
factor (Horio et al., 1993, Cancer Res. 53:1-4).
[0326] The study disclosed herein describes the occurrence of
abnormalities in transcripts of the FHIT gene, located at 3p14.2,
in at least 80% of SCLC and 40% of NSCLC with 88% of the cases also
exhibiting loss of one FHIT allele. Since the RT-PCR nested
amplification would detect only internal alterations in the FHIT
gene transcripts, this is a conservative estimate of the
involvement of the FHIT gene in SCLC and NSCLC.
[0327] The lung tumor transcripts were missing two or more exons of
the FHIT gene. While in NSCLCs a varying pattern of abnormal
transcripts was detected, in SCLCs the amplified transcripts were
either missing exons 4 to 6 or exons 4 to 8. Both types result in
loss of exon 5, containing the initial methionine codon (see
Section 6), with the second type also showing loss of exon 8
containing the highly conserved HIT domain (see Section 6). The
consequence of the loss of these exons is that no in-frame Fhit
protein could be produced.
[0328] Two cases of primary SCLC and a tumor cell line lacked the
normal-sized transcript, which was also underrepresented in the
remaining cases. In addition, one primary tumor exhibited two
abnormal transcripts and a normal transcript, while a normal-sized
product was not amplified from the cell line established from this
tumor. These observations suggest that in the SCLC RNA the
wild-type transcript could have derived from admixed normal
cells.
[0329] In RNAs from NSCLCs the abnormal RT-PCR amplified products
were sometimes less abundant than the normal-sized RT-PCR amplified
products. A possible explanation could be the heterogeneous, often
multifocal nature of these neoplasms, which arise as a consequence
of the chronic exposure of the entire bronchial "field" to
carcinogens, resulting in the presence of different cell clones
carrying different genetic changes (Kim et al., 1993, Am. J.
Pathol. 142:307-317; Barsky et al., 1994, Mod. Pathol. 7:633-640;
Ebina et al., 1994, Cancer Res. 54:2496-2503). In addition, the
tumor samples contained variable amounts of normal stromal tissue
(stromal infiltration is known to occur in non small-cell tumors)
(Rabbitts et al., 1989, Genes Chrom. Cancer 1:95-105). The complete
allele loss seen in the SCLC cell line and in several SCLC primary
specimens and lack of complete loss of alleles in NSCLC supports
this interpretation. It is also possible that the abnormal
transcripts are less stable then the wild type product.
[0330] In the corresponding normal tissue of the patients showing
abnormal tumor transcripts we have observed a normal FHIT product
by PCR and sequence analysis.
[0331] Of particular interest was one NSCLC patient (case 3 of
Table 4) with a mucoepidermoid carcinoma of the lung who
subsequently developed a renal cell carcinoma. In the normal lung
parenchyma of this patient an abnormal transcript missing exons 4
to 8 was detected. This finding raises the possibility that a
constitutional alteration within the FHIT gene could be associated
with a predisposition to develop both lung and renal cancer or
other types of multiple primary tumors. However this alteration
could have been somatically acquired because carcinogen exposure
can induce transformation in several fields of the bronchial
epithelium through the induction of different genetic changes which
are also detectable in early preneoplastic lesions.
[0332] The high frequency (88%) of loss of one FHIT allele observed
in lung tumors of both small cell and non small cell type is
noteworthy. Although we did not determine a minimal region of loss
in our cases, these findings support the idea that inactivation of
the FHIT gene could have occurred by a mechanism of loss of one
allele and altered expression of the remaining one.
[0333] This model is consistent with the observation that the FHIT
gene spans a common fragile region, FRA3B, where abnormalities such
as deletions could be more frequent than point mutations. Tumors
associated with carcinogen exposure, such as cancers of the
aerodigestive tract, could be particularly susceptible to
alterations of the FHIT gene. Due to its etiology, lung cancer is
the likely to be strongly and directly associated with the effects
of agents which interfere with DNA replication, such as nicotine
and mutagens like benzo(a)pyrene contained in cigarette smoke.
Breakage in a fragile site containing gene as a consequence of
physical, chemical and biological agents can thus be expected.
Expressivity of the FRA3B fragile site in peripheral blood
lymphocytes of patients with cancer has been investigated; the
expression of FRA3B appeared to be influenced by habitual tobacco
smoking and significantly higher expression was reported in lung
cancer patients (Murata et al., 1992, Jpn. J. Hum. Genet.
37:205-213).
[0334] High levels of intracellular diadenosine 5',5'''-P1,P4
tetraphosphate (Ap4A) have been detected at the G1-S boundary
(Weinmann-Dorsch et al., 1984, Eur. J. Biochem. 138:179-185) and a
role for Ap4A in the stimulation of DNA polymerase activity has
been proposed (Baxi et al., 1994, Biochemistry 33:14601-14607). It
seems plausible that loss of function of the FHIT gene could result
in the constitutive accumulation of AP4A and in the stimulation of
DNA synthesis and proliferation. Thus loss of FHIT function could
initiate the malignant process by stimulating the proliferation of
the cells that are the precursors of digestive tract cancer and
lung cancer.
7.3. Experimental Procedures
Tumors
[0335] The 59 tumors, including 25 cases of adenocarcinomas, 19
squamous cell carcinomas, 1 mucoepidermoid carcinoma and 14 small
cell lung carcinomas, were obtained from surgically resected lung
cancer patients at Istituto Nazionale Tumori (Milano, Italy). A
cell line (83L) was established from one small-cell tumor (83T).
Twenty-nine NSCLCs were in stage I, 9 in stage II and 6 in stage
III. The tumors were classified histologically according to the
Histological Typing of Lung Tumors by the World Health Organization
(1987) and staged according to the TNM classification of malignant
tumors defined by the International Union Against Cancer (1987).
Most cases (54 out of 59) were from male patients and the mean age
of cases at presentation was 63 years. Matched normal lung
parenchyma tissue samples were taken at a most distant site from
the tumor or in a different segment or lobe, as a source for the
normal RNA and DNA.
RNA Extraction and Reverse Transcription
[0336] Tumor and normal specimens were frozen immediately after
surgical resection and stored at -80.degree. C. Total mRNA was
extracted from frozen tumor and normal lung tissues using
guanidinium-LiCl separation (Sambrook et al., 1989, Molecular
cloning: A Laboratory Manual. Cold Spring Harbor Lab. Press,
Plainview, N.Y.) or the RNA-STAT kit (Tel TEST, Inc., Texas). cDNA
was synthesized from 1 .mu.g of total RNA. Reverse transcription
was performed in a 20 .mu.l volume of 1.times. first strand buffer
(GIBCO), 10 mM DTT (GIBCO), 500 .mu.M dNTPs, 50' ng/.mu.l oligo-dT,
0.3 .mu.g/.mu.l random primers, 16.5 U RNAsin (PROMEGA), 300 U
Superscript II (GIBCO). The samples were first denatured for 5 min
at 95.degree. C. and incubated at 37.degree. C. for 60 min. The
reaction was stopped by inactivating the enzyme at 94.degree. C.
for 5 min. The reaction was diluted to 30 .mu.l and 1 .mu.l was
used for subsequent PCR amplification.
RT-PCR and cDNA Sequencing
[0337] 1 .mu.l of cDNA was used for a first PCR amplification
performed in a volume of 25 .mu.l containing 0.8 .mu.M of primers
5U2 and 3D2 (see Section 6), 50 .mu.M of each dNTP (TAKARA),
1.times.PCR buffer and 1.25 U Taq Polymerase (TAKARA). The PCR
reaction consisted of an initial denaturation at 95.degree. for 3
min and 25 cycles of 15 sec at 94.degree., 30 sec at 62.degree., 45
sec at 72.degree. and a final extension of 5 min at 72.degree.,
using a Perkin Elmer PCR Thermocycler. The amplified product was
diluted 20-fold in TE buffer and 1 .mu.l of the diluted reaction
product was subjected to a second round of PCR amplification using
nested primers 5U1 and 3D1 (see Section 6) for 30 cycles under the
above conditions. The PCR products were resolved on 1.5%
ethidium-bromide stained Metaphor gel (FMC). Bands were cut from
gels and DNA was purified using a QIA quick gel extraction Kit
(QIAGEN). 5-50 ng of cDNA, depending on the size of the PCR
products, were sequenced using primers 5U1 and 3D1 by the
dideoxynucleotide termination reaction chemistry for sequence
analysis on the Applied Biosystems Models 373A and 377 DNA
sequencing systems.
LOH Analysis
[0338] DNAs from frozen tumor and normal tissues were extracted
using standard methods (Sambrook et al., 1989, Molecular cloning: A
Laboratory Manual. Cold Spring Harbor Lab. Press, Plainview, N.Y.).
Analysis of allelic losses was performed using a PCR-based
approach. Primers which amplify polymorphic microsatellite markers
were used for the following loci: D3S4103 (ph13) (3p14.2) internal
to the FHIT gene (see Section 6), D3S1234 (3p14.2), D3S1313
(3p14.3) and D3S1312 (3p14.2) flanking the gene. The sequence of
all oligonucleotide primers will be available through the Genome
Data Base. Twenty cycles of amplification were carried out at
55.degree.-60.degree. C. annealing temperature as appropriate for
each primer.
[0339] For informative cases, allelic loss was scored if the
autoradiographic signal of one allele was approximately 50% reduced
in the tumor DNA, compared to the corresponding normal allele. The
loci displaying microsatellite instability were not scored for
allelic loss.
8. Deposit of Microorganisms
[0340] E. coli strain DH5.alpha. carrying plasmid p7F1, containing
a full-length FHIT cDNA as a BamHI-XbaI insert into the pBluescript
SK.sup.+ vector (Stratagene) was deposited on Jan. 30, 1996, with
the American Type Culture Collection, 1201 Parklawn Drive,
Rockville, Md. 20852, under the provisions of the Budapest Treaty
on the International Recognition of the Deposit of Microorganisms
for the Purposes of Patent Procedures, and assigned accession
number 69977.
[0341] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications
are intended to fall within the scope of the appended claims.
[0342] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
Sequence CWU 1
1
86 1 1095 DNA Homo sapiens 1 tccccgctct gctctgtccg gtcacaggac
tttttgccct ctgttcccgg gtccctcagg 60 cggccaccca gtgggcacac
tcccaggcgg cgctccggcc ccgcgctccc tccctctgcc 120 tttcattccc
agctgtcaac atcctggaag ctttgaagct caggaaagaa gagaaatcca 180
ctgagaacag tctgtaaagg tccgtagtgc tatctacatc cagacggtgg aagggagaga
240 aagagaaaga aggtatccta ggaatacctg cctgcttaga ccctctataa
aagctctgtg 300 catcctgcca ctgaggactc cgaagaggta gcagtcttct
gaaagacttc aactgtgagg 360 acatgtcgtt cagatttggc caacatctca
tcaagccctc tgtagtgttt ctcaaaacag 420 aactgtcctt cgctcttgtg
aataggaaac ctgtggtacc aggacatgtc cttgtgtgcc 480 cgctgcggcc
agtggagcgc ttccatgacc tgcgtcctga tgaagtggcc gatttgtttc 540
agacgaccca gagagtcggg acagtggtgg aaaaacattt ccatgggacc tctctcacct
600 tttccatgca ggatggcccc gaagccggac agactgtgaa gcacgttcac
gtccatgttc 660 ttcccaggaa ggctggagac tttcacagga atgacagcat
ctatgaggag ctccagaaac 720 atgacaagga ggactttcct gcctcttgga
gatcagagga ggaaatggca gcagaagccg 780 cagctctgcg ggtctacttt
cagtgacaca gatgtttttc agatcctgaa ttccagcaaa 840 agagctattg
ccaaccagtt tgaagaccgc ccccccgcct ctccccaaga ggaactgaat 900
cagcatgaaa atgcagtttc ttcatctcac catcctgtat tcttcaacca gtgatccccc
960 acctcggtca ctccaactcc cttaaaatac ctagacctaa acggctcaga
caggcagatt 1020 tgaggtttcc ccctgtctcc ttattcggca gccttatgat
taaacttcct tctctgctgc 1080 aaaaaaaaaa aaaaa 1095 2 147 PRT Homo
sapiens 2 Met Ser Phe Arg Phe Gly Gln His Leu Ile Lys Pro Ser Val
Val Phe 1 5 10 15 Leu Lys Thr Glu Leu Ser Phe Ala Leu Val Asn Arg
Lys Pro Val Val 20 25 30 Pro Gly His Val Leu Val Cys Pro Leu Arg
Pro Val Glu Arg Phe His 35 40 45 Asp Leu Arg Pro Asp Glu Val Ala
Asp Leu Phe Gln Thr Thr Gln Arg 50 55 60 Val Gly Thr Val Val Glu
Lys His Phe His Gly Thr Ser Leu Thr Phe 65 70 75 80 Ser Met Gln Asp
Gly Pro Glu Ala Gly Gln Thr Val Lys His Val His 85 90 95 Val His
Val Leu Pro Arg Lys Ala Gly Asp Phe His Arg Asn Asp Ser 100 105 110
Ile Tyr Glu Glu Leu Gln Lys His Asp Lys Glu Asp Phe Pro Ala Ser 115
120 125 Trp Arg Ser Glu Glu Glu Met Ala Ala Glu Ala Ala Ala Leu Arg
Val 130 135 140 Tyr Phe Gln 145 3 168 PRT S. pombe 3 Met Pro Lys
Gln Leu Tyr Phe Ser Lys Phe Pro Val Gly Ser Gln Val 1 5 10 15 Phe
Tyr Arg Thr Lys Leu Ser Ala Ala Phe Val Asn Leu Lys Pro Ile 20 25
30 Leu Pro Gly His Val Leu Val Ile Pro Gln Arg Ala Val Pro Arg Leu
35 40 45 Lys Asp Leu Thr Pro Ser Glu Leu Thr Asp Leu Phe Thr Ser
Val Arg 50 55 60 Lys Val Gln Ser Ala Ser Ala Ser Asn Ile Gly Ile
Gln Asp Gly Val 65 70 75 80 Asp Ala Gly Gln Thr Val Pro His Val His
Val His Ile Ile Pro Arg 85 90 95 Lys Lys Ala Asp Phe Ser Glu Asn
Asp Leu Val Tyr Ser Glu Leu Glu 100 105 110 Lys Asn Glu Gly Asn Leu
Ala Ser Leu Tyr Leu Thr Gly Asn Glu Arg 115 120 125 Tyr Ala Gly Asp
Glu Arg Pro Pro Thr Ser Met Arg Gln Ala Ile Pro 130 135 140 Arg Thr
Leu Glu Glu Met Glu Lys Glu Ala Gln Trp Leu Lys Gly Tyr 145 150 155
160 Phe Ser Glu Glu Gln Glu Lys Glu 165 4 203 PRT S. cerevisiae 4
Met Ile Leu Ser Lys Thr Lys Lys Pro Lys Ser Met Asn Lys Pro Ile 1 5
10 15 Tyr Phe Ser Lys Phe Leu Val Thr Glu Gln Val Phe Tyr Lys Ser
Lys 20 25 30 Tyr Thr Tyr Ala Leu Val Asn Leu Lys Pro Ile Val Pro
Gly His Val 35 40 45 Leu Ile Val Pro Leu Arg Thr Thr Val Leu Asn
Leu Ser Asp Leu Thr 50 55 60 Met Pro Glu Ser Gln Asp Tyr Phe Lys
Thr Leu Gln Leu Ile His Lys 65 70 75 80 Ala Asp Ser Ile Asn Val Ala
Ile Gln Asp Gly Pro Glu Ala Gly Gln 85 90 95 Ser Val Pro His Leu
His Thr His Ile Ile Pro Arg Tyr Lys Ile Asn 100 105 110 Asn Val Gly
Asp Leu Ile Tyr Asp Lys Leu Asp His Trp Asp Gly Asn 115 120 125 Gly
Thr Leu Thr Asp Trp Gln Gly Arg Arg Asp Glu Tyr Leu Gly Val 130 135
140 Gly Gly Arg Gln Ala Arg Lys Asn Asn Ser Thr Ser Ala Thr Val Asp
145 150 155 160 Gly Asp Glu Leu Ser Gln Gly Pro Asn Val Leu Val Arg
Ala Leu Thr 165 170 175 Glu Met Lys Lys Glu Ala Glu Asp Leu Gln Ala
Arg Leu Glu Glu Phe 180 185 190 Val Ser Ser Asp Pro Gly Leu Thr Gln
Trp Leu 195 200 5 119 PRT B. taurus 5 Ala Asp Glu Ile Ala Lys Ala
Gln Val Ala Arg Pro Gly Gly Asp Thr 1 5 10 15 Ile Phe Gly Lys Ile
Ile Arg Lys Glu Ile Pro Ala Lys Ile Ile Tyr 20 25 30 Glu Asp Asp
Gln Cys Leu Ala Phe His Asp Ile Ser Pro Gln Ala Pro 35 40 45 Thr
His Phe Leu Val Ile Pro Lys Lys Tyr Ile Ser Gln Ile Ser Ala 50 55
60 Ala Glu Asp Asp Asp Glu Ser Leu Leu Gly His Leu Met Ile Val Gly
65 70 75 80 Lys Lys Cys Ala Lys Gly Tyr Arg Met Val Val Asn Glu Gly
Ser Asp 85 90 95 Gly Gly Gln Ser Val Tyr His Val His Leu His Val
Leu Gly Gly Arg 100 105 110 Gln Met Asn Trp Pro Pro Gly 115 6 108
PRT Synechococcus sp. 6 Met Ser Glu Asp Thr Ile Phe Gly Lys Ile Ile
Arg Arg Glu Ile Pro 1 5 10 15 Ala Asp Ile Val Tyr Glu Asp Asp Leu
Cys Leu Ala Phe Arg Asp Val 20 25 30 Ala Pro Gln Ala Pro Val His
Ile Leu Val Ile Pro Lys Gln Pro Ile 35 40 45 Ala Asn Leu Leu Glu
Ala Thr Ala Glu His Gln Ala Leu Leu Gly His 50 55 60 Leu Leu Leu
Thr Val Lys Ala Ile Ala Glu Gly Tyr Arg Thr Val Ile 65 70 75 80 Asn
Thr Gly Pro Ala Gly Gly Gln Thr Val Tyr His Leu His Ile His 85 90
95 Leu Leu Gly Gly Arg Ser Leu Ala Trp Pro Pro Gly 100 105 7 104
PRT M. hyorhinis 7 Met Asn Asn Trp Gln Glu Glu Leu Phe Leu Lys Ile
Ile Lys Arg Glu 1 5 10 15 Glu Pro Ala Thr Ile Leu Tyr Glu Asp Asp
Lys Val Ile Ala Phe Leu 20 25 30 Asp Lys Tyr Ala His Thr Lys Gly
His Phe Leu Val Val Pro Lys Asn 35 40 45 Tyr Ser Arg Asn Leu Phe
Ser Ile Ser Asp Glu Asp Leu Ser Tyr Leu 50 55 60 Ile Val Lys Ala
Arg Glu Phe Ala Gly Ala Thr Gly Phe Lys Leu Leu 65 70 75 80 Ile Asn
Asn Glu Pro Asp Ala Glu Gln Ser Ile Phe His Thr His Val 85 90 95
His Ile Ile Pro Tyr Tyr Lys Lys 100 8 151 PRT S. cerevisiae 8 Met
Glu Pro Leu Ile Ser Ala Pro Tyr Leu Thr Thr Thr Lys Met Ser 1 5 10
15 Ala Pro Ala Thr Leu Asp Ala Ala Cys Ile Phe Cys Lys Ile Ile Lys
20 25 30 Ser Glu Ile Pro Ser Phe Lys Leu Ile Glu Thr Lys Tyr Ser
Tyr Ala 35 40 45 Phe Leu Asp Ile Gln Pro Thr Ala Glu Gly His Ala
Leu Ile Ile Pro 50 55 60 Lys Tyr His Gly Ala Lys Leu His Asp Ile
Pro Asp Glu Phe Leu Thr 65 70 75 80 Asp Ala Met Pro Ile Ala Lys Leu
Asp Thr Tyr Asn Val Leu Gln Asn 85 90 95 Asn Gly Lys Ile Ala His
Gln Glu Val Asp His Val His Phe His Leu 100 105 110 Ile Pro Lys Arg
Asp Glu Lys Ser Gly Leu Ile Val Gly Trp Pro Ala 115 120 125 Gln Glu
Thr Asp Phe Asp Lys Leu Gly Lys Leu His Lys Glu Leu Leu 130 135 140
Ala Lys Leu Glu Gly Ser Asp 145 150 9 455 DNA Homo sapiens
misc_feature 13, 43, 105, 240, 315, 445, 446 n = A,T,C or G 9
aagaaaaccg ganagacttt gaagcacgtt cacgtccacg ttnttcccgg gaaggctgga
60 aaactttcac aggaatgaca gcatctatga ggagctccca gaaanatgac
aaggaggact 120 ttcctgcctc ttggagatca gaggaggaaa tggcagcaga
aagccgcagc tctgcgggtc 180 tactttcagt gacacagatc ctgaattcca
gcaaaagagc tattgccaac cagtttgaan 240 accgcccccc cgcctctccc
caagaggaac tgaatcagca tgaaaatgca gtttcttcat 300 ctcaccatcc
tgtantcttc aaccagtgat cccccacctc ggtcactcca actcccttaa 360
aatacctaga cctaaacggc tcagacaggc agatttgagg tttccccctg tctccttatt
420 cggcagcctt atgattaaac ttccnnctct gctgc 455 10 6 PRT Homo
sapiens VARIANT 4 Xaa = Any Naturally Occurring Amino Acid 10 Ala
Ala Glu Xaa Glu Val 1 5 11 15 PRT Homo sapiens 11 Gly Cys Arg Ile
Arg Arg Gln Gly Glu Thr Ser Asn Leu Pro Val 1 5 10 15 12 19 PRT
Homo sapiens VARIANT 14 Xaa = Any Naturally Occurring Amino Acid 12
Gly Ser Trp Ser Asp Arg Gly Gly Gly Ser Leu Val Glu Xaa Tyr Arg 1 5
10 15 Met Val Arg 13 36 PRT Homo sapiens VARIANT 19 Xaa = Any
Naturally Occurring Amino Acid 13 Arg Asn Cys Ile Phe Met Leu Ile
Gln Phe Leu Leu Gly Arg Gly Gly 1 5 10 15 Gly Ala Xaa Phe Lys Leu
Val Gly Asn Ser Ser Phe Ala Gly Ile Gln 20 25 30 Asp Leu Cys His 35
14 13 PRT Homo sapiens 14 Thr Arg Arg Ala Ala Ala Phe Cys Cys His
Phe Leu Leu 1 5 10 15 35 PRT Homo sapiens VARIANT 11, 32 Xaa = Any
Naturally Occurring Amino Acid 15 Ser Pro Arg Gly Arg Lys Val Leu
Leu Val Xaa Phe Leu Gly Ala Pro 1 5 10 15 His Arg Cys Cys His Ser
Cys Glu Ser Phe Pro Ala Phe Pro Gly Xaa 20 25 30 Thr Trp Thr 35 16
12 PRT Homo sapiens VARIANT 3, 4 Xaa = Any Naturally Occurring
Amino Acid 16 Gln Gln Xaa Xaa Lys Phe Asn His Lys Ala Ala Glu 1 5
10 17 37 PRT Homo sapiens VARIANT 34 Xaa = Any Naturally Occurring
Amino Acid 17 Gly Asp Arg Gly Lys Pro Gln Ile Cys Leu Ser Glu Pro
Phe Arg Ser 1 5 10 15 Arg Tyr Phe Lys Gly Val Gly Val Thr Glu Val
Gly Asp His Trp Leu 20 25 30 Lys Xaa Thr Gly Trp 35 18 8 PRT Homo
sapiens 18 Asp Glu Glu Thr Ala Phe Ser Cys 1 5 19 91 PRT Homo
sapiens VARIANT 12, 57, 78, 88 Xaa = Any Naturally Occurring Amino
Acid 19 Phe Ser Ser Ser Trp Gly Glu Ala Gly Gly Arg Xaa Ser Asn Trp
Leu 1 5 10 15 Ala Ile Ala Leu Leu Leu Glu Phe Arg Ile Cys Val Thr
Glu Ser Arg 20 25 30 Pro Ala Glu Leu Arg Leu Ser Ala Ala Ile Ser
Ser Ser Asp Leu Gln 35 40 45 Glu Ala Gly Lys Ser Ser Leu Ser Xaa
Phe Trp Glu Leu Leu Ile Asp 50 55 60 Ala Val Ile Pro Val Lys Val
Phe Gln Pro Ser Arg Glu Xaa Arg Gly 65 70 75 80 Arg Glu Arg Ala Ser
Lys Ser Xaa Arg Phe Ser 85 90 20 36 PRT Homo sapiens VARIANT 3 Xaa
= Any Naturally Occurring Amino Acid 20 Ser Arg Xaa Gly Ser Leu Ile
Ile Arg Leu Pro Asn Lys Glu Thr Gly 1 5 10 15 Gly Asn Leu Lys Ser
Ala Cys Leu Ser Arg Leu Gly Leu Gly Ile Leu 20 25 30 Arg Glu Leu
Glu 35 21 7 PRT Homo sapiens 21 Pro Arg Trp Gly Ile Thr Gly 1 5 22
32 PRT Homo sapiens VARIANT 2, 27 Xaa = Any Naturally Occurring
Amino Acid 22 Arg Xaa Gln Asp Gly Glu Met Lys Lys Leu His Phe His
Ala Asp Ser 1 5 10 15 Val Pro Leu Gly Glu Arg Arg Gly Gly Gly Xaa
Gln Thr Gly Trp Gln 20 25 30 23 44 PRT Homo sapiens VARIANT 39 Xaa
= Any Naturally Occurring Amino Acid 23 Leu Phe Cys Trp Asn Ser Gly
Ser Val Ser Leu Lys Val Asp Pro Gln 1 5 10 15 Ser Cys Gly Phe Leu
Leu Pro Phe Pro Pro Leu Ile Ser Lys Arg Gln 20 25 30 Glu Ser Pro
Pro Cys His Xaa Ser Gly Ser Ser Ser 35 40 24 5 PRT Homo sapiens 24
Met Leu Ser Phe Leu 1 5 25 22 PRT Homo sapiens VARIANT 8, 18 Xaa =
Any Naturally Occurring Amino Acid 25 Lys Phe Ser Ser Leu Pro Gly
Xaa Asn Val Asp Val Asn Val Leu Gln 1 5 10 15 Ser Xaa Ser Gly Phe
Leu 20 26 25 PRT Homo sapiens VARIANT 5, 15 Xaa = Any Naturally
Occurring Amino Acid 26 Lys Lys Thr Gly Xaa Thr Leu Lys His Val His
Val His Val Xaa Pro 1 5 10 15 Gly Lys Ala Gly Lys Leu Ser Gln Glu
20 25 27 33 PRT Homo sapiens VARIANT 5 Xaa = Any Naturally
Occurring Amino Acid 27 Gly Ala Pro Arg Xaa Met Thr Arg Arg Thr Phe
Leu Pro Leu Gly Asp 1 5 10 15 Gln Arg Arg Lys Trp Gln Gln Lys Ala
Ala Ala Leu Arg Val Tyr Phe 20 25 30 Gln 28 22 PRT Homo sapiens
VARIANT 12 Xaa = Any Naturally Occurring Amino Acid 28 Ile Pro Ala
Lys Glu Leu Leu Pro Thr Ser Leu Xaa Thr Ala Pro Pro 1 5 10 15 Pro
Leu Pro Lys Arg Asn 20 29 28 PRT Homo sapiens VARIANT 14 Xaa = Any
Naturally Occurring Amino Acid 29 Ile Ser Met Lys Met Gln Phe Leu
His Leu Thr Ile Leu Xaa Ser Ser 1 5 10 15 Thr Ser Asp Pro Pro Pro
Arg Ser Leu Gln Leu Pro 20 25 30 20 PRT Homo sapiens 30 Thr Ala Gln
Thr Gly Arg Phe Glu Val Ser Pro Cys Leu Leu Ile Arg 1 5 10 15 Gln
Pro Tyr Asp 20 31 5 PRT Homo sapiens VARIANT 3 Xaa = Any Naturally
Occurring Amino Acid 31 Thr Ser Xaa Ser Ala 1 5 32 6 PRT Homo
sapiens VARIANT 4 Xaa = Any Naturally Occurring Amino Acid 32 Arg
Lys Pro Xaa Arg Leu 1 5 33 28 PRT Homo sapiens VARIANT 7, 28 Xaa =
Any Naturally Occurring Amino Acid 33 Ser Thr Phe Thr Ser Thr Xaa
Phe Pro Gly Arg Leu Glu Asn Phe His 1 5 10 15 Arg Asn Asp Ser Ile
Tyr Glu Glu Leu Pro Glu Xaa 20 25 34 42 PRT Homo sapiens 34 Gln Gly
Gly Leu Ser Cys Leu Leu Glu Ile Arg Gly Gly Asn Gly Ser 1 5 10 15
Arg Lys Pro Gln Leu Cys Gly Ser Thr Phe Ser Asp Thr Asp Pro Glu 20
25 30 Phe Gln Gln Lys Ser Tyr Cys Gln Pro Val 35 40 35 14 PRT Homo
sapiens VARIANT 1 Xaa = Any Naturally Occurring Amino Acid 35 Xaa
Pro Pro Pro Arg Leu Ser Pro Arg Gly Thr Glu Ser Ala 1 5 10 36 57
PRT Homo sapiens VARIANT 11, 54, 55 Xaa = Any Naturally Occurring
Amino Acid 36 Lys Cys Ser Phe Phe Ile Ser Pro Ser Cys Xaa Leu Gln
Pro Val Ile 1 5 10 15 Pro His Leu Gly His Ser Asn Ser Leu Lys Ile
Pro Arg Pro Lys Arg 20 25 30 Leu Arg Gln Ala Asp Leu Arg Phe Pro
Pro Val Ser Leu Phe Gly Ser 35 40 45 Leu Met Ile Lys Leu Xaa Xaa
Leu Leu 50 55 37 108 PRT Homo sapiens VARIANT 4, 14, 35, 80, 105
Xaa = Any Naturally Occurring Amino Acid 37 Glu Asn Arg Xaa Asp Phe
Glu Ala Arg Ser Arg Pro Arg Xaa Ser Arg 1 5 10 15 Glu Gly Trp Lys
Thr Phe Thr Gly Met Thr Ala Ser Met Arg Ser Ser 20 25 30 Gln Lys
Xaa Asp Lys Glu Asp Phe Pro Ala Ser Trp Arg Ser Glu Glu 35 40 45
Glu Met Ala Ala Glu Ser Arg Ser Ser Ala Gly Leu Leu Ser Val Thr 50
55 60 Gln Ile Leu Asn Ser Ser Lys Arg Ala Ile Ala Asn Gln Phe Glu
Xaa 65 70 75 80 Arg Pro Pro Ala Ser Pro Gln Glu Glu Leu Asn Gln His
Glu Asn Ala 85 90 95 Val Ser Ser Ser His His Pro Val Xaa Phe Asn
Gln 100 105 38 22 PRT Homo sapiens 38 Ser Pro Thr Ser Val Thr Pro
Thr Pro Leu Lys Tyr Leu Asp Leu Asn 1 5 10 15 Gly Ser Asp Arg Gln
Ile 20 39 11 PRT Homo sapiens 39 Gly Phe Pro Leu Ser Pro Tyr Ser
Ala Ala Leu 1 5 10 40 7 PRT Homo sapiens VARIANT 4 Xaa = Any
Naturally Occurring Amino Acid 40 Leu Asn Phe Xaa Leu Cys Cys 1 5
41 48 PRT Homo sapiens 41 Glu Gln Ser Val Lys Val Arg Ser Ala Ile
Tyr Ile Gln Thr Val Glu 1 5 10 15 Gly Arg Glu Arg Glu Arg Arg Tyr
Pro Arg Asn Thr Cys Leu Leu Arg 20 25 30 Pro Ser Ile Lys Ala Leu
Cys
Ile Leu Pro Leu Arg Thr Pro Lys Arg 35 40 45 42 8 PRT Homo sapiens
42 Gln Ser Ser Glu Arg Leu Gln Leu 1 5 43 28 PRT Homo sapiens 43
Gly His Val Val Gln Ile Trp Pro Thr Ser His Gln Ala Leu Cys Ser 1 5
10 15 Val Ser Gln Asn Arg Thr Val Leu Arg Ser Cys Glu 20 25 44 50
PRT Homo sapiens VARIANT 44 Xaa = Any Naturally Occurring Amino
Acid 44 Glu Thr Cys Gly Thr Arg Asp Met Ser Leu Cys Ala Arg Cys Gly
Gln 1 5 10 15 Trp Glu Arg Phe His Asp Leu Arg Pro Asp Glu Val Gly
Arg Phe Val 20 25 30 Ser Asp Asp Pro Glu Ser Ser Gly Gln Trp Leu
Xaa Lys His Phe Pro 35 40 45 Gly Asp 50 45 6 PRT Homo sapiens 45
Ser Thr Glu Asn Ser Leu 1 5 46 30 PRT Homo sapiens 46 Arg Ser Val
Val Leu Ser Thr Ser Arg Arg Trp Lys Gly Glu Lys Glu 1 5 10 15 Lys
Glu Gly Ile Leu Gly Ile Pro Ala Cys Leu Asp Pro Leu 20 25 30 47 7
PRT Homo sapiens 47 Lys Leu Cys Ala Ser Cys His 1 5 48 93 PRT Homo
sapiens VARIANT 88 Xaa = Any Naturally Occurring Amino Acid 48 Gly
Leu Arg Arg Gly Ser Ser Leu Leu Lys Asp Phe Asn Cys Glu Asp 1 5 10
15 Met Ser Phe Arg Phe Gly Gln His Leu Ile Lys Pro Ser Val Val Phe
20 25 30 Leu Lys Thr Glu Leu Ser Phe Ala Leu Val Asn Arg Lys Pro
Val Val 35 40 45 Pro Gly Thr Cys Pro Cys Val Pro Ala Ala Ala Ser
Gly Ser Ala Ser 50 55 60 Met Thr Cys Val Leu Met Lys Trp Ala Asp
Leu Phe Gln Thr Thr Gln 65 70 75 80 Arg Val Arg Asp Ser Gly Trp Xaa
Asn Ile Phe Leu Gly 85 90 49 9 PRT Homo sapiens 49 Pro Leu Arg Thr
Val Cys Lys Gly Pro 1 5 50 17 PRT Homo sapiens 50 Cys Tyr Leu His
Pro Asp Gly Gly Arg Glu Arg Lys Arg Lys Lys Val 1 5 10 15 Ser 51 5
PRT Homo sapiens 51 Glu Tyr Leu Pro Ala 1 5 52 20 PRT Homo sapiens
52 Thr Leu Tyr Lys Ser Ser Val His Pro Ala Thr Glu Asp Ser Glu Glu
1 5 10 15 Val Ala Val Phe 20 53 20 PRT Homo sapiens 53 Lys Thr Ser
Thr Val Arg Thr Cys Arg Ser Asp Leu Ala Asn Ile Ser 1 5 10 15 Ser
Ser Pro Leu 20 54 11 PRT Homo sapiens 54 Cys Phe Ser Lys Gln Asn
Cys Pro Ser Leu Leu 1 5 10 55 22 PRT Homo sapiens 55 Ile Gly Asn
Leu Trp Tyr Gln Gly His Val Leu Val Cys Pro Leu Arg 1 5 10 15 Pro
Val Gly Ala Leu Pro 20 56 23 PRT Homo sapiens VARIANT 18 Xaa = Any
Naturally Occurring Amino Acid 56 Ser Gly Pro Ile Cys Phe Arg Arg
Pro Arg Glu Phe Gly Thr Val Val 1 5 10 15 Gly Xaa Thr Phe Ser Trp
Gly 20 57 17 PRT Homo sapiens VARIANT 7 Xaa = Any Naturally
Occurring Amino Acid 57 Val Pro Arg Lys Met Phe Xaa Gln Pro Leu Ser
Arg Thr Leu Trp Val 1 5 10 15 Val 58 122 PRT Homo sapiens 58 Asn
Lys Ser Ala His Phe Ile Arg Thr Gln Val Met Glu Ala Leu Pro 1 5 10
15 Leu Ala Ala Ala Gly Thr Gln Gly His Val Pro Gly Thr Thr Gly Phe
20 25 30 Leu Phe Thr Arg Ala Lys Asp Ser Ser Val Leu Arg Asn Thr
Thr Glu 35 40 45 Gly Leu Met Arg Cys Trp Pro Asn Leu Asn Asp Met
Ser Ser Gln Leu 50 55 60 Lys Ser Phe Arg Arg Leu Leu Pro Leu Arg
Ser Pro Gln Trp Gln Asp 65 70 75 80 Ala Gln Ser Phe Tyr Arg Gly Ser
Lys Gln Ala Gly Ile Pro Arg Ile 85 90 95 Pro Ser Phe Ser Phe Ser
Pro Phe His Arg Leu Asp Val Asp Ser Thr 100 105 110 Thr Asp Leu Tyr
Arg Leu Phe Ser Val Asp 115 120 59 60 PRT Homo sapiens VARIANT 6
Xaa = Any Naturally Occurring Amino Acid 59 Ser Pro Gly Lys Cys Xaa
Ser Asn His Cys Pro Glu Leu Ser Gly Ser 1 5 10 15 Ser Glu Thr Asn
Arg Pro Thr Ser Ser Gly Arg Arg Ser Trp Lys Arg 20 25 30 Ser His
Trp Pro Gln Arg Ala His Lys Asp Met Ser Leu Val Pro Gln 35 40 45
Val Ser Tyr Ser Gln Glu Arg Arg Thr Val Leu Phe 50 55 60 60 6 PRT
Homo sapiens 60 Glu Thr Leu Gln Arg Ala 1 5 61 5 PRT Homo sapiens
61 Asp Val Gly Gln Ile 1 5 62 6 PRT Homo sapiens 62 Thr Thr Cys Pro
His Ser 1 5 63 44 PRT Homo sapiens 63 Ser Leu Ser Glu Asp Cys Tyr
Leu Phe Gly Val Leu Ser Gly Arg Met 1 5 10 15 His Arg Ala Phe Ile
Glu Gly Leu Ser Arg Gln Val Phe Leu Gly Tyr 20 25 30 Leu Leu Ser
Leu Ser Leu Pro Ser Thr Val Trp Met 35 40 64 12 PRT Homo sapiens 64
Ile Ala Leu Arg Thr Phe Thr Asp Cys Ser Gln Trp 1 5 10 65 102 PRT
Homo sapiens VARIANT 6 Xaa = Any Naturally Occurring Amino Acid 65
Pro Gln Glu Asn Val Xaa Pro Thr Thr Val Pro Asn Ser Leu Gly Arg 1 5
10 15 Leu Lys Gln Ile Gly Pro Leu His Gln Asp Ala Gly His Gly Ser
Ala 20 25 30 Pro Thr Gly Arg Ser Gly His Thr Arg Thr Cys Pro Trp
Tyr His Arg 35 40 45 Phe Pro Ile His Lys Ser Glu Gly Gln Phe Cys
Phe Glu Lys His Tyr 50 55 60 Arg Gly Leu Asp Glu Met Leu Ala Lys
Ser Glu Arg His Val Leu Thr 65 70 75 80 Val Glu Val Phe Gln Lys Thr
Ala Thr Ser Ser Glu Ser Ser Val Ala 85 90 95 Gly Cys Thr Glu Leu
Leu 100 66 5 PRT Homo sapiens 66 Ala Gly Arg Tyr Ser 1 5 67 15 PRT
Homo sapiens 67 Asp Thr Phe Phe Leu Phe Leu Ser Leu Pro Pro Ser Gly
Cys Arg 1 5 10 15 68 11 PRT Homo sapiens 68 His Tyr Gly Pro Leu Gln
Thr Val Leu Ser Gly 1 5 10 69 1409 DNA S. pombe 69 gaaacttgtg
tgcatacgaa taataaaatt cgataatatt ggaattttta gtccgcttta 60
tctgttccat gatactgtta cttacatata tgcaagacgc tattttctca tagtctgttt
120 gttttttaag tatatcaatc tttcttatta tattccatag acactttcgc
acatgactct 180 ccagggactc cgcgatatgg gttgtgagca tcgtgaagct
gaattcaacc aacaacttag 240 attcttacaa tattcgtaag ccagaatgcc
aaaacagcta tatttctcca agtttcctgt 300 tggaagtcaa gttttttatc
gtactaaggt aagttaacgg tctcatgtgt gtagatattg 360 gtgtttgcaa
acttttgttt gtcattctta tttattctat aacggcagac agtttgtgat 420
ttttctttgg ttgaggtcag ctgctaacga ttttagttat ctgccgcgtt tgtaaacctg
480 aaaccaattt taccaggtca tgttttggta attccgcaac gggcggtccc
tagattgaaa 540 gatttgacac cttcagaggt aggattctta tgctattcga
aaaaataatg gaatctgcat 600 acgctaacta atgaaaactt agttgacgga
tttgtttact tctgttcgca aagtgcaaca 660 ggtaatcgaa aaggtgtttt
cggcatctgc atcaaacatt ggtattcaag taagtacttt 720 gatagtcaag
gaataaataa aaaaacatat tccttttcac attcaaaata aaaaatcgtt 780
ttaatttaga agctgacatt ttgcttttaa ctcaatagga tggtgtagac gctggtcaaa
840 cagttcctca tgtacatgtt cacattatcc ctcgtaaaaa ggcagatttt
tcagaaaacg 900 atctagtcta cagtgagttg gaaaaaaacg aaggaaatct
tgcttccctt tatcttacgg 960 gaaatgagcg gtatgcagga gatgagagac
cgccaaccag tatgaggcaa gctattccta 1020 aggacgagga tcgtaagcca
agaacacttg aggaaatgga aaaggaagct cagtggttga 1080 aagggtactt
ttccgaagag caagagaagg aataaaaagt tgaagtacct caataccaca 1140
ggggtagtgt ttacgtatga attaagctaa atattatatg accctttttt tttatttcac
1200 ccaaggttac aagaaaaatt tccttttttc tctctaccct gcttacattg
catctgtctg 1260 ctgagcttta gcaacacaac gtaaccatac atattgtgat
gaacccttct acaattcgat 1320 cgaattagct tcagttccct attttgattt
tgctctcttt ctttcatcct ttcctcataa 1380 ccctactaga tatccatctt
tttgaattc 1409 70 19 DNA Homo sapiens 70 tctgctctgt ccggtcaca 19 71
19 DNA Homo sapiens 71 atgtccttgt gtgcccgct 19 72 24 DNA Homo
sapiens 72 tgaggacatg tcgttcagat ttgg 24 73 21 DNA Homo sapiens 73
ctgtgtcact gaaagtagac c 21 74 18 DNA Homo sapiens 74 ctgtaaaggt
ccgtagtg 18 75 21 DNA Homo sapiens 75 ctgtgtcact gaaagtagac c 21 76
8 PRT Homo sapiens VARIANT 5 Xaa = Any Naturally Occurring Amino
Acid 76 Cys Phe Lys Val Xaa Pro Val Phe 1 5 77 420 DNA Homo sapiens
misc_feature 402 n = A,T,C or G 77 atccactgag aacagtctgt aaaggtccgt
agtgctatct acatccagac ggtggaaggg 60 agagaaagag aaagaaggta
tcctaggaat acctgcctgc ttagaccctc tataaaagct 120 ctgtgcatcc
tgccactgag gactccgaag aggtagcagt cttctgaaag acttcaactg 180
tgaggacatg tcgttcagat ttggccaaca tctcatcaag ccctctgtag tgtttctcaa
240 aacagaactg tccttcgctc ttgtgaatag gaaacctgtg gtaccaggga
catgtccttg 300 tgtgcccgct gcggccagtg ggagcgcttc catgacctgc
gtcctgatga agtgggccga 360 tttgtttcag acgacccaga gagttcggga
cagtggttgg anaaacattt tcctggggac 420 78 41 DNA Homo sapiens 78
gaagggagag aaagagaaag aaggtaccta ggtaatacct g 41 79 46 DNA Homo
sapiens 79 agggagagac agagaaagaa agatggcccc gaagccggac agactg 46 80
21 DNA Homo sapiens 80 aagagaaaga actccagaaa c 21 81 24 DNA Homo
sapiens 81 agcagtcttc tgaaagactt caac 24 82 33 DNA Homo sapiens 82
ggagagaaag agaaagaagt acctaggaat acc 33 83 31 DNA Homo sapiens 83
agcagtcttc tgaaagactt caactgtgag g 31 84 26 DNA Homo sapiens 84
agcagtcttc tgaaagctcc tcaaac 26 85 21 DNA Homo sapiens 85
agagaaagaa cacgttcacg t 21 86 31 DNA Homo sapiens 86 agtcttctga
aagcacgttc acgtccatgt t 31
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