U.S. patent application number 12/902722 was filed with the patent office on 2011-05-19 for mct-1, a human oncogene.
Invention is credited to Ronald Gartenhaus.
Application Number | 20110117576 12/902722 |
Document ID | / |
Family ID | 22189020 |
Filed Date | 2011-05-19 |
United States Patent
Application |
20110117576 |
Kind Code |
A1 |
Gartenhaus; Ronald |
May 19, 2011 |
MCT-1, A HUMAN ONCOGENE
Abstract
A novel gene, designated MCT-1 (for Multiple Copies in T-cell
malignancy), is provided. A protein encoded by MCT-1, designated
MCT-1, is also provided. Antisense oligonucleotides complementary
to or homologous with a portion of MCT-1, substantially purified
MCT-1, and methods of determining whether a cell is a tumor cell
are also provided. The invention also includes monoclonal and
polyclonal antibody preparations which bind with specificity to
MCT-1. The invention further includes methods of determining
whether a compound or a gene product is a modulator of MCT-1
expression, a method of reducing MCT-1 expression in a cell, and a
method of conferring a growth advantage on a cell.
Inventors: |
Gartenhaus; Ronald;
(Pikesville, MD) |
Family ID: |
22189020 |
Appl. No.: |
12/902722 |
Filed: |
October 12, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09709131 |
Nov 10, 2000 |
7811561 |
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12902722 |
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PCT/US99/10184 |
May 10, 1999 |
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09709131 |
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60085029 |
May 11, 1998 |
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Current U.S.
Class: |
435/7.23 ;
435/7.21 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/82 20130101; C07K 14/4738 20130101 |
Class at
Publication: |
435/7.23 ;
435/7.21 |
International
Class: |
G01N 33/566 20060101
G01N033/566 |
Claims
1. A method of determining whether a cell expresses MCT-1 protein
comprising the sequence of SEQ ID NO:8, the method comprising:
contacting the cell with a composition comprising isolated
polyclonal antibodies which bind with specificity to the MCT-1
protein and detecting binding of the polyclonal antibodies to the
protein, wherein the detecting the binding of the polyclonal
antibodies is indicative that the cell expresses the MCT-1
protein.
2. The method of claim 1, wherein the cell exhibits deregulated
cell cycle progression.
3. The method of claim 2, wherein the cell is a cancer cell.
Description
[0001] This application is a divisional application of U.S. Pat.
No. 09/709,131, filed on Nov. 10, 2000, which is in turn a
continuation of international patent application PCT/US99/10184,
filed on May 10, 1999, which is in turn entitled to priority to
U.S. provisional patent application 60/085,029, filed on May 11,
1998, the disclosures of each of which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The field of the invention is oncogenes and
tumorigenesis.
BACKGROUND OF THE INVENTION
[0003] Tumorigenesis is a multi-step process wherein a causal
relationship exists between accumulation of genetic abnormalities
and aggressive clinical behavior of tumor cells (Fearon et al.,
1990, Cell 61:759-767; Callfano et al., 1996, Cancer Res
56:2488-2492). In many tumors, amplification of critical
growth-inducing genes is observed, coupled with deregulated
expression of G1 cyclins and their cognate cdk partners (Lammie et
al, 1991, Oncogene 6:439-444; Motokura et al., 1991, Nature
350:512-515).
[0004] Distinct complexes are formed between cyclins and one or
more cognate cyclin dependent kinases (cdks) at different phases of
the cell cycle, and activation of the cognate kinases occurs
following complex formation. Progression of cells through the late
G1 phase of the cell cycle is controlled by G1 cyclins, including D
and E type cyclins and their cognate cdks (Sherr, 1994, Cell
79:551-555). Phosphorylation of the retinoblastoma gene product
(Rb) and related gene products facilitates entry into S phase. The
D type cyclins are known to form complexes with either cdk4 or cdk6
(Baldin et al., 1993, Genes Develop. 7:812-821).
[0005] Expression of G1 cyclins and their cognate cdk partners is
often deregulated in human tumor cells. Overexpression of cyclin D1
can shorten the G1 interval of the cell cycle, and thereby reduce
cell size and/or transform cells, both in vitro and in vivo (Jiang
et al., 1993, Oncogene 8:3447-3457; Lovec et al., 1994, Oncogene
9:323-326).
[0006] Primary cutaneous lymphomas are among the more common
presentations of extra-nodal non-Hodgkins lymphomas (NHLs). NHLs
include adult T-cell leukemia/lymphoma (ATLL) and cutaneous T-cell
lymphoma (CTCL). The etiologic agent of ATLL, HTLV-1, has been
known for years. In contrast, the molecular pathogenesis of CTCL,
the most frequent type of cutaneous lymphoma, is not well
characterized at present. A recent report demonstrated
rearrangement of the tal-1 and lyt-10 genes in a small subset of
CTCL cells, but no consistent molecular lesions were detected (Neri
et al., 1995, Blood 86:3160-3172).
[0007] Development of effective anti-cancer or anti-tumorigenic
treatments would be facilitated by identification of one or more
genes, mal-expression of which is associated with the onset or
progression of tumorigenesis. The present invention provides the
identity and the coding sequence of such a gene.
BRIEF SUMMARY OF THE INVENTION
[0008] The invention relates to an isolated nucleic acid which
binds with high specificity with a portion of the mRNA-coding
region of a human MCT-1 gene. In one embodiment, the mRNA-coding
region of the gene has the nucleotide sequence SEQ ID NO: 7. For
example, the portion may have a nucleotide sequence selected from
the group consisting of at least about 20 or 25 consecutive
nucleotide residues of SEQ ID NO: 7 and at least about 20 or 25
consecutive nucleotide residues of the sequence complementary to
SEQ ID NO: 7. In another embodiment, the isolated nucleic acid has
a sequence which is either at least about 75% homologous with the
portion or at least about 75% complementary to the portion. For
example, the isolated nucleic acid may have a sequence which is
either at least about 95% or 100% homologous with the portion or at
least about 95% or 100% complementary to the portion.
[0009] The isolated nucleic acid of the invention may comprises one
or more modified internucleoside linkages.
[0010] The invention includes a vector comprising the isolated
nucleic acid of the invention. In the vector, the isolated nucleic
acid may be operably linked with a promoter. For example, a portion
comprising nucleotide residues 258-800 of SEQ ID NO: 7 may be
operably linked with a promoter.
[0011] The invention also includes a pair of isolated nucleic acids
of the invention wherein one of the pair is complementary to a
first portion of the mRNA-encoding region and the other of the pair
is homologous with a second portion of the mRNA-encoding
region.
[0012] The invention further includes an isolated molecular beacon
nucleic acid comprising a first portion and a second portion. The
first portion binds with high specificity with a region of the
mRNA-coding region of a human MCT-1 gene. The second portion
anneals with the first portion to a lesser degree when the first
portion is not annealed with the region than when the first portion
is annealed with the region. The first portion has one of a
fluorophore-quencher pair associated therewith, and the second
portion has the other of the fluorophore-quencher pair associated
therewith. The molecular beacon nucleic acid of the invention
fluoresces in the presence of the region to a greater degree than
in the absence of the region.
[0013] In another aspect, the invention relates to an isolated
polypeptide having an amino acid sequence which comprises at least
about ten or fifteen consecutive amino acid residues of SEQ ID NO:
8. The amino acid sequence of the isolated polypeptide may, of
course, be the entirety of SEQ ID NO: 8. In one embodiment, the
polypeptide is substantially purified.
[0014] The invention also relates to a method of reducing MCT-1
expression in a cell. This method comprises providing an isolated
nucleic acid which binds with high specificity with a portion of
the mRNA-coding region of a human MCT-1 gene to the cell.
Expression of MCT-1 in the cell is thereby reduced.
[0015] The invention further relates to a method of increasing
MCT-1 production in a cell. This method comprises providing an
isolated nucleic acid to the cell. The isolated nucleic acid
comprises a promoter operably linked with a portion of the
mRNA-coding region of an MCT-1 gene. Production of MCT-1 in the
cell is increased by providing the isolated nucleic acid to the
cell. In one embodiment of this method, the portion comprises
nucleotide residues 258-800 of SEQ ID NO: 7.
[0016] The invention still further relates to a method of
determining whether a test compound is a modulator of MCT-1
expression. This method comprises culturing a first cell which
overexpresses MCT-1 in the presence of the test compound and
comparing MCT-1 expression in the first cell with MCT-1 expression
in a second cell of the same type cultured in the absence of the
test compound. A difference between MCT-1 expression in the first
cell and MCT-1 expression in the second cell is an indication that
the test compound is a modulator of MCT-1 expression.
[0017] The invention also includes a method of determining whether
a gene product is a modulator of MCT-1 expression. This method
comprises expressing an isolated nucleic acid encoding the gene
product in a first cell which overexpresses MCT-1 and comparing
MCT-1 expression in the first cell with MCT-1 expression in a
second cell of the same type. The isolated nucleic acid is not
expressed in the second cell. A difference between MCT-1 expression
in the first cell and MCT-1 expression in the second cell is an
indication that the gene product is a modulator of MCT-1
expression.
[0018] The invention further includes a method of determining
whether a cell is a tumor cell. This method comprises comparing
MCT-1 expression in the cell and MCT-1 expression in a non-tumor
cell. A difference between MCT-1 expression in the cell and MCT-1
expression in the non-tumor cell is an indication that the cell is
a tumor cell.
[0019] In another aspect, the invention relates to a method of
determining whether a cell is a tumor cell. This method comprises
comparing MCT-1 copy number in the cell and MCT-1 copy number in a
non-tumor cell. A difference between MCT-1 copy number in the cell
and MCT-1 copy number in the non-tumor cell is an indication that
the cell is a tumor cell.
[0020] The invention also includes a method of conferring a growth
advantage on a cell. This method comprises providing an isolated
nucleic acid to the cell. The isolated nucleic acid comprises a
promoter operably linked to a portion of the mRNA-coding region of
an MCT-1 gene. By providing this isolated nucleic acid to the cell,
a growth advantage is conferred on the cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1, comprising FIGS. 1A and 1B is a pair of sequences.
FIG. 1A is a nucleotide sequence (SEQ ID NO: 1) of a 944 nucleotide
residue human cDNA molecule described in Example 1, which was
originally thought to encode MCT-1. It is now known that the
cytosine residue at position 599 is not present in the sequence, as
indicated in the corrected sequence listed in FIG. 5A. FIG. 1B is
the deduced amino acid sequence (SEQ ID NO: 2) of the protein
encoded by the nucleotide sequence listed in FIG. 1A. Single-letter
codes are used to identify amino acid residues. It is now known
that the amino acid sequence of residues 115+ of MCT-1 differs from
that listed here, as indicated in the corrected sequence listed in
FIG. 5B.
[0022] FIG. 2 is a diagram which depicts various polynucleotides.
The top line is a scale which indicates polynucleotide length,
measured in nucleotide residues. The second line depicts MCT-1
mRNA; the region of the molecule corresponding to the coding region
of MCT-1 is indicated by a thick bar. The third line depicts a
3'-RACE (rapid amplification of cDNA ends) product prepared using
MCT-1 mRNA. The fourth line depicts a 5'-RACE product prepared
using MCT-1 mRNA. The fifth through fourteenth lines represent
individual expressed sequence tags (ESTs) which exhibited homology
with MCT-1.
[0023] FIG. 3 is a listing of the amino acid sequence of MCT-1 (SEQ
ID NO: 2), wherein putative post-translational modification sites
are indicated. A putative glycosylation site, a putative Tyr
phosphorylation site, two putative PKC phosphorylation sites, and a
putative CK2 phosphorylation site are indicated.
[0024] FIG. 4 is a comparison of a portion (SEQ ID NO: 9) of the
amino acid sequence of MCT-1 with a similar portion (SEQ ID NO: 10)
of the amino acid sequence of Cyclin H. A solid bar between
adjacent amino acid residues indicates identity; double dots
between adjacent amino acid residues indicates conservative
replacement; single dots between adjacent amino acid residues
indicates amino acid residues which are structurally similar.
[0025] FIG. 5, comprising FIGS. 5A and 5B, is a pair of sequence
listings. The nucleotide sequence (SEQ ID NO: 7) of the cDNA
encoding MCT-1 is listed in FIG. 5A. The amino acid sequence (SEQ
ID NO: 8) of MCT-1 is listed in FIG. 5B. The underlined sequence is
the polyadenylation signal.
DETAILED DESCRIPTION
[0026] The invention relates to the discovery of a gene, herein
designated MCT-1 (for Multiple Copies in T-cell malignancy), which
is overexpressed in certain tumor cells. For example, MCT-1 is
overexpressed in T-cell tumor cells, such as cells obtained from a
patient afflicted with cutaneous T-cell leukemia (CTCL; i.e. the
Hut 78 cell line). Genomic analysis of such cells indicated that
the MCT-1 gene is present in an increased copy number in tumor cell
lines established from a patient. The MCT-1 gene comprises an open
reading frame (ORF) which encodes a 181-amino acid residue
polypeptide, herein designated MCT-1. A cDNA molecule comprising
the ORF of MCT-1 has been isolated (SEQ ID NO: 7; listed in FIG.
5A). MCT-1 has the amino acid sequence SEQ ID NO: 8, and is listed
in FIG. 5B.
Definitions
[0027] As used herein, each of the following terms has the meaning
associated with it in this section.
[0028] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e. to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0029] The terms "nucleic acid" and "polynucleotide" are used
interchangeably herein and mean any nucleic acid, whether composed
of deoxyribonucleosides or ribonucleosides, and whether composed of
phosphodiester linkages or modified linkages such as
phosphotriester, phosphoramidate, siloxane, carbonate,
carboxymethylester, acetamidate, carbamate, thioether, bridged
phosphoramidate, bridged methylene phosphonate, bridged
phosphoramidate, bridged phosphoramidate, bridged methylene
phosphonate, phosphorothioate, methylphosphonate,
phosphorodithioate, bridged phosphorothioate or sulfone linkages,
and combinations of such linkages. The term nucleic acid also
specifically includes nucleic acids composed of bases other than
the five biologically occurring bases (adenine, guanine, thymine,
cytosine and uracil).
[0030] A nucleic acid has a "modified internucleoside linkage" if
at least one phosphodiester bond in the nucleic acid is replaced by
an alternative chemical linkage such as, for example, a
phosphoramidate linkage.
[0031] An "isolated nucleic acid" refers to a nucleic acid segment
or fragment which has been separated from sequences which flank it
in a naturally occurring state, e.g., a DNA fragment which has been
removed from the sequences which are normally adjacent to the
fragment, e.g., the sequences adjacent to the fragment in a genome
in which it naturally occurs. The term also applies to nucleic
acids which have been substantially purified from other components
which naturally accompany the nucleic acid, e.g., RNA or DNA or
proteins, which naturally accompany it in the cell. The term
therefore includes, for example, a recombinant DNA which is
incorporated into a vector, into an autonomously replicating
plasmid or virus, or into the genomic DNA of a prokaryote or
eukaryote, or which exists as a separate molecule (e.g, as a cDNA
or a genomic or cDNA fragment produced by PCR or restriction enzyme
digestion) independent of other sequences. It also includes a
recombinant DNA which is part of a hybrid gene encoding additional
polypeptide sequence.
[0032] By describing two polynucleotides as "operably linked" is
meant that a single-stranded or double-stranded nucleic acid moiety
comprises the two polynucleotides arranged within the nucleic acid
moiety in such a manner that at least one of the two
polynucleotides is able to exert a physiological effect by which it
is characterized upon the other. By way of example, a promoter
operably linked with the coding region of a gene is able to promote
transcription of the coding region.
[0033] As used herein, the term "promoter" means a DNA sequence
which is required for expression of a gene operably linked to the
promoter. In some instances, the promoter may be the core promoter
sequence and in other instances, the promoter may also include an
enhancer sequence and other regulatory elements which are required
for expression of the gene in, for example, an inducible,
suppressible, or tissue-specific manner.
[0034] A "molecular beacon" nucleic acid is a nucleic acid
comprising a pair of complementary regions and having a fluorophore
and a fluorescent quencher associated therewith. The fluorophore
and quencher are associated with different portions of the nucleic
acid in such an orientation that when the complementary regions are
annealed with one another, fluorescence of the fluorophore is
quenched by the quencher. When the complementary regions of the
nucleic acid are not annealed with one another, fluorescence of the
fluorophore is quenched to a lesser degree. Molecular beacon
nucleic acids are described, for example, in U.S. Pat. No.
5,876,930.
[0035] A "vector" is a composition of matter which comprises an
isolated nucleic acid and which can be used to deliver the isolated
nucleic acid to the interior of a cell. Numerous vectors are known
in the art including, but not limited to, linear polynucleotides,
polynucleotides associated with ionic or amphiphilic compounds,
plasmids, and viruses.
[0036] An "mRNA-coding region" of a gene consists of the nucleotide
residues of the coding strand of the gene and the nucleotide
residues of the non-coding strand of the gene which are homologous
with or complementary to, respectively, an mRNA molecule which is
produced by transcription of the gene. It is understood that, owing
to mRNA processing which occurs in certain instances in eukaryotic
cells, the mRNA-coding region of a gene may comprise a single
region or a plurality of regions separated from one another in the
gene as it occurs in the genome. Where the mRNA-coding region of a
gene comprises separate regions in a genome, "mRNA-coding region"
refers both individually and collectively to each of these
regions.
[0037] "Homologous" as used herein, refers to the subunit sequence
similarity between two polymeric molecules, e.g., between two
nucleic acid molecules, e.g., two DNA molecules or two RNA
molecules, or between two polypeptide molecules. When a subunit
position in both of the two molecules is occupied by the same
monomeric subunit, e.g., if a position in each of two DNA molecules
is occupied by adenine, then they are homologous at that position.
The homology between two sequences is a direct function of the
number of matching or homologous positions, e.g., if half (e.g.,
five positions in a polymer ten subunits in length) of the
positions in two compound sequences are homologous then the two
sequences are 50% homologous, if 90% of the positions, e.g., 9 of
10, are matched or homologous, the two sequences share 90%
homology. By way of example, the DNA sequences 3'-ATTGCC-5' and
3'-TATGGC-5' share 50% homology. When every subunit position in the
two molecules is occupied by the same monomeric subunit, the two
molecules are said to be "completely" homologous.
[0038] "Complementary" refers to the broad concept of subunit
sequence complementarity between regions of two nucleic acid
strands or between two regions of the same nucleic acid strand. It
is known that an adenine residue of a first nucleic acid region is
capable of forming specific hydrogen bonds ("base pairing") with a
residue of a second nucleic acid region which is antiparallel to
the first region if the residue is thymine or uracil. Similarly, it
is known that a cytosine residue of a first nucleic acid strand is
capable of base pairing with a residue of a second nucleic acid
strand which is antiparallel to the first strand if the residue is
guanine. A first region of a nucleic acid is complementary to a
second region of the same or a different nucleic acid if, when the
two regions are arranged in an antiparallel fashion, at least one
nucleotide residue of the first region is capable of base pairing
with a residue of the second region. Preferably, the first region
comprises a first portion and the second region comprises a second
portion, whereby, when the first and second portions are arranged
in an antiparallel fashion, at least about 50%, and preferably at
least about 75%, at least about 90%, or at least about 95% of the
nucleotide residues of the first portion are capable of base
pairing with nucleotide residues in the second portion. More
preferably, all nucleotide residues of the first portion are
capable of base pairing with nucleotide residues in the second
portion, in which instance the two portions are described as being
"completely" complementary.
[0039] A first oligonucleotide anneals with a second
oligonucleotide "with high stringency" if the two oligonucleotides
anneal under high stringency hybridization conditions.
[0040] By "high stringency hybridization conditions" is meant those
oligonucleotide hybridizing conditions that (1) employ low ionic
strength and high temperature for washing, for example, 0.015 molar
NaCl, 1.5 millimolar sodium citrate, and 0.1% (w/v) sodium dodecyl
sulfate (SDS) at 50.degree. C.; (2) employ during hybridization a
denaturing agent such as formamide, for example, 50% (v/v)
formamide, 0.1% (w/v) bovine serum albumin, 0.1% (w/v) Ficoll, 0.1%
(w/v) polyvinylpyrrolidone, and 50 millimolar sodium phosphate
buffer at pH 6.5 with 750 millimolar NaCl, 75 millimolar sodium
citrate at 42.degree. C.; or (3) employ 50% (v/v) formamide,
5.times.SSC (0.75 molar NaCl, 75 millimolar sodium pyrophosphate,
5.times.Denhardt's solution, sonicated salmon sperm DNA (50
micrograms per milliliter), 0.1% (w/v) SDS, and 10% (w/v) dextran
sulfate at 42.degree. C., with washes at 42.degree. C. in
0.2.times.SSC and 0.1% (w/v) SDS. Under stringent hybridization
conditions, only highly complementary nucleic acids hybridize.
Preferably, such conditions prevent hybridization of nucleic acids
having 1 or 2 mismatches out of 20 contiguous nucleotides.
[0041] A "portion" and a "region" of a polynucleotide are used
interchangeably to mean at least at least about twenty sequential
nucleotide residues of the polynucleotide. It is recognized that a
portion of a polynucleotide may include every nucleotide residue of
the polynucleotide.
[0042] As used herein, an "amplified genomic sequence" is a
sequence of nucleotide residues in the genome of a mammal such as a
human which is present in the genome in a greater number of copies
than the number of copies normally present in the genome.
[0043] The term "substantially purified" describes a compound,
e.g., a protein or polypeptide which has been separated from
components which naturally accompany it. Typically, a compound is
substantially pure when at least 10%, more preferably at least 20%,
more preferably at least 50%, more preferably at least 60%, more
preferably at least 75%, more preferably at least 90%, and most
preferably at least 99% of the total material (by volume, by wet or
dry weight, or by mole percent or mole fraction) in a sample is the
compound of interest. Purity can be measured by any appropriate
method, e.g., in the case of polypeptides by column chromatography,
gel electrophoresis or HPLC analysis. A compound, e.g., a protein,
is also substantially purified when it is essentially free of
naturally associated components or when it is separated from the
native contaminants which accompany it in its natural state.
[0044] "Malexpression" of a gene means expression of a gene in a
cell of a patient afflicted with a disease or disorder, wherein the
level of expression (including non-expression), the portion of the
gene expressed, or the timing of the expression of the gene with
regard to the cell cycle, differs from expression of the same gene
in a cell of a patient not afflicted with the disease or disorder.
It is understood that malexpression may cause or contribute to the
disease or disorder, be a symptom of the disease or disorder, or
both.
[0045] As used herein, the term "pharmaceutically acceptable
carrier" means a chemical composition with which the active
ingredient(s) may be combined and which, following the combination,
can be used to administer the active ingredient(s) to a
subject.
[0046] As used herein, the term "physiologically acceptable" ester
or salt means an ester or salt form of the active ingredient(s)
which is compatible with any other ingredients of the
pharmaceutical composition and which is not deleterious to the
subject to which the composition is to be administered.
The Isolated Nucleic Acid of the Invention
[0047] The invention relates to an isolated nucleic acid which
binds with high specificity with a portion of the mRNA-coding
region of a human MCT-1 gene. This region is listed in FIG. 5A (SEQ
ID NO: 7). The isolated nucleic acid may be homologous with or
complementary to a portion of SEQ ID NO: 7, and includes nucleotide
residues 258-800 of SEQ ID NO: 7, which encode the amino acid
sequence of MCT-1 (SEQ ID NO: 8). The portion may be homologous
with or complementary to the entire mRNA-coding region, or at least
about twenty, twenty-five, thirty, fifty, or more nucleotide
residues thereof. It is understood that the isolated nucleic acid
of the invention need not be completely homologous with or
completely complementary to the mRNA-coding region in order for it
to bind with high specificity therewith. Instead, the isolated
nucleic acid need only be mostly complementary or homologous. For
example, the isolated nucleic acid may be 75%, 90%, 95%, or,
preferably, completely complementary to or homologous with the
mRNA-coding region.
[0048] In an important embodiment of the present invention, the
isolated nucleic acid has a sequence which is homologous with
nucleotide residues 258-800 of SEQ ID NO: 7. This portion of the
mRNA-coding region encodes the amino acid sequence of MCT-1
protein. By operably linking this portion, or at least most
residues thereof (e.g. the nucleotide residues encoding amino acid
residues 8-65 of MCT-1) with a transcriptional promoter, the
resulting nucleic acid may be used to generate MCT-1 protein,
either by providing the nucleic acid to an organism capable of
expressing it or by using an in vitro transcription/translation
mixture. Because this portion does not include the terminator codon
(i.e. nucleotide residues 801-803 of SEQ ID NO: 7), a termination
sequence should be operably associated with the portion if the
portion is to be expressed. Of course, it is understood that
additional amino acid-specifying codons may be inserted between the
portion and either or both of the promoter region or the
termination sequence, whereby a fusion protein comprising at least
a portion of the amino acid sequence of MCT-1 is generated upon
expression of the nucleic acid. The fusion protein may, for
example, comprise a portion of a hemagglutinin (as described herein
in Example 2) or a hexa-histidine polypeptide sequence for
facilitating purification of the expressed protein by metal-(e.g.
nickel-)affinity chromatography.
[0049] Given the nucleotide sequence of the mRNA-coding region of
MCT-1 provided herein, one skilled in the art can generate a
polynucleotide capable of annealing with either the coding or the
non-coding strand of MCT-1 or with RNA, such as mRNA, transcribed
therefrom. Such oligonucleotides are useful for binding to mRNA and
single-stranded DNA to modulate transcription and translation
thereof, to facilitate specific detection thereof, or to amplify a
sequence using a PCR method, for example.
[0050] For example, an antisense oligonucleotide capable of
annealing with the coding strand of the mRNA-coding region of MCT-1
(and therefore also capable of annealing with an mRNA molecule
generated by transcribing MCT-1) is made by generating an
oligonucleotide having a nucleotide sequence complementary to a
portion of SEQ ID NO: 7. An antisense oligonucleotide capable of
annealing with the non-coding strand of the mRNA-coding region of
MCT-1 is made by generating an oligonucleotide having a nucleotide
sequence homologous with a portion of SEQ ID NO: 7. Preferably, the
antisense oligonucleotide is either complementary to or homologous
with at least about twenty sequential nucleotide residues of SEQ ID
NO: 7, and preferably to or with about twenty-five, thirty, fifty,
or more sequential nucleotide residues. As is understood in the
art, regions of homology between two polynucleotides may be
interrupted by one or more non-homologous base pairs. Thus, the
antisense oligonucleotide may comprise one or more nucleotide
residues which are not homologous or complementary to the portion
of SEQ ID NO: 7. An antisense oligonucleotide complementary to a
portion of SEQ ID NO: 7 is useful for inhibiting translation of an
mRNA molecule generated by transcribing MCT-1. An antisense
oligonucleotide homologous with a portion of SEQ ID NO: 7 is useful
for inhibiting transcription of MCT-1.
[0051] In another important embodiment, the isolated nucleic acids
of the invention are supplied in pairs which are useful for
amplification of all or part of the sequence of the human MCT-1
gene. Design and use of such primer pairs for amplification of
nucleic acids are well known in the art and are fully enabled by
the listing herein of SEQ ID NOs: 1 and 7. Such primer pairs will
generally comprise a first isolated nucleic acid which is
complementary to a first portion of SEQ ID NO: 7 and a second
isolated nucleic acid which is complementary to a second portion of
SEQ ID NO: 7.
[0052] It is understood that the sole sequence difference between
SEQ ID NO: 7 and SEQ ID NO: 1 is the presence of an additional
cytosine residue in SEQ ID NO: 1 at position 599 (i.e. between
residues 598 and 599 of SEQ ID NO: 7). Thus, SEQ ID NO: 1 may be
used in place of SEQ ID NO: 7 herein, except that use of SEQ ID NO:
7 is preferred when the identity of the residue at position 599 is
critical.
[0053] The isolated nucleic acid of the invention may be delivered
to a cell using a vector. The vector may comprise the isolated
nucleic acid operably linked with one or more of a promoter
sequence, a transcriptional or translational regulatory sequence, a
membrane-directing or "signal" sequence, and a terminator sequence,
or it may not be operably linked with any of these. Thus, the
isolated nucleic acid may be merely delivered to a cell by the
vector, be delivered to the cell in a form in which it is
transcribed in the cell, be delivered in a form in which it is
transcribed and translated in the cell, or be delivered in a form
in which it is transcribed, translated, and directed to a
particular location relative to the cell (e.g. to the nucleus or to
the exterior of the cell). All of these sequences and their use are
well known in the art. The vector may, for example, be a viral
vector or a non-viral vector such as a plasmid.
[0054] Numerous modifications of oligonucleotides are known in the
art, and these modifications, as applied to the isolated nucleic
acid of the invention, are also included in the invention. For
example, oligonucleotides comprising altered sugar moieties,
non-natural inter-sugar linkages, phosphorothioate moieties, methyl
phosphonate moieties, short chain alkyl moieties, cycloalkyl
moieties, and the like are known.
[0055] It is not intended that the present invention be limited by
the nature of the nucleic acid employed. The isolated nucleic acid
of the invention may be native or synthesized nucleic acid. The
isolated nucleic acid may be obtained from a viral, bacterial,
animal, plant, or synthetic source. The nucleic acid may be DNA or
RNA and may exist in a double-stranded, single-stranded or
partially double-stranded form. Furthermore, the nucleic acid may
be found as part of a virus or other macromolecule. See, e.g.,
Fasbender et al., 1996, J. Biol. Chem. 272:6479-89 (polylysine
condensation of DNA in the form of adenovirus).
[0056] Nucleic acids useful in the present invention include, by
way of example and not limitation, oligonucleotides and
polynucleotides such as antisense DNAs and/or RNAs; ribozymes; DNA
for gene therapy; viral fragments including viral DNA and/or RNA;
DNA and/or RNA chimeras; mRNA; plasmids; cosmids; genomic DNA;
cDNA; gene fragments; various structural forms of DNA including
single-stranded DNA, double stranded DNA, supercoiled DNA and/or
triple-helical DNA; Z-DNA; and the like. The nucleic acids may be
prepared by any conventional means typically used to prepare
nucleic acids in large quantity. For example, DNAs and RNAs may be
chemically synthesized using commercially available reagents and
synthesizers by methods that are well-known in the art (see, e.g.,
Gait, 1985, Oligonucleotide Synthesis: A Practical Approach (IRL
Press, Oxford, England)). RNAs may be produce in high yield via in
vitro transcription using plasmids such as SP65 (Promega
Corporation, Madison, Wis.).
[0057] In some circumstances, as where increased nuclease stability
is desired, isolated nucleic acids having modified internucleoside
linkages may be preferred. Isolated nucleic acids containing
modified internucleoside linkages may also be synthesized using
reagents and methods that are well known in the art. For example,
methods for synthesizing nucleic acids containing phosphonate
phosphorothioate, phosphorodithioate, phosphoramidate methoxyethyl
phosphoramidate, formacetal, thioformacetal, diisopropylsilyl,
acetamidate, carbamate, dimethylene-sulfide
(--CH.sub.2--S--CH.sub.2--), dimethylene-sulfoxide
(--CH.sub.2--SO--CH.sub.2--), dimethylene-sulfone
(--CH.sub.2--SO.sub.2--CH.sub.2--), 2'-O-alkyl, and
2'-deoxy-2'-fluoro phosphorothioate internucleoside linkages are
well known in the art (e.g. Uhlmann et al., 1990, Chem. Rev.
90:543-584; Schneider et al., 1990, Tetrahedron Lett. 31:335).
[0058] The isolated nucleic acid of the invention may be purified
by any suitable means, as are well known in the art. For example,
the nucleic acids can be purified by reverse phase or ion exchange
HPLC, size exclusion chromatography or gel electrophoresis. Of
course, the skilled artisan will recognize that the method of
purification will depend in part on the size and type of the
nucleic acid to be purified and on the characteristics of any
molecules, structure, or organisms with which it may be associated.
It is furthermore contemplated that the nucleic acid may comprise
nucleotide residues other than the five naturally occurring bases,
adenine, guanine, thymine, cytosine, and uracil.
[0059] Also contemplated is a manufacture comprising a plurality of
isolated nucleic acids (i.e. probes) of the invention fixed in an
ordered array on a surface. Each of the plurality of probes anneals
with high stringency with a portion of the human MCT-1 gene. By
including probes which differ by a single nucleotide residue within
the corresponding portion of the MCT-1 gene, nucleic acids which
comprise different nucleotide residues at that position within the
MCT-1 gene may be differentiated. Thus, using methods well known in
the art, missense and deletion mutations in the MCT-1 sequence may
be detected. Furthermore, by incorporating into the array probes
which bind with high affinity with sequential portions of the wild
type MCT-1 gene, wherein each sequential portion includes one
nucleotide residue not included within the previous sequential
portion, the nucleotide sequence of all, or any portion, of the
MCT-1 gene may be determined. Preferably, the wild type human MCT-1
cDNA sequence which is used is SEQ ID NO: 7). Manufactures of this
type are analogous to the GeneChip.TM. devices manufactured by
Affymetrix, Inc. (Santa Clara, Calif.), which comprise pluralities
of primers which bind with high stringency to, for example,
portions of the human p53 gene or to portions of the HIV-1 protease
or reverse transcriptase genes. Methods for making and using such
manufactures have been described elsewhere, and need only be
modified by the skilled artisan to include the MCT-1 gene sequences
described in the present disclosure (Wallraff et al., February
1997, Chemtech 22-23; Lockhart et al., 1996, Nature Biotechnol.
14:1675-1680; Pease et al., 1994, Proc. Natl. Acad. Sci. USA
91:5022-5026; Fodor et al., 1993, Nature 364:555-556).
The Isolated Polypeptide of the Invention
[0060] The invention further relates to an isolated polypeptide
which is homologous with at least a portion of MCT-1. The isolated
polypeptide of the invention is preferably homologous with at least
about ten, fifteen, twenty, or more amino acid residues of SEQ ID
NO: 8, which is listed in FIG. 5B. It is understood that SEQ ID
NOs: 2 and 8 are identical at amino acid residues 1 to 114.
Therefore, these two sequences may be used interchangeably, except
that use of SEQ ID NO: 8 is preferred when the identity of one or
more of amino acid residues 115 to 181 is critical. In one
embodiment, the isolated polypeptide of the invention comprises
amino acid residues 8 to 65 of SEQ ID NO: 8 (or SEQ ID NO: 2). This
portion is highly similar to a region of cyclin H protein which has
been implicated in protein-protein interactions. Thus, it is
recognized that this region of MCT-1 is likely to be at least a
significant portion of MCT-1 which interacts with the proteins by
means of which MCT-1 exerts its biological effect.
[0061] The isolated polypeptide of the invention may have a
sequence which comprises all or part of SEQ ID NO: 8. For example,
the isolated polypeptide of the invention may be MCT-1 protein,
preferably in a substantially purified form. In addition, the
sequence of the isolated polypeptide of the invention may further
comprise additional amino acid residues (i.e. it may be a fusion
protein) or comprise amino acid substitutions, particularly in the
random coil portions of MCT-1.
[0062] As described herein, MCT-1 has been purified by expressing a
GST-MCT-1 fusion protein in an Escherichia coli vector and
isolating the fusion protein from the vector using known methods.
Monoclonal or polyclonal antibodies which bind with specificity to
MCT-1 may be generated using known methods and are included in the
invention. Antibodies which bind with specificity to MCT-1 are
useful for detecting the presence of MCT-1 in a cell, and thus can
be used to determine whether a cell is a tumor cell. To determine
whether a cell is a tumor cell, an antibody which binds with
specificity to MCT-1 is used to detect the presence of MCT-1 in an
extract prepared using the cell, using any immunoblotting,
immunosorption, or immunoprecipitation technique. The presence of
MCT-1 in the cell is an indication that the cell is a tumor
cell.
[0063] The locations of regions of random coil in the amino acid
sequence of MCT-1 may be predicted using standard sequence analysis
algorithms (e.g. Gamier-Robson analysis). In random coil regions of
proteins, the amino acid sequence is relatively unimportant with
regard to the biological activity of the protein. Thus, individual
amino acid residues in regions of random coil in SEQ ID NO: 8 may
be substituted with substantially any amino acid residue. Other
amino acid residues in SEQ ID NO: 8 should only be substituted with
conservative amino acid residues. Conservative amino acid residue
substitutions are listed on rows of the following table.
TABLE-US-00001 TABLE glycine, alanine valine, isoleucine, leucine
aspartic acid, glutamic acid asparagine, glutamine serine,
threonine lysine, arginine phenylalanine, tyrosine
[0064] It will be appreciated, of course, that the peptides may
incorporate amino acid residues which are modified without
affecting biological activity. For example, the termini may be
derivatized to include blocking groups, i.e. chemical substituents
suitable to protect and/or stabilize the N- and C-termini from
"undesirable degradation", a term meant to encompass any type of
enzymatic, chemical or biochemical breakdown of the compound at its
termini which is likely to affect the function of the compound as
an anti-inflammatory agent, i.e. sequential degradation of the
compound at a terminal end thereof.
[0065] Blocking groups include protecting groups conventionally
used in the art of peptide chemistry which will not adversely
affect the in vivo activities of the peptide. For example, suitable
N-terminal blocking groups can be introduced by alkylation or
acylation of the N-terminus. Examples of suitable N-terminal
blocking groups include C.sub.1-C.sub.5 branched or non-branched
alkyl groups, acyl groups such as formyl and acetyl groups, as well
as substituted forms thereof, such as the acetamidomethyl (Acm)
group. Desamino analogs of amino acids are also useful N-terminal
blocking groups, and can either be coupled to the N-terminus of the
peptide or used in place of the N-terminal reside. Suitable
C-terminal blocking groups, in which the carboxyl group of the
C-terminus is either incorporated or not, include esters, ketones
or amides. Ester or ketone-forming alkyl groups, particularly lower
alkyl groups such as methyl, ethyl and propyl, and amide-forming
amino groups such as primary amines (--NH.sub.2), and mono- and
di-alkylamino groups such as methylamino, ethylamino,
dimethylamino, diethylamino, methylethylamino and the like are
examples of C-terminal blocking groups. Descarboxylated amino acid
analogues such as agmatine are also useful C-terminal blocking
groups and can be either coupled to the peptide's C-terminal
residue or used in place of it. Further, it will be appreciated
that the free amino and carboxyl groups at the termini can be
removed altogether from the peptide to yield desamino and
descarboxylated forms thereof without affect on peptide
activity.
[0066] Other modifications can also be incorporated without
adversely affecting anti-tumorigenic activity and these include,
but are not limited to, substitution of one or more of the amino
acids in the natural L-isomeric form with amino acids in the
D-isomeric form. Thus, the peptide may include one or more D-amino
acid resides, or may comprise amino acids which are all in the
D-form. Retro-inverso forms of peptides in accordance with the
present invention are also contemplated, for example, inverted
peptides in which all amino acids are substituted with D-amino acid
forms.
[0067] Acid addition salts of the present invention are also
contemplated as functional equivalents. Thus, an isolated
polypeptide of the invention may be treated with an inorganic acid
such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric,
and the like, or an organic acid such as an acetic, propionic,
glycolic, pyruvic, oxalic, malic, malonic, succinic, maleic,
fumaric, tartaric, citric, benzoic, cinnamic, mandelic,
methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicylic and
the like, to provide a water soluble salt of the polypeptide is
suitable for use in the methods described herein.
Pharmaceutical Compositions
[0068] The invention encompasses the preparation and use of
medicaments and pharmaceutical compositions comprising either or
both of an isolated nucleic acid of the invention and an isolated
polypeptide of the invention as an active ingredient. Such a
pharmaceutical composition may consist of the active ingredient(s)
alone, in a form suitable for administration to a subject, or the
pharmaceutical composition may comprise the active ingredient(s)
and one or more pharmaceutically acceptable carriers, one or more
additional ingredients, or some combination of these.
Administration of one of these pharmaceutical compositions to a
subject is useful for preventing or inhibiting tumorigenesis in the
subject or for treating a pre-existing tumor, as described
elsewhere in the present disclosure. The active ingredient(s) may
be present in the pharmaceutical composition in the form of a
physiologically acceptable ester or salt, such as in combination
with a physiologically acceptable cation or anion, as is well known
in the art.
[0069] The formulations of the pharmaceutical compositions
described herein may be prepared by any method known or hereafter
developed in the art of pharmacology. In general, such preparatory
methods include the step of bringing the active ingredient(s) into
association with a carrier or one or more other accessory
ingredients, and then, if necessary or desirable, shaping or
packaging the product into a desired single- or multi-dose
unit.
[0070] Although the descriptions of pharmaceutical compositions
provided herein are principally directed to pharmaceutical
compositions which are suitable for ethical administration to
humans, it will be understood by the skilled artisan that such
compositions are generally suitable for administration to animals
of all sorts. Modification of pharmaceutical compositions suitable
for administration to humans in order to render the compositions
suitable for administration to various animals is well understood,
and the ordinarily skilled veterinary pharmacologist can design and
perform such modification with merely ordinary, if any,
experimentation. Subjects to which administration of the
pharmaceutical compositions of the invention is contemplated
include, but are not limited to, humans.
[0071] Pharmaceutical compositions that are useful in the methods
of the invention may be prepared, packaged, or sold in formulations
suitable for oral, rectal, vaginal, parenteral, topical, pulmonary,
intranasal, buccal, ophthalmic, or another route of administration.
Other contemplated formulations include projected nanoparticles,
liposomal preparations, resealed erythrocytes containing the active
ingredient(s), and immunologically-based formulations.
[0072] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in bulk, as a single unit dose, or as a
plurality of single unit doses. As used herein, a "unit dose" is
discrete amount of the pharmaceutical composition comprising a
predetermined amount of the active ingredient(s). The amount of the
active ingredient(s) is generally equal to the dosage of the active
ingredient(s) which would be administered to a subject or a
convenient fraction of such a dosage such as, for example, one-half
or one-third of such a dosage.
[0073] The relative amounts of the active ingredient(s), the
pharmaceutically acceptable carrier, and any additional ingredients
in a pharmaceutical composition of the invention will vary,
depending upon the identity, size, and condition of the subject
treated and further depending upon the route by which the
composition is to be administered. By way of example, the
composition may comprise between 0.1% and 100% (w/w) active
ingredient(s).
[0074] Controlled- or sustained-release formulations of a
pharmaceutical composition of the invention may be made using
conventional technology.
[0075] A formulation of a pharmaceutical composition of the
invention suitable for oral administration may be prepared,
packaged, or sold in the form of a discrete solid dose unit
including, but not limited to, a tablet, a hard or soft capsule, a
cachet, a troche, or a lozenge, each containing a predetermined
amount of the active ingredient(s). Other formulations suitable for
oral administration include, but are not limited to, a powdered or
granular formulation, an aqueous or oily suspension, an aqueous or
oily solution, or an emulsion.
[0076] As used herein, an "oily" liquid is one which comprises a
carbon-containing liquid molecule and which exhibits a less polar
character than water.
[0077] A tablet comprising the active ingredient(s) may, for
example, be made by compressing or molding the active
ingredient(s), optionally with one or more additional ingredients.
Compressed tablets may be prepared by compressing, in a suitable
device, the active ingredient(s) in a free-flowing form such as a
powder or granular preparation, optionally mixed with one or more
of a binder, a lubricant, an excipient, a surface active agent, and
a dispersing agent. Molded tablets may be made by molding, in a
suitable device, a mixture of the active ingredient(s), a
pharmaceutically acceptable carrier, and at least sufficient liquid
to moisten the mixture. Pharmaceutically acceptable excipients used
in the manufacture of tablets include, but are not limited to,
inert diluents, granulating and disintegrating agents, binding
agents, and lubricating agents. Known dispersing agents include,
but are not limited to, potato starch and sodium starch glycolate.
Known surface active agents include, but are not limited to, sodium
lauryl sulphate. Known diluents include, but are not limited to,
calcium carbonate, sodium carbonate, lactose, microcrystalline
cellulose, calcium phosphate, calcium hydrogen phosphate, and
sodium phosphate. Known granulating and disintegrating agents
include, but are not limited to, corn starch and alginic acid.
Known binding agents include, but are not limited to, gelatin,
acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and
hydroxypropyl methylcellulose. Known lubricating agents include,
but are not limited to, magnesium stearate, stearic acid, silica,
and talc.
[0078] Tablets may be non-coated or they may be coated using known
methods to achieve delayed disintegration in the gastrointestinal
tract of a subject, thereby providing sustained release and
absorption of the active ingredient(s). By way of example, a
material such as glyceryl monostearate or glyceryl distearate may
be used to coat tablets. Further by way of example, tablets may be
coated using methods described in U.S. Pat. Nos. 4,256,108;
4,160,452; and 4,265,874 to form osmotically-controlled release
tablets. Tablets may further comprise a sweetening agent, a
flavoring agent, a coloring agent, a preservative, or some
combination of these in order to provide pharmaceutically elegant
and palatable preparation.
[0079] Hard capsules comprising the active ingredient(s) may be
made using a physiologically degradable composition, such as
gelatin. Such hard capsules comprise the active ingredient(s), and
may further comprise additional ingredients including, for example,
an inert solid diluent such as calcium carbonate, calcium
phosphate, or kaolin.
[0080] Soft gelatin capsules comprising the active ingredient(s)
may be made using a physiologically degradable composition, such as
gelatin. Such soft capsules comprise the active ingredient(s),
which may be mixed with water or an oil medium such as peanut oil,
liquid paraffin, or olive oil.
[0081] Oral compositions may be made, using known technology, which
specifically release orally-administered agents in the small or
large intestines of a human patient. For example, formulations for
delivery to the gastrointestinal system, including the colon,
include enteric coated systems, based, e.g., on methacrylate
copolymers such as poly(methacrylic acid, methyl methacrylate),
which are only soluble at pH 6 and above, so that the polymer only
begins to dissolve on entry into the small intestine. The site
where such polymer formulations disintegrate is dependent on the
rate of intestinal transit and the amount of polymer present. For
example, a relatively thick polymer coating is used for delivery to
the proximal colon (Hardy et al., 1987 Aliment. Pharmacol. Therap.
1:273-280). Polymers capable of providing site-specific colonic
delivery can also be used, wherein the polymer relies on the
bacterial flora of the large bowel to provide enzymatic degradation
of the polymer coat and hence release of the drug. For example,
azopolymers (U.S. Pat. No. 4,663,308), glycosides (Friend et al.,
1984, J. Med. Chem. 27:261-268) and a variety of naturally
available and modified polysaccharides (PCT GB 89/00581) may be
used in such formulations.
[0082] Pulsed release technology such as that described in U.S.
Pat. No. 4,777,049 may also be used to administer the active agent
to a specific location within the gastrointestinal tract. Such
systems permit drug delivery at a predetermined time and can be
used to deliver the active agent, optionally together with other
additives that my alter the local microenvironment to promote agent
stability and uptake, directly to the colon, without relying on
external conditions other than the presence of water to provide in
vivo release.
[0083] Liquid formulations of a pharmaceutical composition of the
invention which are suitable for oral administration may be
prepared, packaged, and sold either in liquid form or in the form
of a dry product intended for reconstitution with water or another
suitable vehicle prior to use.
[0084] Liquid suspensions may be prepared using conventional
methods to achieve suspension of the active ingredient(s) in an
aqueous or oily vehicle. Aqueous vehicles include, for example,
water and isotonic saline. Oily vehicles include, for example,
almond oil, oily esters, ethyl alcohol, vegetable oils such as
arachis, olive, sesame, or coconut oil, fractionated vegetable
oils, and mineral oils such as liquid paraffin. Liquid suspensions
may further comprise one or more additional ingredients including,
but not limited to, suspending agents, dispersing or wetting
agents, emulsifying agents, demulcents, preservatives, buffers,
salts, flavorings, coloring agents, and sweetening agents. Oily
suspensions may further comprise a thickening agent. Known
suspending agents include, but are not limited to, sorbitol syrup,
hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone,
gum tragacanth, gum acacia, and cellulose derivatives such as
sodium carboxymethylcellulose, methylcellulose,
hydroxypropylmethylcellulose. Known dispersing or wetting agents
include, but are not limited to, naturally-occurring phosphatides
such as lecithin, condensation products of an alkylene oxide with a
fatty acid, with a long chain aliphatic alcohol, with a partial
ester derived from a fatty acid and a hexitol, or with a partial
ester derived from a fatty acid and a hexitol anhydride (e.g.
polyoxyethylene stearate, heptadecaethyleneoxycetanol,
polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan
monooleate, respectively). Known emulsifying agents include, but
are not limited to, lecithin and acacia. Known preservatives
include, but are not limited to, methyl, ethyl, or
n-propyl-para-hydroxybenzoates, ascorbic acid, and sorbic acid.
Known sweetening agents include, for example, glycerol, propylene
glycol, sorbitol, sucrose, and saccharin. Known thickening agents
for oily suspensions include, for example, beeswax, hard paraffin,
and cetyl alcohol.
[0085] Liquid solutions of the active ingredient(s) in aqueous or
oily solvents may be prepared in substantially the same manner as
liquid suspensions, the primary difference being that the active
ingredient(s) is dissolved, rather than suspended in the solvent.
Liquid solutions of the pharmaceutical composition of the invention
may comprise each of the components described with regard to liquid
suspensions, it being understood that suspending agents will not
necessarily aid dissolution of the active ingredient(s) in the
solvent. Aqueous solvents include, for example, water and isotonic
saline. Oily solvents include, for example, almond oil, oily
esters, ethyl alcohol, vegetable oils such as arachis, olive,
sesame, or coconut oil, fractionated vegetable oils, and mineral
oils such as liquid paraffin.
[0086] Powdered and granular formulations of a pharmaceutical
preparation of the invention may be prepared using known methods.
Such formulations may be administered directly to a subject, used,
for example, to form tablets, to fill capsules, or to prepare an
aqueous or oily suspension or solution by addition of an aqueous or
oily vehicle thereto. Each of these formulations may further
comprise one or more of dispersing or wetting agent, a suspending
agent, and a preservative. Additional excipients, such as fillers
and sweetening, flavoring, or coloring agents, may also be included
in these formulations.
[0087] A pharmaceutical composition of the invention may also be
prepared, packaged, or sold in the form of oil-in-water emulsion or
a water-in-oil emulsion. The oily phase may be a vegetable oil such
as olive or arachis oil, a mineral oil such as liquid paraffin, or
a combination of these. Such compositions may further comprise one
or more emulsifying agents such as naturally occurring gums such as
gum acacia or gum tragacanth, naturally-occurring phosphatides such
as soybean or lecithin phosphatide, esters or partial esters
derived from combinations of fatty acids and hexitol anhydrides
such as sorbitan monooleate, and condensation products of such
partial esters with ethylene oxide such as polyoxyethylene sorbitan
monooleate. These emulsions may also contain additional ingredients
including, for example, sweetening or flavoring agents.
[0088] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for rectal
administration. Such a composition may be in the form of, for
example, a suppository, a retention enema preparation, and a
solution for rectal or colonic irrigation.
[0089] Suppository formulations may be made by combining the active
ingredient(s) with a non-irritating pharmaceutically acceptable
excipient which is solid at ordinary room temperature (i.e. about
20.degree. C.) and which is liquid at the rectal temperature of the
subject (i.e. about 37.degree. C. in a healthy human). Suitable
pharmaceutically acceptable excipients include, but are not limited
to, cocoa butter, polyethylene glycols, and various glycerides.
Suppository formulations may further comprise various additional
ingredients including, but not limited to, antioxidants and
preservatives.
[0090] Retention enema preparations or solutions for rectal or
colonic irrigation may be made by combining the active
ingredient(s) with a pharmaceutically acceptable liquid carrier. As
is well known in the art, enema preparations may be administered
using, and may be packaged within, a delivery device adapted to the
rectal anatomy of the subject. Enema preparations may further
comprise various additional ingredients including, but not limited
to, antioxidants and preservatives.
[0091] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for vaginal
administration. Such a composition may be in the form of, for
example, a suppository, an impregnated or coated
vaginally-insertable material such as a tampon, a douche
preparation, or a solution for vaginal irrigation.
[0092] Methods for impregnating or coating a material with a
chemical composition are known in the art, and include, but are not
limited to methods of depositing or binding a chemical composition
onto a surface, methods of incorporating a chemical composition
into the structure of a material during the synthesis of the
material (i.e. such as with a physiologically degradable material),
and methods of absorbing an aqueous or oily solution or suspension
into an absorbent material, with or without subsequent drying.
[0093] Douche preparations or solutions for vaginal irrigation may
be made by combining the active ingredient(s) with a
pharmaceutically acceptable liquid carrier. As is well known in the
art, douche preparations may be administered using, and may be
packaged within, a delivery device adapted to the vaginal anatomy
of the subject. Douche preparations may further comprise various
additional ingredients including, but not limited to, antioxidants,
antibiotics, antifungal agents, and preservatives.
[0094] As used herein, "parenteral administration" of a
pharmaceutical composition includes any route of administration
characterized by physical breaching of a tissue of a subject and
administration of the pharmaceutical composition through the breach
in the tissue. Parenteral administration thus includes, but is not
limited to, administration of a pharmaceutical composition by
injection of the composition, by application of the composition
through a surgical incision, by application of the composition
through a tissue-penetrating non-surgical wound, and the like. In
particular, parenteral administration is contemplated to include,
but is not limited to, subcutaneous, intraperitoneal, intravenous,
intraarterial, intramuscular, or intrasternal injection and
intravenous, intraarterial, or kidney dialytic infusion
techniques.
[0095] Formulations of a pharmaceutical composition suitable for
parenteral administration comprise the active ingredient(s)
combined with a pharmaceutically acceptable carrier, such as
sterile water or sterile isotonic saline. Such formulations may be
prepared, packaged, or sold in a form suitable for bolus
administration or for continuous administration. Injectable
formulations may be prepared, packaged, or sold in unit dosage
form, such as in ampules, in multi-dose containers containing a
preservative, or in single-use devices for auto-injection or
injection by a medical practitioner. Formulations for parenteral
administration include, but are not limited to, suspensions,
solutions, emulsions in oily or aqueous vehicles, pastes, and
implantable sustained-release or biodegradable formulations. Such
formulations may further comprise one or more additional
ingredients including, but not limited to, suspending, stabilizing,
or dispersing agents. In one embodiment of a formulation for
parenteral administration, the active ingredient(s) is provided in
dry (i.e. powder or granular) form for reconstitution with a
suitable vehicle (e.g. sterile pyrogen-free water) prior to
parenteral administration of the reconstituted composition.
[0096] The pharmaceutical compositions may be prepared, packaged,
or sold in the form of a sterile injectable aqueous or oily
suspension or solution. This suspension or solution may be
formulated according to the known art, and may comprise, in
addition to the active ingredient(s), additional ingredients such
as the dispersing agents, wetting agents, or suspending agents
described herein. Such sterile injectable formulations may be
prepared using a non-toxic parenterally-acceptable diluent or
solvent, such as water or 1,3-butane diol, for example. Other
acceptable diluents and solvents include, but are not limited to,
Ringer's solution, isotonic sodium chloride solution, and fixed
oils such as synthetic mono- or di-glycerides. Other
parentally-administrable formulations which are useful include
those which comprise the active ingredient(s) in microcrystalline
form, in a liposomal preparation, or as a component of a
biodegradable polymer systems. Compositions for sustained release
or implantation may comprise pharmaceutically acceptable polymeric
or hydrophobic materials such as an emulsion, an ion exchange
resin, a sparingly soluble polymer, or a sparingly soluble
salt.
[0097] Formulations suitable for topical administration include,
but are not limited to, liquid or semi-liquid preparations such as
liniments, lotions, oil-in-water or water-in-oil emulsions such as
creams, ointments or pastes, and solutions or suspensions.
Topically-administrable formulations may, for example, comprise
from about 1% to about 10% (w/w) active ingredient(s), although the
concentration of the active ingredient(s) may be as high as the
solubility limit of the active ingredient(s) in the solvent.
Formulations for topical administration may further comprise one or
more of the additional ingredients described herein.
[0098] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for pulmonary
administration via the buccal cavity. Such a formulation may
comprise dry particles which comprise the active ingredient(s) and
which have a diameter in the range from about 0.5 to about 7
nanometers, and preferably from about 1 to about 6 nanometers. Such
compositions are conveniently in the form of dry powders for
administration using a device comprising a dry powder reservoir to
which a stream of propellant may be directed to disperse the powder
or using a self-propelling solvent/powder-dispensing container such
as a device comprising the active ingredient(s) dissolved or
suspended in a low-boiling propellant in a sealed container.
Preferably, such powders comprise particles wherein at least 98% of
the particles by weight have a diameter greater than 0.5 nanometers
and at least 95% of the particles by number have a diameter less
than 7 nanometers. More preferably, at least 95% of the particles
by weight have a diameter greater than 1 nanometer and at least 90%
of the particles by number have a diameter less than 6 nanometers.
Dry powder compositions preferably include a solid fine powder
diluent such as sugar and are conveniently provided in a unit dose
form.
[0099] Low boiling propellants generally include liquid propellants
having a boiling point of below 65.degree. F. at atmospheric
pressure. Generally the propellant may constitute 50 to 99.9% (w/w)
of the composition, and the active ingredient(s) may constitute 0.1
to 20% (w/w) of the composition. The propellant may further
comprise additional ingredients such as a liquid non-ionic or solid
anionic surfactant or a solid diluent (preferably having a particle
size of the same order as particles comprising the active
ingredient(s)).
[0100] Pharmaceutical compositions of the invention formulated for
pulmonary delivery may also provide the active ingredient(s) in the
form of droplets of a solution or suspension. Such formulations may
be prepared, packaged, or sold as aqueous or dilute alcoholic
solutions or suspensions, optionally sterile, comprising the active
ingredient(s), and may conveniently be administered using any
nebulization or atomization device. Such formulations may further
comprise one or more additional ingredients including, but not
limited to, a flavoring agent such as saccharin sodium, a volatile
oil, a buffering agent, a surface active agent, or a preservative
such as methylhydroxybenzoate. The droplets provided by this route
of administration preferably have an average diameter in the range
from about 0.1 to about 200 nanometers.
[0101] The formulations described herein as being useful for
pulmonary delivery are also useful for intranasal delivery of a
pharmaceutical composition of the invention.
[0102] Another formulation suitable for intranasal administration
is a coarse powder comprising the active ingredient(s) and having
an average particle from about 0.2 to 500 micrometers. Such a
formulation is administered in the manner in which snuff is taken
i.e. by rapid inhalation through the nasal passage from a container
of the powder held close to the nares.
[0103] Formulations suitable for nasal administration may, for
example, comprise from about as little as 0.1% (w/w) and as much as
100% (w/w) of the active ingredient(s), and may further comprise
one or more of the additional ingredients described herein.
[0104] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for buccal
administration. Such formulations may, for example, be in the form
of tablets or lozenges made using conventional methods, and may,
for example, 0.1 to 20% (w/w) active ingredient(s), the balance
comprising an orally dissolvable or degradable composition and,
optionally, one or more of the additional ingredients described
herein. Alternately, formulations suitable for buccal
administration may comprise a powder or an aerosolized or atomized
solution or suspension comprising the active ingredient(s). Such
powdered, aerosolized, or aerosolized formulations, when dispersed,
preferably have an average particle or droplet size in the range
from about 0.1 to about 200 nanometers, and may further comprise
one or more of the additional ingredients described herein.
[0105] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for
ophthalmic administration. Such formulations may, for example, be
in the form of eye drops including, for example, a 0.1-1.0% (w/w)
solution or suspension of the active ingredient(s) in an aqueous or
oily liquid carrier. Such drops may further comprise buffering
agents, salts, or one or more other of the additional ingredients
described herein. Other ophthalmalmically-administrable
formulations which are useful include those which comprise the
active ingredient(s) in microcrystalline form or in a liposomal
preparation.
[0106] As used herein, "additional ingredients" include, but are
not limited to, one or more of the following: excipients; surface
active agents; dispersing agents; inert diluents; granulating and
disintegrating agents; binding agents; lubricating agents;
sweetening agents; flavoring agents; coloring agents;
preservatives; physiologically degradable compositions such as
gelatin; aqueous vehicles and solvents; oily vehicles and solvents;
suspending agents; dispersing or wetting agents; emulsifying
agents, demulcents; buffers; salts; thickening agents; fillers;
emulsifying agents; antioxidants; antibiotics; antifungal agents;
stabilizing agents; and pharmaceutically acceptable polymeric or
hydrophobic materials. Other "additional ingredients" which may be
included in the pharmaceutical compositions of the invention are
known in the art and described, for example in Genaro, ed., 1985,
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa., which is incorporated herein by reference.
[0107] A pharmaceutical composition of the invention may be
administered to deliver a dose of between 1 ng/kg/day and 100
mg/kg/day to a subject.
[0108] It is understood that the ordinarily skilled physician or
veterinarian will readily determine and prescribe an effective
amount of the compound to inhibit or treat a tumor in the subject.
In so proceeding, the physician or veterinarian may, for example,
prescribe a relatively low dose at first, subsequently increasing
the dose until an appropriate response is obtained. It is further
understood, however, that the specific dose level for any
particular subject will depend upon a variety of factors including
the activity of the specific compound employed, the age, body
weight, general health, gender, and diet of the subject, the time
of administration, the route of administration, the rate of
excretion, any drug combination, and the stage of progression of
the tumor being treated or inhibited.
[0109] Another aspect of the invention relates to a kit comprising
a pharmaceutical composition of the invention and an instructional
material. As used herein, an "instructional material" includes a
publication, a recording, a diagram, or any other medium of
expression which is used to communicate the usefulness of the
pharmaceutical composition of the invention for inhibiting or
treating a tumor in a subject. The instructional material may also,
for example, describe an appropriate dose of the pharmaceutical
composition of the invention. The instructional material of the kit
of the invention may, for example, be affixed to a container which
contains a pharmaceutical composition of the invention or be
shipped together with a container which contains the pharmaceutical
composition. Alternatively, the instructional material may be
shipped separately from the container with the intention that the
instructional material and the pharmaceutical composition be used
cooperatively by the recipient.
[0110] The invention also includes a kit comprising a
pharmaceutical composition of the invention and a delivery device
for delivering the composition to a subject. By way of example, the
delivery device may be a squeezable spray bottle, a metered-dose
spray bottle, an aerosol spray device, an atomizer, a dry powder
delivery device, a self-propelling solvent/powder-dispensing
device, a syringe, a needle, a tampon, or a dosage measuring
container. The kit may further comprise an instructional material
as described herein.
The Methods of the Invention
[0111] The invention also relates to a method of reducing MCT-1
expression in a cell. The method comprises providing an isolated
nucleic acid of the invention to the cell. Because the isolated
nucleic acid is complementary to or homologous with at least about
twenty consecutive nucleotides of the mRNA-coding region of an
MCT-1 gene to the cell, the nucleic acid anneals with DNA or mRNA
in the cell and reduces expression of MCT-1 in the cell. In one
embodiment, the isolated nucleic acid is complementary to at least
about twenty, and preferably at least about twenty-five, thirty,
fifty, or more nucleotide residues of SEQ ID NO: 7, so that the
isolated nucleic acid binds with high specificity to mRNA generated
by transcription of the genome of the cell and thereby prevents
translation of the mRNA, blocking MCT-1 synthesis. The isolated
nucleic acid may, for example be complementary to a portion of the
mRNA-coding region near or including the translational start site
(i.e. nucleotide residues 258-260 of SEQ ID NO: 7). By way of
example, the isolated nucleic acid may have a sequence which is
75%, 90%, 95%, or completely complementary to nucleotide residues
240-270 of SEQ ID NO: 7.
[0112] The invention also relates to a method of increasing MCT-1
production in a cell. This method comprises providing to the cell
an isolated nucleic acid comprising a promoter operably linked with
a portion of the mRNA-coding region of a human MCT-1 gene. The
region may, for example, include the entire coding sequence of
MCT-1 (i.e. nucleotide residues 258-800 of SEQ ID NO: 7), or it may
include additional (i.e. a fusion protein) or fewer amino acid
residues (i.e. a fragment of MCT-1 such as one including residues
8-65). Of course, the portion of the mRNA-coding region may have
amino acid substitutions, as described elsewhere herein. Production
of MCT-1 in the cell is increased by expression of the coding
sequence of MCT-1 of the isolated nucleic acid. The promoter which
is operably linked may be an inducible, suppressible,
tissue-specific, or constitutive promoter, each of which is well
known in the art.
[0113] The invention further relates to a method of determining
whether a compound is a modulator of MCT-1 expression. This method
of the invention comprises providing a first cell which
overexpresses MCT-1, culturing the first cell in the presence of
the compound, and comparing MCT-1 expression in the first cell with
MCT-1 expression in a second cell of the same type cultured in the
absence of the compound. A difference between MCT-1 expression in
the first cell and MCT-1 expression in the second cell is an
indication that the compound is a modulator of MCT-1 expression.
The first cell and the second cell may each be, for example, a
leukocyte which has been obtained from a patient afflicted with
CTCL and which overexpresses MCT-1.
[0114] Expression of MCT-1 in a first cell and in a second cell may
be detected and compared using known methods, such as those
described herein in the Example. It is understood that this method
may also be practiced using a first cell which does not normally
express MCT-1 or one which normally expresses MCT-1 at a relatively
constant level. When a second cell of the same type is cultured in
the presence of a test compound, comparing expression of MCT-1 in
the first and second cells will indicate whether the test compound
modulates expression of MCT-1. For example, if the first and second
cells do not normally express MCT-1, but the second cell expresses
MCT-1 in the presence of a test compound, then this is an
indication that the test compound is an inducer of MCT-1
expression. The test compound may therefore be considered a
potential carcinogen.
[0115] The invention also includes a method of determining whether
a gene product is a modulator of MCT-1 expression. This method
comprises providing a first cell which overexpresses MCT-1,
expressing an isolated nucleic acid encoding the gene product in
the first cell, and comparing MCT-1 expression in the first cell
with MCT-1 expression in a second cell of the same type, wherein
the isolated nucleic acid is not expressed in the second cell. A
difference between MCT-1 expression in the first cell and MCT-1
expression in the second cell is an indication that the gene
product is a modulator of MCT-1 expression. It is understood that
this method may also be practiced using a first cell which does not
normally express MCT-1 or one which normally expresses MCT-1 at a
relatively constant level.
[0116] The invention further includes a method of determining
whether a cell is a tumor cell. This method of the invention
comprises comparing MCT-1 expression in the cell and MCT-1
expression in a non-tumor cell, preferably of the same type (e.g.
by comparing MCT-1 expression in a T-cell suspected of being
cancerous and in a T-cell not suspected of being cancerous). A
difference between MCT-1 expression in the cell and MCT-1
expression in the non-tumor cell is an indication that the cell is
a tumor cell. The method can be used, for example, to determine
whether the cell is a cutaneous T-cell lymphoma cell. The non-tumor
cell may, for example, be a normal human lymphocyte.
[0117] The invention includes another method of determining whether
a cell is a tumor cell. This alternate method comprises comparing
MCT-1 copy number in the cell and MCT-1 copy number in a non-tumor
cell. A difference between MCT-1 copy number in the cell and MCT-1
copy number in the non-tumor cell is an indication that the cell is
a tumor cell. The method can be used, for example, to determine
whether the cell is a cutaneous T-cell lymphoma cell. The non-tumor
cell may, for example, be a normal human lymphocyte. The copy
number of MCT-1 in a first cell and in a second cell may be
detected and compared using known methods, such as those described
herein in the Example.
[0118] The invention also relates to a method of conferring a
growth advantage on a cell. This method comprises providing the
cell with an isolated nucleic acid comprising a promoter operably
linked to a portion of the mRNA-coding region of an MCT-1 gene, the
region including the coding sequence of MCT-1 (i.e. nucleotide
residues 258-623 of SEQ ID NO: 1). By providing this isolated
nucleic acid to the cell, MCT-1 expression is enhanced, as
described in the Example, and a growth advantage is conferred upon
the cells, relative to cells not provided the isolated nucleic
acid.
[0119] The invention is now described with reference to the
following Examples. These Examples are provided for the purpose of
illustration only and the invention should in no way be construed
as being limited to these Examples, but rather should be construed
to encompass any and all variations which become evident as a
result of the teaching provided herein.
EXAMPLE 1
Molecular Cloning and Characterization of a Novel Gene, MCT-1,
which is Amplified in a Cutaneous T-Cell Lymphoma Cell Line
[0120] The experiments presented in this Example demonstrate that
overexpression of MCT-1 protein in NIH 3T3 fibroblasts shortens the
G1 phase of the cell cycle and promotes anchorage independent
growth.
[0121] Genetic abnormalities of malignant T-cells associated with
cutaneous T-cell leukemia (CTCL) were identified using arbitrarily
primed-polymerase chain reaction (AP-PCR) assay. AP-PCR has been
used by others to detect sub-microscopic genetic alterations which
are associated with tumorigenesis, as described (Welsh et al.,
1990, Nucl. Acids Res. 18:7213-7218; Peinado et al., 1992, Proc.
Natl. Acad. Sci. USA 89:10065-10069; Kohno et al., 1993, Oncogene
7:103-108).
[0122] A panel of T-cell leukemia and lymphoma cell lines was
screened for amplified genomic sequences. An amplified genomic
sequence was identified in a cell line designated HUT 78. This
genomic sequence was cloned and sequenced, and the coding sequence
encoded by the genomic sequence was determined to have no
significant homology to any known gene sequence in the BLAST genome
data base, as determined using the Basic Local Alignment Search
Tool. It was therefore concluded that the amplified genomic
sequence comprised at least part of a novel gene.
[0123] The novel gene was designated MCT-1 (multiple copies in
T-cell malignancies). The amplified genomic sequence encoded a
polypeptide of 181 amino acids (SEQ ID NO: 8), designated MCT-1.
The MotifFinder computer program was used to identify three
putative post-translational modification sites in MCT-1, as
indicated in FIG. 3. PCR primers were designed using the MacVector
program, and these primers were used to generate a 457 nucleotide
residue amplification product when genomic DNA was subjected to
PCR. The primers which were used were designated HuT 78F, which had
the nucleotide sequence 5'-CATTGGAGAC CGCTACACAC AGGAC-3' (SEQ ID
NO: 3), and HuT 78R, which had the nucleotide sequence
5'-CTGTCAAAAT AGTCCGATGC CACG-3' (SEQ ID NO: 4). The same primers
were also useful for amplifying cDNA in an RT-PCR assay, confirming
that the amplified band obtained during the AP-PCR assay was an
exon.
[0124] Northern blot analysis revealed ubiquitous low-level
expression of a 943 nucleotide residue MCT-1 message in normal
human tissues. The full length cDNA sequence corresponding to the
MCT-1 message (hereinafter, "the MCT-1 cDNA") was determined using
a rapid amplification of cDNA ends (RACE) method, as described
(Frohman, 1993, Meth. Enzymol. 218:340-356). The nucleotide
sequence (SEQ ID NO: 7) of the MCT-1 cDNA is listed in FIG. 5A, and
the amino acid sequence (SEQ ID NO: 8) of the 181-amino acid
residue polypeptide encoded thereby is listed in FIG. 5B.
[0125] Due to a sequencing error, the nucleotide sequence was
initially believed to comprise an additional cytosine residue
located between the cytosine residue at position 598 and the
adenine residue a position 599, as indicated in the sequence listed
in FIG. 1A (SEQ ID NO: 1). The putative amino acid sequence (SEQ ID
NO: 2) encoded by the erroneous sequence is listed in FIG. 1A, and
differs from the amino acid sequence of MCT-1 at amino acid
residues 115 to 181.
[0126] The dbEST database was compared with the sequence of the
MCT-1 cDNA, and several overlapping ESTs having homology therewith
were identified. These overlapping regions are indicated in FIG. 2.
MCT-1 was localized to the long arm of chromosome Xq22-24 using
bacterial artificial chromosome (BAC) clones which contained either
the 5'-region, the 3'-region, or both regions of the MCT-1
cDNA.
[0127] Although there was no significant homology between MCT-1 and
any known protein at the primary sequence level, there was one
interesting alignment at the structural protein level, as assessed
using the NRL-3D Database (Amino Acid Sequence Extraction,
Brookhaven Structural Database). A potentially important region
(SEQ ID NO: 9) of MCT-1 is the amino terminal half of the protein,
which has a sequence identity of 32% with a 58-amino-acid-residue
domain (SEQ ID NO: 10) of cyclin H, as indicated in FIG. 4. It is
understood that proteins of the immunoglobulin superfamily have
only, on average, about 23% sequence identity. It is known from
crystallographic studies that the basic three dimensional folding
patterns among this superfamily of proteins is extremely well
conserved.
[0128] The region of homology between MCT-1 and cyclin H covers a
region of cyclin H that spans a surface domain of the protein that
is putatively involved in protein-protein interactions (Andersen et
al., 1997, EMBO J. 16:958-967). The non-homologous regions of MCT-1
and cyclin H correspond to regions of random coil within the cyclin
H molecule. Therefore, MCT-1 may be predicted to exhibit high
structural homology with cyclin H. Thus, the homologous region of
MCT-1 may also be involved in protein-protein interactions, and
MCT-1 was hypothesized to have a role in cell cycle regulation.
[0129] In order to test this hypothesis, MCT-1 was overexpressed in
NIH 3T3 fibroblasts, and the effect on growth of those cells was
observed. Following transfection of the fibroblasts, an
approximately two-fold decrease in the duration of the G1/S phase
of the cell cycle was observed, relative to non-transfected
fibroblasts. Over expression of MCT-1 increases the proliferative
rate of cells by decreasing the length of G1 phase without causing
a reciprocal increase in the durations of the S or G2-M phases.
[0130] The transforming ability of MCT-1 was assessed by soft agar
growth assays. Soft agar culture assays were performed essentially
as described (Hsiao et al., 1989, Mol. Cell Biol. 9:2641-2647).
Using limiting dilution, clonal cell lines of pcDNA3 and
pCMV-MCT-1-transfected cells were established. Briefly, NIH 3T3
fibroblast monolayers transfected with pcDNA3 or pCMV-MCT-1 were
seeded into 0.3% (w/v) Bacto-Agar suspension supplemented with DMEM
and 20% (v/v) fetal calf serum. This suspension was overlaid above
a layer of 0.5% (w/v) agar in the same medium on a 90 millimeter
diameter plate. Samples were made in triplicate and re-fed every
four days. Colonies were scored both by naked eye and by
microscopy. Neither the parent cell line nor cells transfected with
pCMV proliferated. Growth of pCMV-MCT-1-transfected cells was
observed after four weeks. Thus, it was demonstrated that only
MCT-1-overexpressing cells remained viable and continued to
proliferate
[0131] Some cell cycle regulatory proteins are directly involved in
oncogenesis, and cyclins have specifically been implicated in
tumorigenesis. Amplification of cyclin E has been demonstrated in
both breast and colon cancer cell lines (Keyomarsi et al., 1993,
Proc. Natl. Acad. Sci. USA 90:1112-1116; Leach et al., 1993, Cancer
Res. 53:1986-1989). The strongest evidence to date for
participation of cyclins in oncogenesis it that cyclin D1
amplification and overexpression occurs in primary human breast
tumors (Buckley et al., 1993, Oncogene 8:2127-2133) and that cyclin
D1 overexpression leads to transformation in vitro (Jiang et al.,
1993, Oncogene 8:3447-3457). Furthermore, a D-type cyclin, CCND2,
has been implicated in cell cycle progression, and has recently
been demonstrated to be amplified in non-Hodgkin's lymphoma (Sherr,
1993, Cell 73:1059-1065; Hoglund et al., 1996, Blood
87:324-330).
[0132] The HUT 78 cell line in which MCT-1 amplification was
observed was derived from peripheral blood cells obtained from a
patient afflicted with Sezary syndrome. Because MCT-1 was localized
to chromosomal bands Xq22-24, primary samples from patients
afflicted with either CTCL (n=40) or chronic lymphocytic leukemia
(n=20). Amplification of MCT-1 was not detected in these samples.
Nonetheless, it appears that MCT-1 overexpression contributes to
deregulated cell cycle progression and proliferation in vitro.
Further support for the hypothesis that MCT-1 is an oncogene is
provided by the observation that the gene supports soft agar growth
in fibroblasts which over express it and by the observed structural
homology between MCT-1 and cyclin H.
[0133] The materials and methods used in the experiments presented
in this Example are now described.
[0134] Chromosomal localization of MCT-1 was ascertained by
fluorescent in situ hybridization (FISH) analysis. Briefly, a BAC
library was screened using PCR primers homologous with or
complementary to the 5'- and 3'-ends of the cDNA listed herein in
FIG. 1A (SEQ ID NO: 1). Two BAC clones (BAC 5839 and BAC 5841)
hybridized with both the 5'- and the 3'-primer. Each BAC probe was
labeled with dioxigenin dUTP by nick translation. The labeled probe
was hybridized to normal metaphase chromosomes from
phytohemagglutinin-stimulated peripheral blood lymphocytes.
Specific hybridization signals were observed on the long arm of the
X chromosome (Xq22-24) using BAC 5839.
[0135] Stable cell lines which overexpressed human MCT-1 were
established by transfecting NIH 3T3 fibroblasts either with vector
pcDNA3 (comprising promoter pCMV) alone or with vector comprising
the full length MCT-1 cDNA (pCMV-MCT-1). Geneticin (G418)
resistance was conferred by pCMV-MCT-1. pCMV-MCT-1 was made using
specific restriction enzymes. To generate pCMV-MCT-1, the vector
comprised the T7 promoter operably linked to the coding sequence of
MCT-1 was generated using primers having nucleotide sequences (+)
5'-GCTGAGGATC CGGTTGCCTA AAAG-3' (SEQ ID NO: 5) and (-)
5'-TCTGGTGAAT TCATTCAGCA TAA-3' (SEQ ID NO: 6), digested with BamHI
and EcoRI, and ligated to pCMV. These constructs were verified by
DNA sequence analysis.
[0136] Transfected cells were grown in selection medium for 2
weeks. Selection medium comprised Dulbecco's modified Eagle's
medium (DMEM) complete plus 1 milligram per milliliter G418. In
this medium, 100% of mock transfected cells exhibited cell death.
Controls included cells transfected with vector (pcDNA3) alone and
non-transfected 3T3. Population doubling times were calculated by
counting cells every 72 hours for 12 days.
[0137] After serum depletion for 48 hours, cells were re-plated at
3.times.10.sup.5 cells per 10-centimeter diameter dish in DMEM plus
10% (v/v) fetal calf serum (FCS). Every two hours cells were
collected and analyzed for DNA content by flow cytometry.
Fluorescence data was collected using an Epics Coulter flow
cytometer, and the percentage of cells in each of the G1, S, and
G2-M phases of the cell cycle were determined by analysis with the
software program, MultiCycle (Phoenix).
AP-PCR
[0138] Genomic DNA was prepared from all T-cell lines and normal
PBL samples. All reactions were carried out in a 25 microliter
volume containing 10 millimolar Tris-HCl, 200 millimolar each dNTP,
50 millimolar KCl, 5 millimolar MgCl.sub.2, 25 picomoles of 10-mer
arbitrary primer, 0.1 milligram DNA template and 1 Unit Taq DNA
Polymerase (Fisher Biotech) at pH 8.3. A panel of 10-mer to 20-mer
primers were labeled using T4 polynucleotide kinase and
[.gamma.-.sup.32P]-ATP. All reactions were performed using a
GeneAMP PCR System 9600 (Perkin-Elmer). The profile was as follows.
The first 5 cycles of the temperature profile: [0139] denaturation
at 95.degree. C. for 30 seconds, then [0140] primer annealing at
25.degree. C. for 1 minute, and then [0141] extension for 1 minute
at 72.degree. C. The last 25 cycles: [0142] denaturation at
95.degree. C. for 30 seconds, then [0143] primer annealing at
30.degree. C. for 30 seconds, and then [0144] extension for 1
minute at 72.degree. C. PCR products were separated by
electrophoresis in denaturing 8 molar urea/polyacrylamide gels
followed by autoradiography.
Cloning and Sequencing of Genomic MCT-1 Sequences Amplified by
AP-PCR
[0145] The band that appeared to be amplified in the HUT 78 lane
relative to the corresponding bands from other cell lines and
normal lymphocytes was isolated for cloning and sequencing. This
band was excised from gels and incubated in 10 milliliters of
1.times.Assay Buffer A (Fisher Biotech catalog no. FB6000-10) at
90.degree. C. for 10 minutes. Five microliter of eluted DNA was
re-amplified using the same AP-PCR primer as before with MgCl.sub.2
concentration of 5 millimolar for 30 cycles at 30.degree. C. The
PCR product was analyzed by electrophoresis in a polyacrylamide gel
to confirm its size and purity. Amplified DNA was cloned into a
compatible thymidine pMOSBlue T-vector (Amersham, Arlington
Heights, Ill.). The presence of an appropriate insert was
determined using direct colony PCR using T7 and U19-mer pMOSBlue
specific primers. Sequencing was performed using an established
methodology. Sequences obtained from several clones was compared to
known sequences in the GenBank data base using the BLASTn and
BLASTx computer programs (Altschul et al., 1990, J. Mol. Biol.
215:403-410).
Isolation and Sequencing of MCT-1 cDNA
[0146] Full length MCT-1 cDNA sequence was obtained by the RACE
method as previously described using normal peripheral blood
lymphocyte (PBL) cDNA (Frohman, 1993, Meth. Enzymol.
218:340-356).
Southern Blot Analysis
[0147] 5 To 10 micrograms of genomic DNA of cells obtained from
humans afflicted with CTCL or CLL was digested with either HindIII
or EcoRI and electrophoresed in 1.0% (w/v) agarose gels. Transfer
to nitrocellulose membrane and subsequent hybridization was
performed using standard methods. An MCT-1 cDNA probe was random
primed with PRIME-IT kit (Stratagene, La Jolla, Calif.) and
purified using PrimeErase Quik columns (Stratagene) according to
the supplier's directions. The occurrence of gene amplification was
assessed by comparing the ratio of MCT-1 to .beta.-actin signals.
Quantification was carried out using a STORM phosphorimager 860
(Molecular Dynamics, Sunnyvale, Calif.).
EXAMPLE 2
Increased G1 Cyclin/cdk Activity in Cells Overexpressing MCT-1
[0148] The materials and methods used in the experiments presented
in this Example are now described.
NIH 3T3 Cell Culture and DNA Transfection
[0149] Stably transfected NIH 3T3 cell lines which constitutively
expressed MCT-1 (i.e. which comprised plasmid pCMV-MCT-1) and
stably transfected cell lines which comprised a control (pCMV)
vector were generated as described in Example 1. Individual clones
of transfected cells were obtained by limiting dilution.
[0150] Transient assays of the level of MCT-1 protein expression
were performed using the Lipofectamine method according to the
supplier's instructions (GIBCO, Grand Island, N.Y.). Briefly, an
expression vector (pcDNA-HA-MCT-1, which encodes a protein having
an HA tag fused at the amino terminus of MCT-1) was constructed by
cloning cDNA encoding MCT-1 into BamHI and EcoRI site of pcDNA3-HA.
Transfected cells were analyzed 48 hours later for production of
the fusion protein HA-MCT-1 using an anti-HA antibody described
herein.
Lymphocyte Cell Lines
[0151] PBL (peripheral blood lymphocytes) were prepared from whole
fresh blood of healthy donors. Mononuclear cells were isolated by
centrifugation in the presence of Ficoll (Organon Teknika
Corporation, Durham, N.C.), cultured for 48 hours in RPMI 1640
medium containing 20% (v/v) FCS, 100 Units per milliliter
penicillin, 100 micrograms per milliliter streptomycin, 2
millimolar L-glutamine, and 1% (w/v) phytohemagglutinin (PHA;
GIBCO, Grand Island, N.Y.). Non-adherent PBL were viably frozen for
further analysis.
[0152] Interleukin-2- (IL-2-)independent cell lines which were used
included cell lines C10MJ, MT-2, Hut 78, H-9, HUT 102, DA 202, and
C91PL (Advanced Biotechnologies Inc., Columbia, Md.). Cells of
these lines were cultured in RPMI 1640 medium containing 10% FCS,
100 Units per milliliter penicillin, 100 micrograms per milliliter
streptomycin and 2 millimolar glutamine (GIBCO, Grand Island,
N.Y.). IL-2-dependent cell lines N1185 and N1186 (described by
Berneman et al., 1992, Proc. Natl. Acad. Sci. USA 89:3005-3009)
were cultured using the same culture conditions as above with the
addition to the medium of 40 Units per milliliter of recombinant
IL-2 (GIBCO, Grand Island, N.Y.).
Immunoprecipitation and Immunoblotting
[0153] Cell pellets were lysed in lysate buffer, which comprised 10
millimolar Tris, 150 millimolar NaCl, 1 millimolar EDTA, 0.1% (w/v)
SDS, and 150 millimolar phenylmethylsulfonyl fluoride (PMSF) at a
pH of 7.4. Total protein concentration in each sample was
determined using a commercial bicinchoninic acid assay kit
(micro-BCA.TM. kit; Pierce, Rockford, Ill.) according to the
manufacturer's instructions. Equal amounts of whole cell lysate (50
to 100 micrograms) were resuspended in 5 milliliters of TB S
containing (final concentrations) 1 microgram per milliliter
leupeptin, 1 microgram per milliliter aprotinin, 0.01% (w/v) PMSF,
0.01% (w/v) n-tosyl-L-phenylalanine-chloromethyl ketone (TPCK),
0.01% (w/v) n-alpha-p-tosyl-L-lysine-chloromethyl ketone (TLCK),
0.1% (w/v) sodium azide and 1% (w/v) nonyl phenoxy polyethoxy
ethanol (NP-40; SIGMA, Saint Louis, Mo.). TBS was Tris-buffered
saline containing 0.1% (w/v) sodium dodecyl sulfate. Samples were
pre-cleared with Protein-G beads (GIBCO, Grand Island, N.Y.) and
either normal rabbit or mouse serum (each at a 1:1000 dilution).
Immunoprecipitation of cyclin D1 was carried out for 12 hours at
4.degree. C. Immune complexes were precipitated using 1 to 5
micrograms of antibody and Protein-G agarose, and were then heated
at 95.degree. C. for 5 minutes in IP buffer, which comprised 93
millimolar Tris, 3% (w/v) SDS, 1.1 millimolar
.beta.-mercaptoethanol, 0.03% (w/v) bromophenol blue (BPB), and 15%
(v/v) glycerol (SIGMA, Saint Louis, Mo.) at a pH of 6.8. The eluant
was analyzed using a denaturing, reducing SDS-PAGE gel, and the
contents of the gel were transferred to supported nitrocellulose
filter by electroblotting. Filters were incubated with 1 to 5
micrograms of an antibody which binds specifically with one of the
following: cyclin D1, cdk4, cdk6, PCNA, and p21. Horseradish
peroxidase-linked anti-mouse or anti-rabbit whole antibody was used
as a secondary antibody. Chemiluminescence was detected using an
ECL.TM. kit (Amersham Life Science, Arlington Heights, Ill.)
according to the manufacturer's instruction.
Immune Complex Protein Kinase (CDK4 AND CDK6) Assays
[0154] NIH 3T3 cell pellets were lysed in lysate buffer. Total
protein concentration in each sample was determined using the micro
BCA.TM. kit (Pierce, Rockford, Ill.) according to the
manufacturer's instructions. 50 Microgram aliquots of cell extract
were transferred to individual microfuge tubes, and the total
volume was brought to 500 microliters with IP buffer.
Immunoprecipitation was performed by incubating these solutions
overnight at 4.degree. C. in the presence of 2.5 micrograms of
mouse monoclonal anti-cdk4 antibody or anti-cdk6 antibody, followed
by incubation for 4 hours in the presence of 25 microliters of
protein G-agarose beads. This amount of beads provided an excess of
G-agarose, relative to antibody. Precipitated protein pellets were
washed 3 times using ice-cold lysate buffer and then resuspended in
20 milliliters of ice-cold kinase buffer, which comprised 50
millimolar HEPES buffer, 80 millimolar .beta.-glycerophosphate, 2.5
millimolar ethylene glycol bis (.beta.-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 10 millimolar MgCl.sub.2,
1 millimolar dithiothreitol (DTT), 2.5 millimolar PMSF, 60 KIU per
milliliter aprotinin, 10 milligrams per milliliter leupeptin, and
10 millimolar cyclin AMP-dependent protein kinase-inhibitory
peptide (SIGMA, Saint Louis, Mo.) at a pH of 7.5. 12 Milliliters of
a reaction mix which comprised 10 microcuries of
.gamma.[.sup.32P]ATP (about 3,000 Curies per millimole; Amersham,
Arlington Heights, Ill.), 25 millimolar non-labeled ATP, and 200
nanograms of Rb protein as substrate were added to each sample, and
the mixed samples were incubated at 30.degree. C. for 15 minutes.
GST-RB was expressed and purified as previously described (Meyerson
and Harlow, 1994) as a source of Rb protein for the reaction mix.
Kinase reactions were stopped by adding a volume of 2.times.SDS
sample buffer equal to the volume of the mixed sample and boiling
this diluted sample for 5 minutes. 2.times.SDS sample buffer
comprised 4% (w/v) SDS, 150 millimolar Tris chloride, 20% (v/v)
glycerol, 0.02% (w/v) BPB, and 2 millimolar sodium vanadate at a pH
of 6.8. Proteins in the boiled, diluted sample were separated by
SDS-PAGE in a 10% (w/v) gel. After drying, the gel was assayed by
autoradiography to detect labeled proteins.
Antibodies
[0155] Monoclonal and polyclonal antibodies designated HD11
(anti-cyclin D1), PC10 (anti-PCNA), F-5 (anti-p21), H-303
(anti-cdk4), and H-230 (anti-cdk6) were obtained from Santa Cruz
Biotechnology (Santa Cruz, Calif.). Polyclonal MCT-1 antibodies
were generated by inoculating rabbits with a synthetic peptide
corresponding to the first 20 amino acids at the amino terminus of
MCT-1. Immune sera was provided by Research Genetics (Huntsville,
Ala.).
Western Blot
[0156] Cultured cells (5.times.10.sup.6 to 10.times.10.sup.6) were
washed three times with phosphate buffered saline (PBS). Cells were
collected by centrifugation, and the pellet was lysed in lysate
buffer. The total protein concentration in each sample was
determined using the micro BCA.TM. kit (Pierce, Rockford, Ill.)
according to the manufacturer's instructions. 25 Micrograms of
total protein per sample was fractionated by electrophoresis in a
Tris-glycine PAGE gel (Novex, San Diego, Calif.) under denaturing,
reducing conditions. The proteins in the gel were transferred to
supported nitrocellulose filters using an electroblotting apparatus
(Millipore, Marlborough, Mass.). Replicate filters were incubated
either with anti-cyclin D1 antibody or with MCT-1 immune sera.
Chemiluminescence was performed using an ECL.TM. kit (Amersham Life
Science, Arlington Heights, Ill.) according to the manufacturer's
instruction. The filters were then exposed to x-ray film and
labeled proteins were quantitated by laser densitometry using a
personal densitometer (Molecular Dynamics, Sunnyvale, Calif.).
Focus Forming Assay
[0157] Stable transfectants and cells of the parent cell lines were
grown to near confluence, and were then plated in 100-millimeter
tissue culture dishes at a density of about 0.5.times.10.sup.6
cells per dish. The cultures were re-fed every 5 to 6 days, and the
number of transformed foci was determined after 2 weeks. Focus
formation and morphologic changes were visualized both by the naked
eye and by microscopy after incubation for 1 hour with Coomassie
blue. All experiments were reproduced at least three times for each
DNA transfected.
[0158] The results of the experiments presented in this Example are
now described. These experiments were designed to examine the
impact of MCT-1 overexpression on protein kinase-mediated G1 phase
checkpoints. The kinase activity of two cyclin D1-associated cdks,
cdk4 and cdk6, was examined in NIH 3T3 cells which constitutively
expressed MCT-1. The cellular levels of cyclin D1 protein, and the
association of cyclin D1 with cdk4, cdk6, PCNA, and p21 were also
assessed.
[0159] Steady state cellular protein levels of MCT-1 in NIH 3T3
cells stably transfected with pCMV-MCT-1 or with control vector
(pCMV), as well as non-transfected 3T3 cells, were assessed by
Western blot analysis. An approximately 20 kilodalton band was
detected with a greater intensity in cell lysates obtained from
cells transfected with pCMV-MCT-1 than in lysates obtained from
non- or control-transfected cells. Similar results were obtained
using a transient transfection assay involving HA-tagged MCT-1
protein. HA-tagged MCT-1 protein could be immunoprecipitated from
lysates of cells transfected with pCMV-HA-MCT-1, and HA-tagged
MCT-1 was detected by Western blotting at a position corresponding
to a size of about 20 kilodaltons.
[0160] An increased level of cyclin D1 protein was detected by
immunoprecipitation in lysates from NIH 3T3 cells transfected with
PCMV-MCT-1, relative to asynchronously grown control cells which
were not transfected or which were transfected with control vector.
Because cdk4 and cdk6 associate with cyclin D1 during cell cycle
progression, complex formation among these molecules was
investigated using co-immunoprecipitation analysis. Increased
subunit complex formation in NIH 3T3 cells constitutively
expressing MCT-1, relative to cells which were not transfected with
a vector encoding MCT-1. Physical interaction of PCNA with cyclin
D1-cdk4/cdk6 complexes was also investigated.
Co-immunoprecipitation of cyclin D1 and PCNA was detected at an
increased level, relative to cells which were not transfected with
a vector encoding MCT-1. Direct physical interaction was not
detected between MCT-1 and any of proteins cyclin D1, cdk4, cdk6,
and PCNA under these assay conditions.
[0161] Previous studies demonstrated that ectopic expression of
cyclin D1 can induce transcriptional activation of the p21 gene
(Hiyama et al., 1997, Oncogene 14: 2533-2542). In normal human
fibroblasts, the cdk inhibitory protein p21 can be detected in
association with various cyclin/cdk complexes in combination with
PCNA (Zhang et al., 1993, Mol. Biol. Cell 4:897-906). Therefore,
these G1 cyclin/cdk complexes were examined for the presence p21.
Protein p21 was determined to be associated with these complexes in
cell lines which constitutively expressed MCT-1.
[0162] Previous studies establish that p21 can act as a universal
inhibitor of cyclin/cdk kinase activity (Xiong et al., 1993, Nature
366:701-704). Because increased G1 cyclin/cdk complexes were
observed in cells which overexpressed MCT-1, as described in this
Example, we analyzed the catalytic activity of cdk4 and cdk6 in
extracts made from these cells were assessed. A markedly increased
ability to phosphorylate Rb protein (a substrate of both cdk4 and
cdk6) was observed in in vitro immune complex kinase assays
performed using either cdk4 or cdk6 immunoprecipitated from
MCT-1-overexpressing cells. Without wishing to be bound by any
particular theory of operation, these data suggest that association
of a single p21 molecule with a cyclin/cdk complex permit stables
complex formation among cyclin D1, cdk and PCNA.
[0163] Using a focus forming assay, the ability of cells
overexpressing MCT-1 to form foci comprising smaller cells that
grew in clusters was demonstrated. Focus formation could not be
detected among non- or control-transfected cells. These results are
consistent with earlier work showing that fibroblasts
overexpressing cyclin D1 exhibit morphological changes and grow in
clusters (Jiang et al., 1993, Oncogene 8:3447-3457; Wang et al.,
1994, Nature 369:669-671).
[0164] MCT-1 and cyclin D1 protein levels were assessed in
asynchronously-grown T-cell tumor cells and PBL control cells. A
number of the T-cell tumor cell lines exhibited elevated MCT-1
protein levels relative to PBL controls. Increased MCT-1 protein
expression correlated with increased levels of cyclin D1. The two
IL-2 dependent cell lines N1185 and N1186 exhibited increased
levels of cyclin D1, but contained no detectable MCT-1 protein. The
HUT 78 cell line had the highest level of MCT-1 protein. This
result is consistent with gene amplification as described in
Example 1 herein. None of the other tumor cell lines analyzed in
this study exhibited MCT-1 gene amplification.
[0165] A striking finding of the experiments described in this
Example is the strong correlation between MCT-1 overexpression and
elevated cyclin D1 protein levels, both in transfected NIH 3T3
cells and in a panel of T-cell tumor cell lines. These experiments
demonstrate for the first time that MCT-1 protein is endogenously
expressed in tumor cells. Furthermore, these experiments also
highlight the biological significance of genomic amplification of
MCT-1 in the HUT 78 cell line, since the level of MCT-1 protein is
greatly increased relative to the other cell lines which do not
exhibit genomic amplification.
[0166] Without wishing to be bound by any particular theory of
operation, it is recognized that these data are consistent with
MCT-1 acting through an upstream mechanism(s) involving cyclin D1
resulting in dys-regulation of G1-associated cdk activity. MCT-1
overexpression induces cells to pass through cell cycle phase G1.
It has been demonstrated by others that when cyclin D1 levels are
elevated, as they are in cells constitutively expressing MCT-1 (as
described herein), expression of several genes involved in growth
regulation are induced (Jiang et al., 1993, Oncogene 8:3447-3457).
Furthermore, the amino terminus of MCT-1 shares a region of
homology with the carboxyl terminal region of cyclin H. As
described in Example 1, this region of cyclin H is known to be
involved in protein-protein interactions (Andersen et al., 1997,
EMBO J. 16:958-967). Cyclin H forms a ternary complex with proteins
cdk7 and MAT 1, and together these proteins form the cdk-activating
kinase (CAK). The CAK is responsible for activating cdk1, cdk2 and
cdk4 (Nigg, 1996, Curr. Opin. Cell Biol. 8:312-317).
[0167] Thus, still not wishing to be bound by any particular theory
of operation, a plausible explanation for the rapid progression of
MCT-1 overexpressing cells through the G1 phase is enhancement of
CAK activity and increased cyclin D1 protein expression, which is
coupled with enhanced expression of other growth regulating genes.
The experiments described in this Example demonstrate that
overexpression of MCT-1 results in loss of normal cell cycle
regulatory controls with an increase in G1 cyclin/cdk complex
formation. While the underlying mechanisms are not known at
present, dys-regulation of MCT-1 appears to be a potent
transforming event in vitro, and overexpression is increased in
T-cell tumor cell lines relative to normal lymphocytes. These
observations demonstrate that abnormally high levels of expression
of MCT-1 protein can be used as an indicator of the tumor state of
a cell, and that tumorigenesis may be inhibited by inhibiting
expression of MCT-1, such as by providing an antisense
oligonucleotide which is complementary to or homologous with a
portion of the gene encoding MCT-1 to a cell.
[0168] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety.
[0169] While this invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of this invention may be devised by others skilled in
the art without departing from the true spirit and scope of the
invention. The appended claims are intended to be construed to
include all such embodiments and equivalent variations.
Sequence CWU 1
1
101944DNAHomo sapiens 1gctacctcca actgctgagg aaccggttgc ctaaaaggag
ccggcaaaag cgcctacgtg 60gagtccagag gagcggaagt agtcagattt gactgagagc
cgtaaagcgc ggctggctct 120cgttttccgg ataacgacta cagctccgac
tgtcagtgcc ggccttcctc gtgtgagggg 180atctgccgga cccctgcaaa
ttcaatttct ttcccattcc gggcccttcc ctatcgtcgc 240ccccttcacc
ttggatcatg ttcaagaaat ttgatgaaaa agaaaatgtg tccaactgca
300tccagttgaa aacttcagtt attaagggta ttaagaatca attgatagag
caatttccag 360gtattgaacc atggcttaat caaatcatgc ctaagaaaga
tcctgtcaaa atagtccgat 420gccatgaaca tatagaaatc cttacagtaa
atggagaatt actctttttt agacaaagag 480aagggccttt ttatccaacc
ctaagattac ttcacaaata tccttttatc ctgccacacc 540agcaggttga
taaaggagcc atcaaatttg tactcagtgg agcaaatatc atgtgtccca
600ggcttaactt ctcctggagc taagctttac cctgctgcag tagataccat
tgttgctatc 660atggcagaag gaaaacagca tgctctatgt gttggagtca
tgaagatgtc tgcagaagac 720attgagaaag tcaacaaagg aattggcatt
gaaaatatcc attatttaaa tgatgggctg 780tggcatatga agacatataa
atgagcctca gaaggaatgc acttgggcta aatatggata 840ttgtgctgta
tctgtgtttg tgtctgtgtg tgacagcatg aagataatgc ctgtggttat
900gctgaataaa ttcaccagat gctaaaaaaa aaaaaaaaaa aaaa 9442121PRTHomo
sapiens 2Met Phe Lys Lys Phe Asp Glu Lys Glu Asn Val Ser Asn Cys
Ile Gln1 5 10 15Leu Lys Thr Ser Val Ile Lys Gly Ile Lys Asn Gln Leu
Ile Glu Gln 20 25 30Phe Pro Gly Ile Glu Pro Trp Leu Asn Gln Ile Met
Pro Lys Lys Asp 35 40 45Pro Val Lys Ile Val Arg Cys His Glu His Ile
Glu Ile Leu Thr Val 50 55 60Asn Gly Glu Leu Leu Phe Phe Arg Gln Arg
Glu Gly Pro Phe Tyr Pro65 70 75 80Thr Leu Arg Leu Leu His Lys Tyr
Pro Phe Ile Leu Pro His Gln Gln 85 90 95Val Asp Lys Gly Ala Ile Lys
Phe Val Leu Ser Gly Ala Asn Ile Met 100 105 110Cys Pro Arg Leu Asn
Phe Ser Trp Ser 115 120325DNAArtificial SequencePrimer HuT 78F
3cattggagac cgctacacac aggac 25424DNAArtificial SequencePrimer HuT
78R 4ctgtcaaaat agtccgatgc cacg 24524DNAArtificial
SequencepCMV-MCT-1 Generation Primer (+) 5gctgaggatc cggttgccta
aaag 24623DNAArtificial SequencepCMV-MCT-1 Generation Primer (-)
6tctggtgaat tcattcagca taa 237943DNAHomo sapiens 7gctacctcca
actgctgagg aaccggttgc ctaaaaggag ccggcaaaag cgcctacgtg 60gagtccagag
gagcggaagt agtcagattt gactgagagc cgtaaagcgc ggctggctct
120cgttttccgg ataacgacta cagctccgac tgtcagtgcc ggccttcctc
gtgtgagggg 180atctgccgga cccctgcaaa ttcaatttct ttcccattcc
gggcccttcc ctatcgtcgc 240ccccttcacc ttggatcatg ttcaagaaat
ttgatgaaaa agaaaatgtg tccaactgca 300tccagttgaa aacttcagtt
attaagggta ttaagaatca attgatagag caatttccag 360gtattgaacc
atggcttaat caaatcatgc ctaagaaaga tcctgtcaaa atagtccgat
420gccatgaaca tatagaaatc cttacagtaa atggagaatt actctttttt
agacaaagag 480aagggccttt ttatccaacc ctaagattac ttcacaaata
tccttttatc ctgccacacc 540agcaggttga taaaggagcc atcaaatttg
tactcagtgg agcaaatatc atgtgtccag 600gcttaacttc tcctggagct
aagctttacc ctgctgcagt agataccatt gttgctatca 660tggcagaagg
aaaacagcat gctctatgtg ttggagtcat gaagatgtct gcagaagaca
720ttgagaaagt caacaaagga attggcattg aaaatatcca ttatttaaat
gatgggctgt 780ggcatatgaa gacatataaa tgagcctcag aaggaatgca
cttgggctaa atatggatat 840tgtgctgtat ctgtgtttgt gtctgtgtgt
gacagcatga agataatgcc tgtggttatg 900ctgaataaat tcaccagatg
ctaaaaaaaa aaaaaaaaaa aaa 9438181PRTHomo sapiens 8Met Phe Lys Lys
Phe Asp Glu Lys Glu Asn Val Ser Asn Cys Ile Gln1 5 10 15Leu Lys Thr
Ser Val Ile Lys Gly Ile Lys Asn Gln Leu Ile Glu Gln 20 25 30Phe Pro
Gly Ile Glu Pro Trp Leu Asn Gln Ile Met Pro Lys Lys Asp 35 40 45Pro
Val Lys Ile Val Arg Cys His Glu His Ile Glu Ile Leu Thr Val 50 55
60Asn Gly Glu Leu Leu Phe Phe Arg Gln Arg Glu Gly Pro Phe Tyr Pro65
70 75 80Thr Leu Arg Leu Leu His Lys Tyr Pro Phe Ile Leu Pro His Gln
Gln 85 90 95Val Asp Lys Gly Ala Ile Lys Phe Val Leu Ser Gly Ala Asn
Ile Met 100 105 110Cys Pro Gly Leu Thr Ser Pro Gly Ala Lys Leu Tyr
Pro Ala Ala Val 115 120 125Asp Thr Ile Val Ala Ile Met Ala Glu Gly
Lys Gln His Ala Leu Cys 130 135 140Val Gly Val Met Lys Met Ser Ala
Glu Asp Ile Glu Lys Val Asn Lys145 150 155 160Gly Ile Gly Ile Glu
Asn Ile His Tyr Leu Asn Asp Gly Leu Trp His 165 170 175Met Lys Thr
Tyr Lys 180958PRTHomo sapiens 9Lys Glu Asn Val Ser Asn Cys Ile Gln
Leu Lys Thr Ser Val Ile Lys1 5 10 15Gly Ile Lys Asn Gln Leu Ile Glu
Gln Phe Pro Gly Ile Glu Pro Trp 20 25 30Leu Asn Gln Ile Met Pro Lys
Lys Asp Pro Val Lys Ile Val Arg Cys 35 40 45His Glu His Ile Glu Ile
Leu Thr Val Asn 50 551050PRTHomo sapiens 10Lys Glu Asn Arg Thr Cys
Leu Ser Gln Leu Leu Asp Ile Met Lys Ser1 5 10 15Met Arg Asn Leu Val
Lys Lys Tyr Glu Pro Pro Arg Ser Glu Glu Val 20 25 30Ala Val Leu Lys
Gln Lys Leu Glu Arg Cys His Ser Ala Glu Leu Ala 35 40 45Leu Asn
50
* * * * *