U.S. patent number 5,747,261 [Application Number 07/786,598] was granted by the patent office on 1998-05-05 for protein related to but distinct from egf receptor and antibodies reactive therewith.
This patent grant is currently assigned to The United States of America as represented by the Department of Health. Invention is credited to Stuart A. Aaronson, C. Richter King, Matthias H. Kraus.
United States Patent |
5,747,261 |
King , et al. |
May 5, 1998 |
Protein related to but distinct from EGF receptor and antibodies
reactive therewith
Abstract
The isolation, cloning and characterization of a human gene
related to but distinct from EGF receptor gene has been described.
Nucleotide sequence of the gene and amino acid sequence of the
polypeptide encoded by the gene have been determined. The use of
the nucleic acid probes and antibodies having specific binding
affinity with said polypeptide for diagnostic and therapeutic
purposes have also been described.
Inventors: |
King; C. Richter (Washington,
DC), Kraus; Matthias H. (Bethesda, MD), Aaronson; Stuart
A. (Great Falls, VA) |
Assignee: |
The United States of America as
represented by the Department of Health (Washington,
DC)
|
Family
ID: |
26808394 |
Appl.
No.: |
07/786,598 |
Filed: |
November 1, 1991 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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110791 |
Oct 21, 1987 |
|
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836414 |
Mar 5, 1986 |
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Current U.S.
Class: |
435/7.1; 435/805;
436/501; 530/300; 530/324; 530/387.1; 530/389.2; 530/399;
530/387.9; 530/350; 530/302; 436/813; 435/6.16 |
Current CPC
Class: |
G01N
33/57484 (20130101); G01N 33/68 (20130101); C07K
14/71 (20130101); G01N 33/566 (20130101); G01N
2333/82 (20130101); Y02A 50/30 (20180101); Y10S
435/805 (20130101); Y10S 436/813 (20130101); Y10S
435/81 (20130101); Y02A 50/473 (20180101); A61K
38/00 (20130101) |
Current International
Class: |
C07K
14/71 (20060101); G01N 33/566 (20060101); C07K
14/435 (20060101); G01N 33/574 (20060101); G01N
33/68 (20060101); A61K 38/00 (20060101); G01N
033/53 () |
Field of
Search: |
;435/6,7.1,805
;436/501,813 ;530/300,302,324,350,387.1,387.9,389.2,399 |
References Cited
[Referenced By]
U.S. Patent Documents
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4968603 |
November 1990 |
Slamon et al. |
|
Other References
Land et al., Science 222:771-778, 1983. .
Cohen, et al., J. Biol. Chem 255:4834-4842, 1980. .
Nishimura, et al. Proc. Natl Acad. Sci. USA 79:4303-4307, 1982.
.
Kasuga et al. Nature 298:667-669, 1982. .
Rubin, et al. Nature 305:438-440, 1983. .
Yamamoto, et al. Cell 35:71-78, 1983. .
de Klein, et al., Nature 300:765, 1982. .
Collins, et al. Proc. Natl. Acad. Sci. USA 80:4813, 1983. .
Liberman, et al., Nature 313:144, 1985. .
Lin, et al. Science 224:843, 1984. .
Rigby, et al., J. Mol. Biol. 113:237, 1977. .
Wahl, et al. Proc. Natl. Acad. Sci USA 76:3683-3687, 1979. .
Downward, et al. Nature 307:521-527, 1984. .
Ullrich, et al., Nature 309:418-425, 1984. .
Doolittle, et al. Science 221:275-277, 1983. .
Schechter et al., Nature 312:513-516 (1984). .
Paik, S. et al. "Pathologic Findings From the national Surgical
Adjuvant Breast and Bowel Project: Prognostic Significance of
erbB-2 Protein Overexpression in Primary Breast Cancer" J. of
Clinical Oncology (1990) 8:103-112. .
King, C.R. et al. "Heterogeneous Expression of erbB-2 Messenger RNA
in Human Breast Cancer" Cancer Research(1989) 49:4185-4191. .
Park, J-B. et al. "Amplification, Overexpression, and Rearrangement
of erbB-2 Protooncogene in Primary Human Stomach Carcinomas" Cancer
Research (1989) 49:6605-6609. .
King, C.R. et al. "Implications of erbB-2 overexpression for basic
science and clinical medicine" seminars in Cancer Biology (1990)
1:329-337. .
Berger, M.S. et al. "Correlation of c-erbB-2 Gene Amplification and
Protein Expression in Human Breast Carcinoma with Nodal Status and
Nuclear Grading" Cancer Research (1988) 48:1238-1243. .
King et al., Amplification of a Novel v-erbB-Related Gene in a
Human Mammary Carcinoma, Science, (1985) 229:974-976. .
Kraus et al., Overexpression of the EGF Receptor-Related
Proto-Oncogene erbB-2 in Human Mammary Tumor Cell Lines . . . The
EMBO Journal, (1987) 6:605-610. .
Di Foire et al., erbB-2 Is a Potent Oncogene When Overexpressed in
NIH/3T3 Cells, Science., (1987) 237:178-182. .
Lacroix et al., Overexpression of erbB-2 or EGF Receptor Proteins
Present in Early Stage Mammary . . . Oncogen,(1989) 4:145-151.
.
Slamon et al., Human Breast Cancer: Correlation of Relapse and
Survival with . . . , Science, (1987), 235:177-182. .
Schechter, A.L. et al. Science 229:976-8 (1985). .
Chemical Abstracts 104 No. 17, Issued Apr. 28, 1986, p. 143,
142890e, King, C.R. et al, "Oncogenes as Growth Factors . . .
erbB-Related Gene". .
King, C.R. et al, Cell Memb. Cancer Proc. Int. Workshop, 2nd, 1985,
pp. 411-416. .
Sedlak (1994) Genetic Engineering News of May 15, 1994, pp. 8-9.
.
Brison (1993) Biochimica et Biophysica Acta, vol. 1155, pp. 25-41.
.
Semba et al. (1985) Proc. Natl. Acad Sci (USA), vol. 82, pp.
6497-6501. .
Yamamoto et al. (1986) Nature, vol. 319, pp. 230-234..
|
Primary Examiner: Marschel; Ardin H.
Attorney, Agent or Firm: Needle & Rosenberg, P.C.
Parent Case Text
This is a division of application Ser. No. 07/110,791, filed Oct.
21, 1987 which is a C.I.P. of Ser. No. 06/836,414, filed Mar. 5,
1986, now abandoned.
Claims
We claim:
1. A purified MAC117 polypeptide having at least in part the
following amino acid sequence: ##STR4##
2. A purified, complete MAC117 protein.
Description
BACKGROUND OF THE INVENTION
1. Technical Field
The present invention is related to the cloning, isolation and
partial characterization of a hitherto unidentified human gene.
More particularly, the present invention is related to the
preparation and identification of a v-erbB related human gene that
is a new member of the tyrosine kinase encoding family of genes and
is amplified in a human mammary carcinoma.
2. State of the Art
A number of genes have been identified as retroviral oncogenes that
are responsible for inducing tumors in vivo and transforming cells
in vitro (Land et al., Science 222:771-778, 1983). Some of them
apparently encode transforming proteins that share a kinase domain
homologous to that of pp60.sup.src, a tyrosine-specific protein
kinase. The cellular cognate, encoded by the c-src gene, also
exhibits tyrosine-specific kinase activity. Of particular interest
is the fact that tyrosine-specific kinases are also encoded by
other genes for several receptors for polypeptide growth factors,
including the receptors for epidermal growth factor (EGF) (Cohen et
al., J. Biol. Chem. 255:4834-4842, 1980), platelet-derived growth
factor (PDGF) (Nishimura et al., Proc. Natl. Acad. Sci. USA
79:4303-4307, 1982), insulin (Kasuga et al., Nature 298:667-669,
1982), and insulin-like growth factor I (Rubin et al., Nature
305:438-440, 1983). This implies a possible link between the action
of the growth factor-receptor complex and the oncogene products
with tyrosine-specific kinase activity.
Recent analysis of the v-erbB gene and the EGF receptor gene
indicates that the v-erbB gene is a part of the EGF receptor gene
and codes for the internal domain and transmembrane portion of the
receptor (Yamamoto et al., Cell 35:71-78, 1983; Downward et al.,
Nature 307:521-527, 1984; Ullrich et al., Nature 309:418-425,
1984). These findings, together with the extensive identity of the
amino acid sequences of the v-sis protein and platelet-derived
growth factor (Waterfield et al., Nature 304:35-39, 1983; Doolittle
et al., Science 221:275-277, 1983), suggest that some viral
oncogene products mimic the action of the polypeptide growth
factor-receptor complex in activating a cellular pathway involved
in cell proliferation and tumor formation.
Genetic alterations affecting proto-oncogenes of the tyrosine
kinase family may play a role in spontaneous tumor development. A
specific translocation affecting the c-abl locus, for example, is
associated with chronic myelogenous leukemia (de Klein et al.,
Nature 300:765, 1982; Collins et al., Proc. Natl. Acad. Sci. USA
80:4813, 1983). Several recent studies have also documented
amplification or rearrangement of the gene for the EGF receptor in
certain human tumors (Libermann et al., Nature 313:144, 1985), or
tumor cell lines (Ullrich et al., Nature 309:418, 1984; Lin et al.,
Science 224:843, 1984). However, a gene that is a new member of the
tyrosine kinase family anc is amplified in a human mammary
carcinoma and is closely related to, but distinct from the EGF
receptor gene, has not heretofore been known.
SUMMARY OF THE INVENTION
It is, therefore, an object of the present invention to provide a
novel, clones, human gene having the nucleotide sequence as shown
in FIG. 1 and described more fully herein infra.
It is a further object of the present invention to provide
products, e.g. various RNAs and/or polypeptides encoded by the
clones gene.
It is a still further object of the present invention to provide
antibodies, either polyclonal or monoclonal, directed against the
protein product encoded by said gene and a diagnostic kit
containing said antibodies for the detection of carcinomas.
It is another object of the present invention to provide
complementary DNA (cDNA) clones homologous to the messenger RNA
(mRNA) encoded by the cloned gene, said cDNA clones being capable
of expressing large amounts of corresponding protein in a
heterologous vector system, such as bacteria, yeast, eukaryotes and
the like.
It is yet another object of the present invention to produce a
transformed cell or organism capable of expressing said gene by
incorporating said gene or a part thereof into the genome of said
cell, vector or organism.
It is a still further object of the present invention to provide
nucleic acid probes and/or antibody reagent kits capable of
detecting said gene or a product thereof.
Other objects and advantages of the present invention will become
apparent as the detailed description of the invention proceeds.
BRIEF DESCRIPTION OF DRAWINGS
These and other objects, features and many of the attendant
advantages of the invention will be better understood upon a
reading of the following detailed description when considered in
connection with the accompanying drawings wherein:
FIG. 1 shows a characteristic fragment produced by Eco RI
restriction of the cloned gene of the present invention: the
restriction-site map of .lambda.MAC117 and plasmid pMAC117. A: Acc
I; B: Bam HI; Bg: Bgl I; N: Nco I; R: Eco RI; X: Xba I; Xh: Xho I.
The sites were located by electrophoretic analysis of the products
of single and double digestion. Regions homologous to v-erbB or
human repetitive sequences (region flanked by arrows) were located
by Southern blot hybridization (Southern, J. Mol. Biol. 98:503
(1975)), with the v-erbB probe or total human DNA made radioactive
by nick translation (Rigby et al., J. Mol. Biol. 113:237 (1977)).
Hybridization conditions were as described in FIG. 2. The
nucleotide sequence of pMAC117 between the Acc I site and the Nco I
sites and regions of encoded amino acid sequence homologous to the
EGF receptor are shown. The AG or GT dinucleotides flanking the
putative coding regions are underlines. To determine the sequence,
Nco I, Hinf I and Sau 96 I fragments were labeled at the 3' termini
by means of a large fragment of E. coli DNA polymerase, separated
into single strands by gel electrophoresis and chemically degraded
(Maxam et al., Proc. Natl. Acad. Sci., USA 74:560 (1977)).
FIGS. 2A and 2B shows the gel electrophoretic properties of
specific gene fragments; detection of v-erbB- and pMAC117-specific
gene fragments in normal human placenta, A431 cells or human
mammary carcinoma MAC117. DNA (15 .mu.g) was cleaved with Eco RI,
separated by electrophoresis in agarose gels and transferred to
nitrocellulose paper (Southern, J. Mol. Biol. 98:503 (1975)).
Hybridization to the .sup.32 P-labeled probe (Rigby et al., J. Mol.
Biol. 113:237 (1977)) was conducted in a solution of 40 percent
formamide, 0.75M NaCl and 0.075M sodium citrate at 42.degree. C.
(Wahl et al., Proc. Natl. Acad. Sci., USA 76:3683 (1979)). The
v-erbB probe (A) was a mixture of the 0.5-kbp Bam HI-Bam HI
fragment and the 0.5-kbp Bam HI-Eco RI fragment of avian
erythroblastosis proviral DNA. The pMAC117 probe (B) was a 1-kbp
Bgl I-Bam HI fragment. After hybridization, the blots were washed
first in 0.3M NaCl plus 0.03M sodium citrate at room temperature
and then in 0.015M NaCl, 0.0015M sodium citrate and 0.1 percent
sodium dodecyl sulfate at 42.degree. C. (v-erbB probed blots) or at
52.degree. C. (pMAC117 probed blots). Hybridization was detected by
autoradiography.
FIG. 3 shows a comparison of the putative encoded amino acid
sequence of various polypeptide products, and comparison of the
putative encoded amino acid sequence in pMAC117 with known tyrosine
kinase sequences. Black regions represent homologous amino acids.
Differing amino acid residues are shown in one-letter code (A,
alanine; C. cysteine, D. aspartic acid; E. glutamic acid; F.
phenylalanine; G. glycine; H. histidine; I. isoleucine; K. lysine;
L. leucine; M. methionine; N. asparagine; P. proline; Q. glutamine;
R. arginine; S. serine; T. threonine; V. valine; W. tryptophan; Y.
tyrosine). Amino acid positions conserved in all sequences are
denoted by *. The tyrosine homologous to that autophosphorylated by
the v-src protein (Smart et al., Proc. Natl. Acad. Sci. USA
78:6013, 1981) is shown by an arrow. The v-abl sequence contains a
tyrosine residue in this region displaced by two positions. The
amino acid sequences of human EGF receptor, v-src, v-abl, v-fms,
and human insulin receptor were aligned by the computer program
described by Ullrich et al., Nature 313:756, 1985 which is
incorporated herein by reference. The homology observed with the
predicted amino acid sequences of v-yes and v-fes was 51 percent
and 48 percent, respectively.
FIG. 4 shows the distinction between .lambda.MAC117 and human EGF
receptor genes by the detection of distinct messenger RNA species
derived from the .lambda.MAC117 gene and the human EGF receptor
gene. Polyadenylated messenger RNA of A431 cells was separated by
denaturing gel electrophoresis in formaldehyde (Lehrach et al.,
Biochemistry 16:4743, 1977), transferred to nitrocellulose
(Southern, J. Mol. Biol. 98:503, 1975), and hybridized under
stringent conditions (50 percent formamide, 0.75M NaCl, 0.075M
sodium citrate, at 42.degree. C.) with .sup.32 P-labeled probe from
pMAC117 (Bgl I-Bam HI fragment) or human EGF receptor complementary
DNA (pE7 2-kb Cla I inserted fragment). Filters were washed under
conditions of high stringency (0.015M NaCl plus 0.0015M sodium
citrate at 55.degree. C.). Hybridization was detected by
autoradiography with exposure times of 4 hours for the pMAC117
probe and 1 hour for the human EGF receptor probe.
FIG. 5 shows the restriction map of complementary DNA or MAC117
encompassing the entire coding region of the gene. Clone pMAC137
was isolated from an oligo dT primed normal human fibroblast cDNA
library (Okyama et al., Mol. Cell. Biol. 3, 280, 1983) using a
0.8-kbp Acc I fragment from the 3' terminus cell pMAC117 as probe.
Clones .lambda.MAC30, .lambda.MAC10', and .lambda.MAC14-1 were
subsequently isolated from a randomly primed MCF-7 cDNA library
(Walter et al., Proc. Natl. Acad. Sci. USA, 82, 7889, 1985) using
cDNA fragments as probes. Restriction sites: B-Bam HI, BII-Bst EII,
E-Eco RI, N-NCO I, P-Pst I, Sm-Sma I, Sp-Sph I, and St-Stu I.
FIG. 5B illustrates three probes, a, b and c, representing the 5'
end, a middle portion and the entire coding region, respectively,
which were employed in subsequent studies elucidating the role and
function of this v-erbB-related gene.
FIGS. 6A and 6B show the overexpression of MAC117 in RNA in human
mammary tumor cell lines. (A) Northern blot analysis. Total
cellular RNA (10 .mu.g) of mammary tumor cell lines, normal
fibroblasts M413 and HBL100 was hybridized with a cDNA probe
derived from the 5' end of the coding region (FIG. 5B, probe a).
M413 and HBL100 cells contain specific mRNA detectable after longer
autoradiographic exposures. Similar results were obtained when
probe b or c (FIG. 5B) was employed for hybridization. (B)
Quantitation of mRNA levels. Serial 2-fold dilutions of total RNA
were applied to nitrocellulose. Replicate filters were hybridized
with either a cDNA probe (FIG. 5B, probe b) or human .beta.-actin
which served as control for RNA amounts present on the
nitrocellulose filter. Relative amounts detected with each probe
are indicated in comparison to the hybridization signals observed
in normal human fibroblast M413.
FIGS. 7A and 7B show the 185-kDal protein specific for MAC117 and
its overexpression in human mammary tumor cell lines. 40 .mu.g
cellular protein was separated by electrophoresis and transferred
to nitrocellulose filters. The protein was detected with an
antipeptide antibody coupled to .sup.125 I protein A. The
specificity of antibody detection was determined by pre-incubation
of the antibody with excess amounts of peptide prior to
immunodetection. (+) preincubation with peptide, (-) no peptide. In
panel B, nonspecific bands at 100 kd are observed in longer
exposures of peptide blocked immunoblots (panel A).
FIGS. 8A and 8B show the gene amplification of MAC117 in 4 mammary
tumor cell lines and the absence of MAC117 gene amplification in 4
other mammary tumor cell lines overexpressing MAC117 mRNA. (A)
Southern blot analysis. For each line 10 .mu.g genomic DNA were
restricted with Xba I and hybridized with a probe comprising the
entire coding region of MAC117 (FIG. 5B, probe c). Hind III
restriction fragments of lambda DNA served as mol. wt. standards.
(B) DNA dot-blot analysis. Genomic DNA (10 .mu.g) digested with Eco
RI was applied in serial 2-fold dilutions to nitrocellulose
filters. Filters were hybridized either with a probe specific for
MAC117 (FIG. 5B, probe b) or mos, which served as a control for DNA
amounts applied to replicate nitrocellulose filters. Gene copy
numbers of MAC117 relative to M413 indicate the minimal extent of
gene amplification detected in DNA from mammary tumor cell
lines.
FIG. 9 depicts the construction of expression vectors for the human
MAC117 cDNA. A Nco I-Mst II fragment encompassing the entire open
reading frame was cloned under the transcriptional control of
either the SV40 early promoter or MuLV LTR. Symbols: , erbA-erbB
intergenic region of pAEV11 containing the 3' splice acceptor site;
N=Nco I, Sp=Sph I, M=Mst II, St=Stu I, H=Hind III, Sm=Sma I, P=Pst
I, B=BamH I, X=Xho I. Sites indicated in parenthesis were not
reconstituted after the cloning procedures.
FIG. 10 shows the comparison of the levels of MAC117 proteins in
LTR-1/erbB-2 transformed NIH/3T3 cells and human mammary tumor
lines by immunoblot analysis. Varying amounts of total cellular
protein were separated by electrophoresis and transferred to
nitrocellulose filters. The MAC117 protein was detected with rabbit
anti-peptide serum coupled to .sup.125 I protein A as previously
described.
DETAILED DESCRIPTION OF INVENTION
The above and other objects and advantages of the present invention
are achieved by a cloned human gene having the nucleotide sequence
as shown in FIG. 1. Although any methods and materials similar or
equivalent to those described herein can be used in the practice or
testing of the present invention, the preferred methods and
materials are now described. All publications mentioned under the
"Brief Description of Drawings" and hereunder are incorporated
herein by reference. Unless defined otherwise, all technical or
scientific terms used herein have the same meaning as commonly
understood by one or ordinary skill in the art to which this
invention belongs.
Cells and Tissues
Preparation of High Molecular Weight DNA
1. From A431 Cells:
A431 carcinoma cells were established in culture and maintained in
Dulbecco's modified Eagle's medium with 10% fetal calf serum.
Cells were grown to 90% confluence in four 175 cm.sup.2 tissue
culture flasks, washed twice with phosphate buffered saline (Gibco
Biochemicals), then lysed in 10 mM Tris (pH 7.5), 150 mM NaCl, 50
mM ethylenediaminetetraacetate (EDTA) and 0.5% sodium dodecyl
sulfate (SDS). Proteinase K (Boehinger Mannheim) was added to a
concentration of 0.1 mg/ml and the cell extracts digested for 3
hours at 50.degree. C. DNA was extracted 3 times with phenol and
once with CHCl.sub.3. DNA was precipitated with 2 volumes of
ethanol, spooled and resuspended in 20 ml of 10 mM Tris-HCl (pH
7.5), 1 mM EDTA. The solution was then made 10 .mu.g/ml with (DNase
free) RNase (Boehinger Mannheim) and incubated for 2 hr at
50.degree. C. NaCl was added to 0.5M and the solution extracted
with phenol followed by CHCl.sub.3. DNA was precipitated with 2
volumes of ethanol, spooled, and resuspended in 10 mM Tris, 1 mM
EDTA. The concentration was determined by routine
spectrophotometric procedure at 260 nm wavelength.
2. From Tissues:
Two grams original mass of primary tumor (designated MAC117
obtained from memorial Sloan-Kettering Cancer Center Specimen code
31-26606) were pulverized in a mortar and pestle at liquid nitrogen
temperature, suspended in 10 ml of 10 mM Tris-HCl (pH 7.5), 150 mM
NaCl, 2 mM EDTA, reacted with proteinase K at 500 .mu.g/ml
(Boehinger Mannheim) and SDS at 0.5% at 37.degree. C. for 10 hr.
The solution was then extracted twice with phenol and twice with
the mixture of phenol:CHCl.sub.3 :isoamyl alcohol at 25:24:1 and
once with CHCl.sub.3 :isoamyl alcohol (24:1). DNA was precipitated
by 2 volumes of ethanol removed by spooling, and resuspended in 1
mM Tris-HCl (pH 7.5), 0.2 mM EDTA.
Electrophoretic Analysis of DNA Fragments using "Southern
Hybridization"
1. Restriction Enzyme Cleavage
Each sample of DNA (15 .mu.g) was digested in 0.4 ml of 100 mM
Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl.sub.2, 100 ug/ml bovine
serum albumin and 30 units of restriction enzyme (New England
Biolabs) for 2 hr at 37.degree. C. Following reaction, 10 .mu.g of
tRNA was added and the solution extracted once with an equal volume
of a mixture of phenol and CHCl.sub.3 (1:1). Nucleic acids were
precipitated from the aqueous phase by addition of 2 volumes of
ethanol. Following centrifugation for 10 min at 12,000.times.g
(Eppendorf microfuge) the samples were washed once with 80%
ethanol, dried to remove ethanol, and resuspended in 40 .mu.l
distilled H.sub.2 O.
2. Agarose Gel Electrophoresis
DNA samples were made 40 mM Tris acetate (pH 7.2), 20 mM Na
acetate, 1 mM EDTA, 5.0% glycerol, 0.05% bromophenol blue.
Electrophoresis was conducted in a BRL H4 apparatus containing 400
ml 0.8% agarose, 40 mM Tris acetate (pH 7.2), 20 mM Na acetate, 1
mM EDTA and 1 .mu.g/ml ethidium bromide for about 16 hr at about 50
volts following conventional procedure. DNA was detected by
irradiation with ultraviolet light.
3. Transfer to Nitrocellulose
The agarose gel was treated twice for 15 min in 1 liter of 0.5M
NaOH. 1.5M NaCl, then twice for 30 min with 1M NH.sub.4 Ac, 20 mM
NaOH. The agarose gel was then placed on a stack of filter paper
saturated with 1 liter of 1M NH.sub.4 Ac, 20 mM NaOH. A sheet of
nitrocellulose membrane (0.45 .mu.m pore size Schleicher &
Schuell) was placed on top of the gel followed by dry filter paper.
Transfer was allowed to occur overnight. DNA was fixed to
nitrocellulose by baking at 80.degree. C. in vacuo for 2 hr.
Hybridization to RNA and DNA Blots
Hybridization was conducted in 20 ml of 40% formamide, 0.75M NaCl,
0.075M Na citrate, 0.05% BSA, 0.05% polyvinyl pyrolidone, 0.05%
Ficol 400 and 20 .mu.g/ml sheared denatured calf thymus DNA. All
hybridization was conducted for 16 hr at 42.degree. C. in a water
bath. Following hybridization, nitrocellulose membranes were washed
2 times for 20 min in 1 liter of 0.3M NaCl, 30 mM Na citrate,
followed by washed in 15 mM NaCl, 1.5 mM Na citrate, first with and
then without 0.1% sodium dodecyl sulfate. These final washes were
at 42.degree. C. for v-erbB probes and at 52.degree. C. with
pMAC117 and pE7 probes, vide infra. Autoradiography was conducted
at -70.degree. C. with Kodak XAR5 film. Exposure times were 2 hr
for FIG. 2A and 20 min for FIG. 2B, 40 min for EGF receptor probe
on FIG. 4, and 4 hr for the pMAC117 probe of FIG. 4.
Generation of Probe DNAs
A nucleic acid probe is defined as a fragment of DNA or RNA whose
nucleotide sequence has at least partial identity with the sequence
of the gene or its messenger RNA so as to enable detection or
identification of the gene. Since a gene may have several
fragments, there could be a plurality of probes for detecting the
gene.
The probes used were the 0.5-kb Bam HI to Bam HI fragment combined
with the 0.5-kb Bam HI to Eco RI fragment of the v-erbB gene of AEV
11; the 1-kb BglI to Bam HI fragment of pMAC117; and the 2-kb Cla I
fragment of pE7 as described by Xu, et al., (Nature 309:806,
1984).
DNA fragments were isolated by gel electrophoresis in 1% low
melting point agarose gels (Bethesda Research Labs) in 40 mM Tris
acetate, 20 mM Na acetate, 1 mM EDTA, followed by melting of the
gel at 70.degree. C. and extraction with phenol followed by
CHCl.sub.3 and ethanol precipitation. DNAs were made radioactive by
using a nick-translation kit (Amersham) in which 50 .mu.l reactions
contained 250 .mu.Ci .alpha.P.sup.32 dCTP (Amersham) and 0.5 .mu.g
DNA. Radioactive probe DNA was purified from unincorporated
nucleotides by 2 cycles of ethanol precipitation. Yields were above
2.times.10.sup.8 cpm/.mu.g DNA. Before hybridization all probes
were made single-stranded by treatment with 90% formamide.
RNA Electrophoresis and Transfer to Nitrocellulose
RNA samples (5 .mu.g A431 polyadenylated RNA, obtained from
National Institutes of Health, Bethesda, Md. 21218) were treated
for 5 min at 50.degree. C. in 50% formamide, 6.7% formaldehyde, 20
mM Mops (pH 7.0) (Sigma Biochemicals), 5 mM Na acetate, 1 mM EDTA
in 25 .mu.l total volume. Electrophoresis was conducted in BRL H4
apparatus in 250 ml of 1.5% agarose, 20 mM Mops (pH 7.0), 5 mM Na
acetate, 1 mM EDTA, 1 .mu.g/ml ethidium bromide at 40 volts for 16
hr. RNA was detected using ultraviolet light. The gel was soaked
for 30 min at 20.degree. C. in 50 mM NaOH, followed by two 30 min
washes in 1M Tris (pH 7.5), followed by 30 min in 3M NaCl, 0.3M Na
citrate. Transfer to nitro-cellulose was accomplished by placing
the gel atop a stack of filter paper saturated with 1.5M NaCl,
0.15M Na citrate, followed by 0.45 .mu.M pore size nitrocellulose
(Schleicher and Schuell), followed by dry filter paper. Transfer
was allowed to proceed for 16 hr. The nitrocellulose filter was
washed twice for 20 min in 0.3M NaCl, 30 mM Na citrate. RNA was
fixed to the paper by baking at 80.degree. C. in vacuo for 2
hr.
DNA Sequence Analysis
DNA fragments containing the AccI-NcoI region (FIG. 1) were
digested with either Nco I, Hinf I or Sau 96I (New England
Biolabs). These fragments were end-labeled in reactions of 50 .mu.l
containing 50 mM Tris-HCl (pH 7.2), 10 mM MgCl.sub.2, 0.1 mM
dithiothreitol, 50 .mu.g/ml BSA, 10 .mu.Ci.alpha..sup.= PdXTP
(Amersham--where x represents the correct nucleotide for fill-in),
2 units E. coli DNA polymerase large fragment (New England
Biolabs). Following labeling, single-stranded material was prepared
by electrophoresis. Samples were denatured in 30% dimethyl
sulfoxide, 1 mM EDTA and 0.05% bromophenol blue at 90.degree. C.
for 2 hr. Samples were chilled and electrophoresed in acrylamide
gels in a Bethesda Research Labs apparatus. DNA was detected by
autoradiography and isolated by elution into 10 mM Tris-HCl (pH
7.0), 1 mM EDTA. Chemical degradation of DNA for sequence analysis
was conducted using standard procedures. Cleavage at guanine (G)
residues was conducted by reaction with dimethyl sulfonate at
22.degree. C. for 10 min. Cleavage at adenine (A) residues was
conducted by 12 min reaction at 90.degree. C. in 1.5M NaOH, 1 mM
EDTA. Cleavage at cytosine (C) residues was conducted using
hydrazine in 2M NaCl for 13 min at 22.degree. C. Cleavage at
thymine (T) residues was conducted using hydrazine with no added
NaCl for 10 min at 22.degree. C. Following cleavage, all reactions
were twice precipitated using ethanol and thoroughly dried. All
samples were reacted with 1M piperidine at 90.degree. C. for 30
min. Piperidine was removed by evaporation in a Savant speed vac
concentrator. Fragments were separated by electrophoresis in
acrylamide gels (BRL HO apparatus) in 8M urea, 50 mM Tris-borate
(pH 8.3), 1 mM EDTA. Detection of degraded ladder was by
autoradiography using Kodak XAR5 film at -70.degree. C.
Cloning of .lambda.MAC117
High molecular weight DNA (6 .mu.g) from tumor MAC117 (see above)
was digested with 12 units restriction enzyme Eco RI (New England
Biolabs) in a volume of 100 ul for about one hour at 37.degree. C.
DNA was obtained by phenol CHCl.sub.3 extraction and ethanol
precipitation and resuspended in water at a concentration of 0.1
.mu.g/ml. This DNA (0.2 .mu.g) was ligated to .lambda.wes .lambda.B
arms (Bethesda Research Labs) (1 .mu.g) using T4 DNA ligase (New
England Biolabs) in a total volume of 20 ml [50 mM Tris-HCl pH 7.4,
10 mM MgCl.sub.2 10 mM dithiothreitol, 0.5 mM spermidine, 1 mM
ATP]. This mixture of ligated DNAs was packaged into infectious
bacteriophage particles using the Packagene system (Promega
Biotec). These particles were used to infect bacteria BNN45 and
about 8.times.10.sup.5 individual phage plaques were obtained.
These phage plaques were screened for individual plaques containing
DNA homologous to the v-erbB probes (described above) using
standard procedures. Briefly, bacterial culture plates containing
approximately 15,000 plaques were grown overnight. Sterile
nitrocellulose discs (Scheicher and Schuell) were applied to the
dish, removed and allowed to air dry for about 90 minutes. The
discs were then treated with 0.2M NaOH, 1.5M NaCl followed by 0.4M
Tris-HCl pH 7.5 followed by 0.3M NaCl 0.03M Na citrate and baked in
vacuo for two hours at 80.degree. C. These discs were then exposed
to hybridization and washing conditions identical to those
described for FIG. 2 using the identical v-erbB probe. Washing
conditions were also identical to those for FIG. 2. Hybridization
was detected by autoradiography at -70.degree. C. for 16 hours.
Single hybridizing phage plaques were obtained by three successive
hybridization experiments (as described above) to isolate a pure
phage culture.
DNA from MAC117 was digested with Eco RI, then ligated into
bacteriophage .lambda.gtWES, packaged in vitro, and transferred to
Escherichia coli (E. coli) strain BNN45 by infection following
standard techniques well known in the art. A library of
4.times.10.sup.5 bacteriophages was screened by plaque
hybridization with radioactive v-erbB DNA. Ten of 14 hybridizing
phages contained a 6-kbp Eco RI fragment. FIG. 1 shows the physical
map of one of these phages, .lambda.MAC117, and pMAC117, a PUC12
subclone containing a 2-kbp Bam HI fragment of .lambda.MAC117 that
hybridized with v-erbB probes. The region of pMAC 117 to which
v-erbB hybridized most intensely was flanked by Acc I and Nco I
sites. Human repetitive sequences were also localized (FIG. 1,
region demarcated by arrows).
A deposit of pMAC117 cloned in E. coli has been made at the
American Type Culture Collection (ATCC), Bethesda, Md. under
accession number 53408. Upon issuance of a patent, the culture will
continue to be maintained for at least 30 years and made available
to the public without restriction subject, of course, to the
provisions of the law in this respect.
As shown in FIG. 2A, DNA prepared from tissue of a human mammary
carcinoma, MAC117, shows a pattern of hybridization that differed
both from that observed with DNA of normal human placenta and from
that observed with the A431 squamous-cell carcinoma line, which
contains amplified epidermal growth factor (EGF) receptor genes. In
A431 DNA, four Eco RI fragments were detected that had increased
signal intensities compared to those of corresponding fragments in
placenta DNA (FIG. 2A). In contrast, MAC117 DNA contained a single
6-kilobase pair (kbp) fragment, which appeared to be amplified
compared to corresponding fragments observed in both A431 and
placenta DNA's (FIG. 2A). These findings indicate that the MAC117
tumor contained an amplified DNA sequence related to, but distinct
from, the cellular erbB proto-oncogene.
By digestion of pMAC117 with Bgl I and Bam HI, it was possible to
generate a single-copy probe homologous to v-erbB. This probe
detected a 6-kb Eco RI fragment that was amplified in MAC117 DNA
and apparently increased in A431 cellular DNA relative to normal
DNA (FIG. 2B). The sizes of the fragment corresponded to the
amplified 6-kb Eco RI fragment detected in MAC117 DNA by means of
v-erbB (FIG. 2A). Hybridization to Southern blots containing serial
dilutions of MAC117 genomic DNA indicated an approximate
amplification of 5- to 10-fold when compared to human placenta
DNA.
The nucleotide sequence of the portion of pMAC117 located between
the Nco I and Acc I sites contained two regions of nucleotide
sequence homologous to v-erbB separated by 122 nucleotides (FIG.
1). These regions shared 69 percent nucleotide sequence identity
with both the v-erbB and the human EGF receptor gene. The predicted
amino acid sequence of these regions was 85 percent homologous to
two regions that are contiguous in the EGF receptor sequence.
Furthermore, these two putative coding regions of the MAC117
sequence were each flanked by the AG and GT dinucleotides that
border the exons of eukaryotic genes. These findings suggest that
the sequence shown in FIG. 1 represents two exons, separated by an
intron of a gene related to the erbB/EGF receptor gene.
The predicted amino acid sequence of the .lambda.MAC117 putative
exons is homologous to the corresponding sequences of several
members of the tyrosine kinase family. The most striking homology
was observed with the human EGF receptor or erbB (FIG. 3). In
addition, 42 percent to 52 percent homology with the predicted
amino acid sequences of other tyrosine kinase-encoding genes was
observed. At 25 percent of the positions there was identity among
all the sequences analyzed (FIG. 3). A tyrosine residue in the
.lambda.MAC117 putative coding sequence, conserved among the
tyrosine kinases analyzed, is the site of autophosphorylation of
the src protein (Smart et al., Proc. Natl. Acad. Sci. USA. 78:6013,
1981).
The availability of cloned probes of the MAC117 gene made it
possible to investigate its expression in a variety of cell types.
The MAC117 probe, consisting of the Bgl I to Bam HI restriction
fragment of pMAC117, detected a single 5-kb transcript in A431
cells (FIG. 4). Under the stringent conditions of hybridization
utilized, this probe did not detect any of the three RNA species
recognized by EGF receptor complementary DNA. Thus, MAC117
represents a new functional gene within the tyrosine kinase family,
closely related to, but distinct from the gene encoding the EGF
receptor.
There is precedent for the identification of genes related to known
oncogenes on the basis of their amplification in human tumors. For
example, the high degree of amplification of N-mvc in certain
malignancies made it detectable by means of the mvc gene as a
molecular probe (Schwab, Nature 305:245, 1983; Kohl et al., Cell
35:349, 1983). In the present study, a five- to tenfold
amplification of a v-erbB-related gene in the MAC117 mammary
carcinoma made it possible to identify this sequence against a
complex pattern of EFG receptor gene fragments.
The MAC117 coding sequence, as determined by nucleotide and
predicted amino acid sequence, is most closely related to the
erb3/EGF receptor among known members of the tyrosine kinase
family. The two genes are distinct, however, as evidenced by the
sequence diversity and transcript size. Detailed structural
analysis of the complete coding sequence would further elucidate
the role and function of this v-erbB-related gene.
To this purpose we have isolated cDNAs with a complexity of over
4.5 kb from the MAC117 mRNA (Kraus et al., EMBO Journal 6:605-610,
1987). A restriction map is shown in FIG. 5A. An oligo (dT) primed
normal human fibroblast cDNA library (Okayama and Berg, 1983) was
screened with a 0.8 kbp Acc I DNA fragment from the 3' terminus of
a genomic clone of MAC117 (FIG. 1). The largest plasmid obtained,
pMAC137, carried a 2-kbp insert comprising 1.5 kbp of 3' coding
information and 3' untranslated sequence. The remaining coding
information upstream was obtained from three phage clones,
.lambda.MAC30, .lambda.MAC10' and .lambda.MAC14-1, identified in a
randomly primed MCF-7 cDNA library (Walter et al., 1985; FIG.
5A).
To assess the role of MAC117 in human mammary neoplasia, we
compared mRNAs of 16 mammary tumor cell lines to normal human
fibroblasts, M413, and a human mammary epithelial cell line,
HBL100. Increased expression of an apparently normal size 5-kb
transcript was detected in 8 of 16 tumor cell lines when total
cellular RNA was subjected to northern blot analysis. An aberrantly
sized erbB-2 mRNA was not detected in any of the cell lines
analyzed (Kraus et al., EMBO Journal 6:605-610, 1987). To
quantitate more precisely the amount of MAC117 transcript in eight
mammary tumor cell lines which overexpress MAC117, serial 2-fold
dilutions of total cellular RNA were subjected to dot blot analysis
using human .beta. actin as a control for the amount of RNA applied
to the nitrocellulose filters. The highest levels of MAC117 mRNA,
which ranged from 64- to 128-fold over that of our controls, were
observed in the cell lines MDA-MB453, SK-BR-3, MDA-MB361, and
BT474. Moreover, MAC117 mRNA levels were increased 4- to 8-fold in
four cell lines including BT483, MDA-MB175, ZR-75-30, and ZR-75-1
(FIG. 6).
To determine if the overexpression of MAC117 mRNA resulted in a
steady state increase of its encoded gene product, we developed a
specific immunoblot assay. Antisera were raised against a synthetic
peptide whose sequence corresponded to a portion of the putative
tyrosine kinase domain of MAC117. As this region is partially
conserved between the encoded proteins of the EGFR and MAC117
genes, we tested its specificity using A431 and SK-BR-3 cell lines
which overexpress EGFR or MAC117 mRNA, respectively. As shown in
FIG. 7A, a specific band of .about.185 kd was detected in extracts
of SK-BR-3 but not in A431 cells. This band was not detected when
the antibody was preincubated with the synthetic peptide
corresponding to its antigen. To estimate the relative amounts of
MAC117 protein in different mammary tumor cell lines, immunoblot
analysis was conducted using equivalent amounts of total cellular
protein. As shown in FIG. 7B, an intense band of protein was
detected in extracts of SK-BR-3 and a less intense but readily
detectable band in extracts of ZR-75-1. No MAC117 protein was
detected in extracts of MCF-7, a mammary tumor cell line, that did
not display overexpression of erbB-2 mRNA. We interpret these
results to indicate that substantially more erbB-2 protein is found
in both SK-BR-3 and ZR-75-1 than in MCF-7 cells where the amount of
protein escapes the sensitivity of the assay. These observations
indicted that elevated mRNA levels of MAC117 are translated into
MAC117 proteins. This demonstrated that gene amplification of
MAC117 results in overexpression of mRNA and protein of MAC117 in
human mammary tumor cells. Furthermore, increased mRNA and protein
levels are observed in mammary tumor cells in the absence of gene
amplification suggestive for an additional mechanism as a result of
which mRNA and protein of our novel v-erbB-related gene can be
found overexpressed in tumor cells (Kraus et al., 1987).
To directly assess the effects of MAC117 overexpression on cell
growth properties, we assembled a full length normal human MAC117
clone from overlapping cDNA clones (FIGS. 5A,B) linked to the
transcriptional initiation sequences of either the Moloney murine
leukemia virus long terminal repeat (MuLV LTR) or the SV40 early
promoter in expression vectors in order to express a normal coding
sequence of MAC117 in NIH3T3 cells (FIG. 9) (DiFiore et al.,
Science 237:178-182, 1987). Previous studies have indicated
different strengths of LTR and the SV40 promoters in these cells
(Gorman et al., Proc. Natl. Acad. Sci. USA 79, 6777, 1982). Because
of the presence of the MuLV donor splice site close to the 5' LTR
(Shinnick et al., Nature 293, 543, 1981), we engineered one of the
LTR-based vectors (LTR-1/MAC117) to contain an acceptor splice site
immediately upstream of the translation initiation codon of the
MAC117 coding sequence (FIG. 9). This vector was constructed in
order to ensure correct splicing of the message even if a cryptic
splice acceptor site were present within the MAC117 open reading
frame. In the SV40-based expression vector (SV40/MAC117) the erbB-2
coding sequence replaced the neomycin-resistance gene of pSV2/neo
(Southern et al., J. Mol. Appl. Genet. 1, 327, 1982) (FIG. 9). To
assess the biologic activity of our human MAC117 vectors, we
transfected NIH/3T3 cells with serial dilutions of each DNA. As
shown in Table 1, LTR-1/MAC117 DNAs induced transformed foci at
high efficiency of 4.1.times.10.sup.4 focus-forming units per
picomole of DNA (ffu/pM). In striking contrast, the SV40/erbB-2
construct failed to induce any detectable morphological alteration
of NIH/3T3 cells transfected under identical assay conditions
(Table 1). Since the SV40/erbB-2 construct lacked transforming
activity, these results demonstrated that the higher levels of
MAC117 expression under LTR influence correlated with its ability
to exert transforming activity. To compare the growth properties of
NIH/3T3 cells transfected by these genes, we analyzed the
transfectants for anchorage-independent growth in culture, a
property of many transformed cells. The colony-forming efficiency
of a LTR-1/MAC117 transformant was very high and comparable to that
of cells transformed by LTR-driven v-H-ras and v-erbB (Table 1).
Moreover, the LTR-1/MAC117 transfectants were as malignant in vivo
as cells transformed by the highly potent v-H-ras oncogene and
50-fold more tumorigenic than cells transfected with v-erbB. In
contrast, SV40/MAC117 transfectants lacked anchorage-independent
growth in vitro and did not grow as tumors in nude mice even when
10.sup.5 cells were injected (Table 1).
To compare the level of overexpression of the 185-kd protein
encoded by MAC117 in human mammary tumor cell lines possessing
amplified MAC117 genes with that of NIH/3T3 cells experimentally
transformed by the MAC117 coding sequence, we compared MAC117
specific protein amounts by Western Blotting (DiFiore et al.,
Science 237:178-182, 1987). An anti-MAC117 peptide serum detected
several discrete protein species ranging in size from 150 to 185 kd
in extracts of MDA-MB361 and SK-BR-3 mammary tumor cell lines, as
well as LTR/MAC117 NIH/3T3 transformants (FIG. 10). The relative
levels of the 185-kd MAC117 product were similar in each of the
cell lines and markedly elevated over that expressed by MCF-7
cells, where the 185-kd protein was not detectable under these
assay conditions (FIG. 10). Thus, human mammary tumor cells which
overexpressed the MAC117 gene demonstrated levels of the MAC117
gene product capable of inducing malignant transformation in a
model system.
Overexpression of proto-oncogenes can cause cell transformation in
culture and may function in the development of human tumors.
Amplification of a normal ras gene or its increased expression
under the control of a retroviral long terminal repeat (LTR)
induces transformation of NIH 3T3 cells (Chang et al., Nature
297:479, 1982). Expression of the normal human sis/PDGF-2 coding
sequence in NIH 3T3 cells, which do not normally express their
endogenous sis proto-oncogene, also leads to transformation (Gazit
et al., Cell 39:89, 1984; Clarke et al., Nature 308:464, 1984). In
Burkitt lymphoma, a chromosomal translocation involving myc places
its normal coding sequence under the control of an immunoglobulin
gene regulatory sequence. The resulting alteration in myc
expression is likely to be causally related to tumor development
(Nishikura et al., Science 224:399, 1984). The observation of
amplification of myc or N-myc in more malignant phenotypes of
certain tumors has supported the idea that overexpression of these
genes can contribute to the progression of such tumors. The
erbB/EGF receptor gene is amplified or overexpressed in certain
tumors or tumor cell lines. The five- to tenfold amplification of
the v-erbB-related gene of the present invention in a mammary
carcinoma indicates that increased expression of this gene may have
provided a selective advantage to this tumor. The isolation of a
new member of the tyrosine kinase gene family amplified in a human
mammary carcinoma in accordance with the present invention, makes
possible the elucidation of the role of this gene in human
malignancy.
Use of Specific Nucleic Acid Probes
As demonstrated in FIGS. 2 and 4, the isolation and use of a Bgl I
to Bam HI restriction fragment of pMAC117 to specifically detect
the gene and its mRNA product has been set forth. The importance of
this technique, involving this probe and others like it, is that
the biological functions of the gene described here can be
determined and these functions related to practical application,
some of which are listed below.
1. Isolation of cloned cDNA. This involves the use of probes
specific for the gene described herein; an example is the Bgl I-Bam
HI fragment of pMAC117. These probes are made radioactive by
standard techniques, such as those noted above, and screening of
the libraries of cDNA clones is done using standard methods
analogous to those described in "Cloning of .lambda.MAC117" above.
This approach was employed to clone cDNA comprising the entire
coding region of this gene, the restriction map of which is shown
in FIG. 5A.
2. Use of cDNA clones. Due to the fact that cDNA clones contain
complete information for encoding the protein, these cDNA clones
provide a "second generation" of specific probes for the gene
described herein. Such probes are shown in FIG. 5B. Their
application for hybridization analysis is demonstrated in FIG. 6
and FIG. 8. As shown in FIG. 8, the availability of probes, such as
probe c in FIG. 5B, facilitates the comprehensive hybridization
analysis of the entire coding region of this gene or any defined
part of it. In addition, the complete coding information allows the
expression of the protein product in a heterologous system. Such
systems utilize strong and/or regulated transcription promoters
placed in such a way as to direct overexpression of the gene.
Techniques for accomplishing expression of the gene are well known
in the art and can be found in such publications as Rosenberg et
al., Method sin Enzym. 101, 123 (1983); Guarante, L., Methods in
Enzym. 101, 181 (1983). The coding region of our novel
v-erbB-related gene was overexpressed under the transcriptional
control of MuLV-LTR or SV40 early promoter. Thereby, high
expression levels were achieved with MuLV-LTR which caused the
neoplastic transformation of transfected cells. These cells can be
used as a source to rescue infectious recombinant virus which might
prove useful to infect heterologous cells not susceptible to DNA
transfection. In addition, these cells serve as a source for high
and defined levels of antigen for this novel v-erbB-related
gene.
3. Preparation of antibodies specific for the protein product of
the gene. Of course, the identification and knowledge of the gene
allows its product, protein, for example, to be detected. Poly- or
monoclonal antibodies are prepared against said protein by standard
techniques, often by commercially available services. The critical
reagent in the production of antibodies is the antigen (protein)
used. In this case, the antigens are the peptides chemically
synthesized by standard and commercially available techniques
according to the predicted amino acid sequences derived from the
nucleic acid sequence of the gene or its corresponding cDNA.
Another potential antigen is the protein itself encoded by the gene
and purified from the heterologous expression systems as described
above. The antibodies are then employed by standard immunological
techniques for the specific detection or diagnostic purposes. Such
antibodies were raised against a peptide representing amino acids
35 through 49 of the peptide sequence: ##STR1## The specificity of
these antibodies in detecting the gene product of this novel.
v-erbB-related gene is demonstrated in FIG. 7A. As shown in FIG. 7B
and FIG. 10, these antibodies can be utilized to detect the
overexpression of the protein product of our novel v-erb-B-related
gene in human mammary tumor cells.
Further applications of the Gene
Having the knowledge of the gene allows preparing specific nucleic
acid probes to detect the gene described here or its mRNA product.
The probes are, of course, derived from the gene, such as the Bgl
I-Bam HI fragment of pMAC117 used in FIGS. 2 and 4, or
alternatively such probes are derived from other regions of the
gene or its corresponding cDNA corresponding cDNA, as shown in FIG.
5B. The use of nucleic acid probes in the molecular diagnosis of
human cancer has been documented (Taub et al., Proc. Natl. Acad.
Sci. USA 79, 7837 (1983); Schwab et al., Proc. Natl. Acad. Sci. USA
81, 4940 (1984)). The finding that the gene described here is
amplified in a human mammary carcinoma indicates that alterations
occur to this gene in human disease. Thus, detection of the
amplification or increased expression of this gene provides useful
diagnostic tools for the detection and treatment of human mammary
carcinoma or other malignancies resulting from the v-erbB related
gene. Hence, diagnostic kits which contain as their principal
component specific nucleic acid probes for this gene or its mRNA
transcript are of commercial value. The probe is used in analyses
similar in concept to those shown in FIG. 2 and FIG. 4 for the
detection of gene amplification structure or the expression of
mRNA.
Specific antibody reagents (as described above) capable of
detecting the protein product of the gene described herein are
employed in a way similar to the use of specific nucleic acid
probes. In other words, the expression of aberrant forms and
amounts of a gene product is a measure of the related neoplastic
condition (Nishikura et al., Science 224, 399 (1984); Srivastava,
et al., Proc. Natl. Acad. Sci. USA 82, 38-42 (1985)). The detection
of the aberrant expression of the protein product of the gene is of
importance in the diagnosis of human cancers. As shown in FIG. 7
and FIG. 10, antibodies generated against peptides derived from
parts of the amino acid sequence: ##STR2## specifically detect the
protein product of the gene having the nucleotide sequence:
##STR3## in human tumor cells. Antibody reagent (produced as
described above) is, of course, the critical reagent of the
diagnostic kits for this purpose. Such antibody reagents are then
employed in such standard methodologies as immunoprecipitation,
western blot analysis, immunofluorescence analysis and the like
well known in the art. The determination of amplification in a
human mammary carcinoma of the gene described here indicates that
overexpression (or other abnormality) of the protein product of
this gene is functionally important, thus diagnostically relevant.
This relevance is further substantiated by the observations that
gene amplification of this gene is associated with overexpression
of its mRNA and protein in human mammary tumor cells and that
protein levels observed in human mammary tumor cell lines
exhibiting gene amplification of this gene are sufficient to induce
neoplastic transformation of NIH/3T3 cells in vitro. Furthermore, a
recent report (Slamon et al., Science 235:177-181, 1987) correlates
gene amplification of this novel erbB-related gene with a reduced
disease free survival in breast cancer patients, suggesting the
potential usefulness of analysis of this gene for its gene product
as a diagnostic parameter in the clinical setting management of
breast cancer patients.
A diagnostic test in accordance with the present invention
involves, for example, material obtained by surgical biopsy of
potential tumor material. Such material is then analyzed by one or
more procedures as follows.
1. DNA is isolated from the sample by standard methods (see above).
The DNA is then analyzed by established methods, such as Southern
blot hybridization using standard techniques similar to those used
in the analysis shown in FIG. 2. Gene-specific probes (described
above) are made radioactive by standard techniques and used for
detecting genetic abnormalities. Such abnormalities include gene
amplification, as seen in the MAC117 tumor sample and tumor cell
lines in FIG. 8, or gene rearrangement, as detected by aberrantly
migrating bands of hybridization.
2. RNA is isolated from the tumor sample by standard methods (see
above). This RNA is analyzed by blot hybridization techniques
similar to those described in FIG. 4. Gene-specific probes
(described above) are made radioactive by standard techniques and
used for detecting the mRNA products of the erbB-related gene
described here. Such abnormalities include overexpression or
abnormal forms of RNA. Overexpression of an apparently normal sized
mRNA is shown in 8 human mammary tumor cell lines in FIG. 6. In
addition, mRNA amount may also be quantitated by spot hybridization
procedures in which serial dilutions of RNA are fixed to
nitrocellulose filter and the mRNA of v-erb-B-related gene
described here detected by hybridization. Such a procedure has been
employed in FIG. 6B. The foregoing techniques are standard. This
allows detection of mRNA overexpression or alteration of
structure.
When antigens or proteins (polypeptides) are to be analyzed, the
proteins are separated according to molecular size, for example by
gel electrophoresis, transferred to nitrocellulose membranes and
the protein product of the erbB-related gene described here
detected by reaction with specific antibodies, described above.
Such a test is ale to detect alterations in the quantity of protein
as well as abnormal protein forms. With such an approach protein
levels of the v-erb-B-related gene have been detected in human
mammary tumor cell lines (FIG. 7, FIG. 10).
In addition, specific antibodies may be used in the analysis of
histological sections. These techniques, which are well known for
other antibody specificities, involve the thin sectioning of
biopsied material from a potential tumor, followed by reaction with
specific antibodies. The antibody-antigen reaction is then made
visible by a variety of standard methods including labeling with
fluorescently tagged or ferritin tagged second anitsera and the
like. Such detection systems allow the detection of the localized
aberrant display of the protein product of the erbB-related gene
described here.
In addition, although the demonstrated genetic abnormality (shown
in FIG. 2) of the gene described here occurs in human mammary
carcinoma, genetic abnormalities may also be associated with other
clinically important syndromes of neoplastic or other origin.
Genetic abnormalities have long been known to be involved in
thalassemias, for example.
Knowledge of the erbB-related gene described here also makes
possible a means of cancer treatment. If it is found that some
cancers display abnormally high quantities of the gene product on
their surface, such tumors can be treated with antibodies specific
for the gene product which have been conjugated to a toxic
substance, such as radioactive markers, biological modifiers or
toxins and the like. Another treatment modality involves a similar
assumption of overexpression. In this approach, a specific natural
product, even if unidentified but which has high binding affinity
for the protein product of the gene described here is used to
target toxins to the tumor cells. This treatment modality is
supported by the finding, reported here, of distinct but limited
homology of this gene product to the EGF receptor. If a ligand
analogous to EGF exist for the erbB-related gene described here, it
may serve as such a targeting agent.
Diagnostic kits for the detection of the protein product of the
erbB-related gene. Kits useful for the diagnosis of human cancers
having abnormalities of this gene are now disclosed.
a) Kits designed to detect the protein by immunoblotting These kits
preferably comprise containers containing (a) homogenization
solution (50 mM Tris-HCl pH 7.5, 1% sodium dodecyl sulfate and 0.1%
.beta.-mercaptoethanol) for the extraction of protein sample from
biopsied material from putative tumors; (b) reagents for the
preparation of immunoblots of the protein samples (acrylamide gels
are prepoured and contain 7.5% acrylamide, 0.025% bis acrylamide,
0.38M Tris-HCl pH 8.8, 0.1% sodium dodecyl sulfate; the
nitrocellulose sheets will be formed to the gel size; and transfer
buffer 0.25M Tris-glycine pH 8.8, 30% methanol); specific antibody
reagents for the detection of the protein product of the
erbB-related gene (antisera directed against the protein product of
erbB-related gene described here and reaction buffer containing
0.1M Tris-HCl pH 7.5, 5.0M EDTA, 0.25% gelatin, 0.1% nonidet P-40);
and reagents and instructions for the visualization and
interpretation of antibody-antigen interaction (these include
radioactive protein A; biotin conjugated second antiserum, or
peroxidase conjugated second antiserum). While this kit includes
components ordinarily found and well known in the art, the critical
component is the gene product-specific antibodies and buffers or
media for performing immunological tests. The antibodies are
derived or prepared as described above from either the peptide
sequence predicted from the nucleotide sequence of the gene or its
mRNA or from the protein product itself through standard
immunization procedures.
b) Kits designed for the detection of the protein product of the
erbB-related gene in tissue sections. Such kits include
instructions for preparation of sections; instructions and standard
reagents for the preparation of slides for microscopy; H.sub.2
O.sub.2 for removal of endogenous peroxidase; instructions for
incubation with antibodies specific for the protein product of the
erbB-related gene described here in a buffer solution preferably
containing phosphate buffered saline; and second antibodies for
detection (these may be coupled to peroxidase, biotin, or
ferritin); and instructions for visualization of detection complex.
In addition the kits may include: reagents and instructions for the
preparation of sections from biopsied putative tumor material;
specific antibody reagents for the protein product of erbB-related
gene described here and instructions for its reaction with the
tissue section; and reagents and instructions for the detection of
the protein-antibody interaction either by immunofluorescence,
ferritin conjugated second antibodies or other standard methods
well known in the art.
A method for the Treatment of Human Cancers which Express High
Levels of the Protein Product of the Gene described Herein
This method involves administering to the patient one of two types
of reagent which preferentially binds cells expressing high levels
of the protein product of the erbB-related gene described here.
These reagents are either antibodies directed against the protein
product or a ligand, which is likely to exist because of the
homology of the gene to a growth factor receptor. The ligand is
isolated by standard techniques using the intrinsic protein kinase
activity of the protein product of the erbB-related gene. Extracts
of body fluids and cell culture supernatants are incubated with the
protein and .gamma.-.sup.32 P ATP. The presence of ligand is
inferred by incorporation of .sup.32 P into the protein. The ligand
is then purified by standard techniques such as ion exchange
chromatography, gel permeation chromatography, isoelectric
focusing, gel electrophoresis and the like. The natural ligand or
antibody is tagged with one or more agents which will cause injury
to cells to which they bind. Such tagging systems include
incorporation of radioactive or biological toxins. The present
discovery of amplification of the erbB-related gene makes it likely
that some tumors carry large amounts of the corresponding protein.
Hence, the two type-specific agents will bind in larger amounts to
the protein present in the body and thus direct the toxic effects
of the reagents to these cells.
It is understood that the examples and embodiments described herein
are for illustrative purposes only and that various modifications
or changes in light thereof will be suggested to persons skilled in
the art and are to be included within the spirit and purview of
this application and the scope of the appended claims.
Table 1 compares transformation characteristics of NIH/3T3 cells
transfected with vectors generating different expression levels of
the MAC117 coding sequence.
TABLE 1 ______________________________________ Cell number Specific
Colony-forming required for DNA transforming efficiency in 50%
tumor transfectant.sup.a activity.sup.b (ffu/pM) agar (%).sup.c
incidence.sup.d ______________________________________ LTR-1/MAC117
4.1 .times. 10.sup.4 45 10.sup.3 SV40/MAC117 <10.sup.0 <0.01
>10.sup.6 LTR/erbB 5.0 .times. 10.sup.2 20 5 .times. 10.sup.4
LTR/ras 3.6 .times. 10.sup.4 35 10.sup.3 pSV2/gpt <10.sup.0
<0.01 >10.sup.6 ______________________________________ .sup.a
All transfectants were isolated from plates which received 1 .mu.g
cloned DNA and were selected by their ability to grow in the
presence of killer HAT medium (Mulligan et al., Proc. Natl. Acad.
Sci. U.S.A. 78, 2072, 1981). .sup.b Focusforming units were
adjusted to ffu/pM of cloned DNA added based on the relative
molecular weights of the respective plasmids. .sup.c Cells were
plated at 10fold serial dilutions in 0.33% soft agar medium
containing 10% calf serum. Visible colonies comprising >100
cells were scored at 14 days. .sup.d NFR nude mice were inoculated
subcutaneously with each cell line. Ten mice were tested at cell
concentrations ranging from 10.sup.6 to 10.sup.3 cells/mouse. Tumor
formation was monitored at least twice weekl for up to 30 days.
* * * * *