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Methods of detection and treatment of breast cancer

Abstrict

Novel methods of detecting and treating breast cancer are described.

Claims

What is claimed is:

1. A method of inhibiting breast cancer cell growth comprising supplying Csk Homologous Kinase to breast cells whereby breast cancer cell growth is inhibited.

2. The method of claim 1, wherein a nucleic acid sequence encoding Csk Homologous Kinase, or a biologically active fragment, analog or derivative thereof, is supplied to the breast cells, wherein the nucleic acid sequence encoding Csk Homologous Kinase or a biologically active fragment, analog or derivation thereof, is expressed in the breast cell.

3. The method of claim 2 wherein Csk Homologous Kinase is supplied to the cell by a nucleic acid construct containing a nucleic acid sequence encoding Csk Homologous Kinase or a biologically active fragment, analog or derivative thereof, wherein the construct is introduced into the breast cell and Csk Homologous Kinase is expressed.

4. The method of claim 1, wherein Csk Homologous Kinase protein or a biologically active Csk Homologous Kinase peptide is supplied to breast cells.

5. The method of claim 4, wherein the Csk Homologous Kinase protein or peptide is supplied to the cell by introducing into the cell a liposome containing the Csk Homologous Kinase or Csk Homologous Kinase peptide.

6. The method of claim 5, wherein the liposome is contained in a topical formulation and is introduced to the breast cells by topical application.

7. A Csk Homologous Kinase derivative produced by altering native or variant Csk Homologous Kinase DNA within the Csk Homologous Kinase SH2 domain to optimize its association with ErbB-2.

8. The derivative of claim 7, wherein the mutation is a substitution of one or more amino acids between amino acid 127 and 150 of SEQ ID NO: 1.

9. The derivative of claim 7, wherein the mutation is in an amino acid residue conserved between csk and c-src, but not conserved between csk and Csk Homologous Kinase.

10. The derivative of claim 9, wherein the mutation is a substitution of amino acid 129 of SEQ ID NO: 1.

11. The derivative of claim 9, wherein the mutation is a substitution from said residue to the corresponding residue conserved in csk and c-src.

12. A derivative of Csk Homologous Kinase comprising a mutation which is a substitution of one or more amino acids between amino acid 127 and 150 of SEQ ID NO: 1.

13. The derivative of claim 12, wherein the mutation is in an amino acid residue conserved between csk and c-src, but not conserved between csk and Csk Homologous Kinase.

14. The derivative of claim 12, wherein the mutation is a substitution of amino acid 129 of SEQ ID NO: 1.

15. The derivative of claim 12, wherein the mutation is a substitution from said residue to the corresponding residue conserved in csk and c-src.

16. The derivative of claim 12, wherein the mutation is in an amino acid residue conserved between csk, c-src and Csk Homologous Kinase.

17. The derivative of claim 12 wherein the mutation is a substitution of amino acid 147 of SEQ ID NO: 1

18. A Csk Homologous Kinase derivative produced by altering native or variant Csk Homologous Kinase DNA within the Csk Homologous Kinase SH2 domain to inhibit its association with ErbB-2.

19. A CHK analog that mimics the binding of the derivative of claim 7.

20. A CHK analog that mimics the binding of the derivative of claim 12.

21. A pharmaceutical composition that contains an effective amount of the CHK derivative of claim 7.

22. A pharmaceutical composition that contains an effective amount of the CHK derivative of claim 12.

23. A method of identifying a biologically active CHK analog with greater biological activity than native CHK, comprising: a. contacting ErbB-2 with the CHK analog to be tested for biological activity, under conditions sufficient for binding of ErbB-2 by the CHK analog; b. contacting ErbB-2 with native CHK, under conditions sufficient for binding of ErbB-2 by the native CHK; and c. comparing the binding of ErbB-2 by the CHK analog with the binding of ErB-2 by the native CHK.

24. The method of claim 23, wherein the CHK analog is the analog of claim 19.

25. The method of claim 23, wherein the CHK analog is an analog of claim 20.

26. The CHK analog identified by the method of claim 23.

27. A method of identifying a biologically active CHK derivative with greater biological activity than native CHK, comprising: a. contacting ErbB-2 with the CHK derivative to be tested for biological activity, under conditions sufficient for binding of ErbB-2 by the CHK derivative; b. contacting ErbB-2 with native CHK, under conditions sufficient for binding of ErbB-2 by the native CHK; and c. comparing the binding of ErbB-2 by the CHK derivative with the binding of ErB-2 by the native CHK.

28. The method of claim 27, wherein the CHK derivative is the derivative of claim 7.

29. The method of claim 27, wherein the CHK derivative is the derivative of claim 12.

30. The CHK derivative identified by the method of claim 27.

31. A CHK analog that binds to ErbB-2 and enhances the activity of CHK.

32. A pharmaceutical composition that comprises an effective amount of a CHK analog, wherein the CHK analog binds to ErbB-2 and enhances the activity of CHK.

Description

RELATED APPLICATIONS

[0001] This application is a Continuation of U.S. Ser. No.: 09/315,928, filed May 20, 1999, which is a Divisional of U.S. Ser. No.: 08/876,882, filed Jun. 16, 1997, which claims priority to pending Provisional Application No. 60/035,228, filed Jan. 8, 1997, the entire teachings of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0003] Breast cancer is the second leading cause of cancer death among women in the United States and is the leading cause of death among women aged 30-70. (Abeloff, M. D., Curr. Opin. Oncol., 8:447-448 (1996)). The inheritance of germ-line mutations in autosomal dominant susceptibility genes appears to be responsible for 5-10% of all breast cancer cases (Fitzgerald, M. G., et al., New Engl. J. Med., 334:143-149 (1996)), and up to 36% of the cases diagnosed before age 30. BRCA1 was the first isolated breast cancer susceptibility gene (Langston, A. A., et al., New Engl. J. Med., 334:137-142 (1996); Couch, F. J. and Weber, B. L., Hum. Mutat., 8:8-18 (1996)) and mutations in BRCA1 alone account for approximately 45% of the families with high incidence of breast and ovarian cancer (Chen, Y. M., et al., Science, 272:125-126 (1996); Sully, R., et al., Science, 272:123-126 (1996)). In addition, a second breast cancer susceptibility gene, BRCA2, has been isolated recently (Wooster, R., et al., Nature, 378:789-792 (1995); Tavtigian, S. V., et al., Nat. Genet., 12:333-337 (1996)).

[0004] However, the majority of breast carcinomas appear to be sporadic and have a complex accumulation of molecular and cellular abnormalities that constitute the malignant phenotype. A number of somatic gene alterations, such as loss of expression of specific tumor suppressor genes, have been found to occur in primary human breast tumors (Borg, A., et al., Cancer Res., 52:2991-2994 (1992); Eeles, R. A., et al., Cancer Surveys, 25:101-124 (1995)). Additionally, there is considerable evidence that genetic alterations in growth factor signaling pathways can contribute to human breast malignancies. In this regard, activation of different proto-oncogenes has been found in primary breast tumor (Berns, E. M., et al., Cancer Res., 52:1036-1039 (1992); Borg, A., et al., Brit. J. Cancer, 63:136-142 (1991); Gullick, W. J., et al., Brit. J. Cancer, 63:434-438 (1991)). Thus, there is considerable importance in identifying, at a molecular level, factors that contribute to the progression from normal growth towards malignancy.

SUMMARY OF THE INVENTION

[0005] The present invention relates to the demonstration that a cytoplasmic protein tyrosine kinase, Csk Homologous Kinase or CHK, is expressed in human breast cancer, but not in adjacent normal breast tissue. Specifically, the present invention relates to methods of detecting the presence of cancer in mammalian breast tissue by the detection of the protein tyrosine kinase CHK, or the detection of nucleic acids encoding the CHK in mammalian tissue, specifically breast tissue. The detection of CHK in breast tissue is indicative of cancer.

[0006] The presence of CHK in breast tissue can be determined by detecting the expression of CHK protein, or a protein fragment, in breast tissue samples obtained from the mammal. For example, biopsy tissue can be obtained from the mammal, fixed in a suitable medium and contacted with anti-CHK antibodies, for example rabbit anti-CHK, which specifically bind to the CHK protein if it is present in the tissue sample. The anti-CHK antibody can itself be detectably labeled, or a detectably labeled second antibody, for example, peroxidase-conjugated mouse anti-rabbit antibody, can be used.

[0007] The presence of CHK in breast tissue can also be detected using an immunoblot (e.g., Northern blot) assay. For example, tissue can be obtained from the mammal and a cell lysate prepared which contains proteins released from the tissue cells. The lysate proteins can be separated by electrophetic means, such as by size by SDS polyacrylamide gel electrophoresis, and contacted with anti-CHK antibody which specifically binds to CHK if it is present in the lysate. Again, the anti-CHK antibody can be detectably labeled, or a detectably labeled second antibody can be used. Alternatively, CHK protein present in a cell lysate can be detected by enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or other immunoassays.

[0008] The presence of CHK in breast tissue can also be determined by detecting the presence of a nucleic acid sequence encoding all, or a portion, of the CHK protein. The nucleic acid can be DNA or RNA. For example, genomic DNA, cDNA or RNA can be obtained from a sample of breast tissue and contacted with a polynucleotide (a nucleic acid probe) that forms a stable hybrid with the nucleic acid sequence encoding CHK. The probe can be detectably labeled. The DNA or RNA obtained from the mammal can be amplified prior to assay, for example using the polymerase chain reaction (PCR) or the ligase chain reaction (LCR), using specific nucleic acid primers. Primers useful to amplify the CHK nucleic acid specifically hybridize to the CHK nucleic acid or to nucleic acid sequence that flanks the target CHK nucleic acid sequence region.

[0009] Overexpression of the receptor tyrosine kinase, ErbB-2 (also termed neu/HER-2) has been associated with the development of breast cancer. (Slamon, D. J., et al., Science, 244:707-712 (1989); Olsson, H., et al., J. Natl. Cancer Instit., 83:1483-1487 (1991)). A common pathway linking the activation mechanisms in ErbB-2 amplification in breast cancer is increased tyrosine kinase activity which leads to cellular transformation. The abundance of ErbB-2 receptors and their ligands (e.g., heregulin or HGR) in breast cancer points to a functional role in the pathogenesis of this malignancy. As demonstrated herein, CHK specifically interacts with activated ErbB-2 upon HGR stimulation and results described herein suggest that CHK functions as a negative regulator of ErbB-2 mediated mitogenic signaling.

[0010] Accordingly, the present invention also encompasses methods of inhibiting breast cancer cell growth (also referred to herein as neoplastic cell growth), specifically ErbB-2 mediated neoplastic cell growth, by supplying CHK to cancer cells. For example, CHK protein, peptide or a biologically active fragment thereof, or a CHK analog or derivative, can be supplied to mammalian breast tissue which is abnormal, e.g., neoplastic, or at risk of becoming abnormal. The CHK protein can be supplied to the target breast tissue by introducing into target cells a liposome preparation that contains CHK. Specifically encompassed by this invention is the topical application of such liposomes in a cream or ointment.

[0011] Alternatively, CHK can be supplied to the target tissue by introducing a nucleic acid sequence encoding CHK, or a biologically active fragment, analog, or derivative of CHK which is then expressed in the breast tissue.

[0012] As described herein, for the first time, Csk-homologous Kinase has been identified as playing an important role in signaling in neoplastic breast tissue and as functioning as a negative regulator of ErbB-2. As a result of this work, novel methods of detecting and inhibiting breast cancer are now available.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIGS. 1A-D depict autoradiographs showing the association of the CHK-SH2 domain with ErbB-2 upon stimulation with HRG. Lysates were precipitated with the CHK-SH2 GST fusion protein and immunoblotted with monoclonal antiphosphotyrosine antibody (PY20), (A), or with polyclonal anti-ErbB-2 antibodies (B). Immunoprecipitations of the same lystates were performed using 3E8 monoclonal anti-ErbB-2 antibody and blotted with PY20 (C) or with anti-ErbB-2 antibodies (D). Molecular size markers are indicated on the left (kDa).

[0014] FIGS. 2A-B show the nucleotide (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2) of two overlapping matk cDNA clones representing the full-length cDNA. Nucleotide numbers are shown on the left. The putative initiation codon at nucleotide position 263 is shown in bold type.

[0015] FIG. 3 shows the nucleotide sequence (SEQ ID NO: 3) and deduced amino acid sequence (SEQ ID NO: 4) of a CHK fragment. Specific primer positions are indicated.

[0016] FIG. 4 is a graphic representation showing the results of an in vitro kinase assay of CHK and Csk immunoprecipitates.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The family of protein tyrosine kinases (PTKs) includes oncogenes and growth factor receptors, several of which have been linked to the pathogenesis and progression of certain cancers (Bishop, J. M., Genes. Dev., 9:1309-1315 (1995) Cancer, W. G., et al., Breast Can. Res. & Treat., 35:105-1114 (1995)). Increasing evidence indicates that the c-src proto-oncogene may play an important role in breast cancer. Human breast cancers often show much higher levels of src protein kinase activity than normal adjacent epithelium (Hennipman, A., et al., Cancer Research, 49:516-521 (1989), Ottenhoff-Kalff, A. E., et al, Cancer Research, 52:4773-4778 (1992)). Indeed, about 70% of the elevated total tyrosine kinase activity found in primary breast cancers can be attributed to increased src activity. Involvement of pp60c-src with two major signaling pathways in human breast cancer has been demonstrated. In human breast carcinoma cell lines, the SH2 domain of src binds to activated epidermal growth factor (EGF-R) and p185.sup.ErbB-2, a receptor tyrosine kinase (Luttrell, D. K., et al., Proc. Natl. Acad. Sci. USA, 91:83-87 (1994)).

[0018] Overexpression of the receptor tyrosine kinase ErbB-2 (also termed neu/HER-2) has been also associated with the development of breast cancer (Salmon, D. J., et al., Science, 244:707-712 (1989), Williams, T. M., et al., Pathobiology, 59:45-52 (1991)). A common pathway linking the activation mechanisms in ErbB-2 amplification in breast cancer is increased tyrosine kinase activity which leads to cellular transformation (Olsson, H., et al., J. Natl. Cancer. Inst., 83:1483-1487 (1991)).

[0019] Four members of the ErbB (HER) family are presently know: p170.sup.ErbB-1 (epidermal growth factor receptor, EGR-R), p185.sup.ErbB-2, p180.sup.ErbB-3 and p180.sup.ErbB-4. In particular, the overexpression of the p185.sup.ErbB-2 correlates with a poor clinical prognosis in breast cancer (Beerli, R. R., et al., Mol. Cell. Biol., 15:6496-6505 (1995), Holmes, W. E., et al., Science, 256:1205-1210 (1992), Wen, D., et al., Cell, 69:559-572 (1992)). The overall amino acid homology within this receptor family ranges from 40-50%, and all the family members are characterized by two cysteine-rich regions in the extracellular domain, a single transmembrane region and a large cytoplasmic domain that exhibits tyrosine kinase activity (Wen, D., et al., Cell, 69:559-572 (1992)).

[0020] Several ligands that bind to and stimulate the kinase activity of the ErbB family members have been identified and are classified as EGF-like ligands. EGF, HB-EGF, amphiregulin, betacellulin, epiregulin and transforming growth factor-.alpha. (TGF-.alpha.) are the ligands for the EGF-R (ErbB-1) (Cohen, B. D., et al., J. Biol. Chem., 271:4813-4818 (1996), Johnson, G. R., et al., J. Biol. Chem., 268:2924-2931 (1993)). Heregulin (HRG) and its rat homologue, neu differentiation factor (NDF), are a subfamily of neruegulins which are EGF-like ligands that bind to and activate both ErbB-3 and ErbB-4. Although none of these factors binds directly to the ErbB-2, both EGF and HRG induce its tyrosine phosphorylation, presumably by ligand-driven heterodimerization and cross-phosphorylation. Interestingly, ErbB-2, by heterodimerizing with the EGF-R and ErbB-3, confers high affinity binding sites for EGF and HRG, respectively (Beerli, R. R., et al., Mol. Cell. Biol., 15:6469-6505 (1995), Marchionni, M. A., et al., Nature, 362:312-318 (1993)).

[0021] Recently, a cytoplasmic tyrosine kinase, CHK (Csk Homologous Kinase), previously referred to as MATK (Megakaryocyte Associate Tyrosine Kinase), has been identified. The CHK protein, primarily expressed in hematopoietic cells and in human brain has an apparent molecular weight of 58 kD, and shares 50% homology with the human Csk (c-terminal src kinase). Like Csk, CHK contains SH3, SH2 and tyrosine kinase domains, and lacks the src family N-terminal myristylation and autophosphorylation sites. CHK was found to phosphorylate the inhibitory carboxyl-terminal conserved tyrosine of several src-related enzymes in vitro, including Lck, Fyn and c-src, and to reduce the elevated phosphotyrosine levels of src family kinases in Csk-deficient fibroblasts.

[0022] As described herein, for the first time, the interaction of CHK with ErbB-2 upon the activation of breast cancer cells by HRG has been demonstrated. This interaction occurred via the SH2 domain of CHK and was specific to the activated ErbB-2 receptor upon HRG stimulation. Also described herein for the first time, is the demonstration that CHK is expressed in human breast cancer cells but not in adjacent normal breast tissue cells.

[0023] The present invention relates to methods of detecting and treating breast cancer in mammals wherein the methods encompass the detection or use of CHK protein, or nucleic acids encoding CHK. As defined herein, the term CHK protein encompasses the full-length CHK protein as described in Bennett, B. D., et al., J. Biol. Chem., 269::1068-10741 (1995), the teachings of which are hereby incorporated by reference, and also biologically active CHK fragments, derivatives analogs, variants and mutants.

[0024] The term "biologically active" CHK fragments, derivatives analogs, variants and mutants is defined herein as the activity encompassing the specific association of CHK with the intracellular domain of ErbB-2, or chimeric ErbB-2 molecules, such as EGF-ErbB-2 molecules. As described herein, this association is mediated by the SH2 domain of CHK. Association of CHK with ErbB-2 can be demonstrated, for example, using immunoprecipation experiments as described in the Examples. Because CHK is a tyrosine kinase, biological activity is also defined herein as the ability of CHK to phosphorylate tyrosine, specifically the phosphorylation of the carboxyl-terminal tyrosine of src-related kinases, thereby repressing their activity. Several src-related kinases include Lck, Fyn and c-src. Assays that demonstrate the phosphorylation ability of CHK include immune complex kinase reactions and the ability to phosphorylate kinases in yeast co-expression systems as described in (Avraham, S., et al., J. Biol. Chem., 270:1833-1842 (1995), Chow, L. M., et al., Oncogene, 9:3371-3374 (1994), Klags, S., et al., Proc. Natl. Acad. Sci. USA, 19:2597-2601 (1994) and Davidson, D., et al., J. Bil. Chem., 272:1355-1362 (1997)), the teachings of which are hereby incorporated by reference. Other methods of measuring kinase activity are known to those of skill in the art.

[0025] Another biological activity of CHK is the antigenic property of inducing a specific immunological response as determined using well-known laboratory techniques. For example, a biologically active CHK can induce an immunological response which produces antibodies specific for CHK (anti-CHK antibodies).

[0026] To be "functionally" or "biologically active" a CHK protein fragment, analog, mutant or derivative typically shares substantial sequence (amino acid or nucleic acid) identity (e.g., at least about 65%, typically at least about 80% and most typically about 90-95%) with the corresponding sequences of endogenous, or naturally occurring, CHK and possesses one or more of the functions of endogenous CHK thereof. For example, a biologically active CHK fragment typically shares sequence homology with endogenous CHK protein in the domains, e.g., tyrosine kinase domain, or SH2 domain, important for biological activity.

[0027] CHK of the present invention is understood to specifically include CHK proteins having amino acid sequences analogous to the sequence of the endogenous CHK. Such proteins are defined herein as CHK analogs. An "analog" is defined herein to mean an amino acid sequence with sufficient identity to the amino acid sequence of endogenous CHK protein to possess the biological activity of the protein. For example, an analog of a polypeptide can be introduced with "silent" changes in the amino acid sequence wherein one or more amino acid residues differ form the amino acid sequence of CHK, yet possess e.g., kinase activity or associates with ErbB-2. Examples of such differences include additions, deletions or substitutions of residues. Also encompassed by the present invention are proteins that exhibit greater or lesser biological activity of CHK protein.

[0028] The present invention also encompasses biologically active fragments of CHK protein. Such fragments can include only a part of the full-length amino acid sequence of CHK yet possess iological activity. As used herein, a "biologically active fragment" means a fragment that can exert a biological or physical effect of the full-length protein, or has a biological characteristic, e.g., antigenicity, of the full-length protein. The antigenicity of a peptide fragment can be determined, for example, as described in Geysen, et al., WO 84/03564, the teachings of which are herein incorporated by reference. Such activities and characteristics are described above. Such fragments can be produced by amino and carboxyl terminal deletions as well as internal deletions. Also included are active fragments of the protein as obtained by enzymatic digestion. Such peptide fragments can be tested for biological activity as described herein.

[0029] "Derivatives" and "variants" of CHK are CHK proteins that have been modified. They include CHK proteins that have been modified by alterations in their amino acid sequence. They also include truncated and hybrid forms of CHK. "Truncated" forms are shorter versions of CHK, typically modified so as to remove the C-terminal regions which effect binding or secretion. "Hybrid" or "chimeric" forms are CHK proteins that are composed of one or more CHK proteins combined with one or more other proteins, such as another kinase.

[0030] Variants can be produced using methods discussed below. The CHK gene can be mutated in vitro or in vivo using techniques well known to those of skill in the art, for example, site-specific mutagenesis and oligonucleotide mutagenesis. Manipulations of the CHK protein sequence can be made at the protein level as well. Any of numerous chemical modifications can be carried out by known techniques including, but not limited to, specific chemical cleavage by cyanogen bromide, trypsin and papain. It can also be structurally modified or denatured, for example, by heat or by being immobilized on a solid surface.

[0031] The amino acid sequences of the CHK proteins of the present invention can be altered to optimize CHK association with ErbB-2 by methods known in the art by introducing appropriate nucleotide changes into native or variant DNA encoding the CHK, or by in vitro synthesis of the desired CHK. Alterations can be created outside or within the CHK SH2 domain.

[0032] In general, mutations can be conservative or non-conservative amino acid substitutions, amino acid insertions or amino acid deletions. The mutations can be at or near (within 5 or 10 amino acids) the SH2 binding domain. More preferably, DNA encoding an CHK amino acid sequence variant is prepared by site-directed mutagenesis of DNA that encodes a variant or a nonvariant version of CHK. Site-directed (site-specific) mutagenesis allows the production of CHK variants through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.

[0033] Typically, a primer of about 20 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered. In general, the techniques of site-specific mutagenesis are well known in the art, as exemplified by publications such as Edelman et al., DNA 2, 183 (1983). The site-specific mutagenesis technique typically employs a phage vector that exists in both a single-stranded and double-stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage, for example, as disclosed by Messing et al., Third Cleveland Symposium on Macromolecules and Recombinant DNA, A. Walton, ed., Elsevier, Amsterdam (1981). This and other phage vectors are commercially available and their use is well-known to those skilled in the art. A versatile and efficient procedure for the construction of oligonucleotide directed site-specific mutations in DNA fragments using M13-derived vectors was published by Zoller, M. J. and Smith, M., Nucleic Acids Res. 10:6487-6500 (1982)). Also, plasmid vectors that contain a single-stranded phage origin of replication can be employed to obtain single-stranded DNA. Veira et al., Meth. Enzymol. 153:3 (1987). Alternatively, nucleotide substitutions can be introduced by synthesizing the appropriate DNA fragment in vitro, and amplifying it by PCR procedures known in the art.

[0034] In general, site-specific mutagenesis can be performed by first obtaining a single-stranded vector that includes within its sequence a DNA sequence that encodes the relevant protein. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example, by the method of Crea et al., Proc. Natl. Acad. Sci. USA 75, 5765 (1978). This primer can then be annealed with the single-stranded protein sequence-containing vector, and subjected to DNA-polymerizing enzymes such as E. coli polymerase I Klenow fragment, to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector can then be used to transform appropriate host cells such as JM 101 cells, and clones can be selected that include recombinant vectors bearing the mutated sequence arrangement. Thereafter, the mutated region can be removed and placed in an appropriate expression vector for protein production.

[0035] The PCR technique can also be used in creating amino acid sequence variants of an CHK. When small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template. For introduction of a mutation into a plasmid DNA, one of the primers can be designed to overlap the position of the mutation and to contain the mutation; the sequence of the other primer is preferably identical to a stretch of sequence of the opposite strand of the plasmid, but this sequence can be located anywhere along the plasmid DNA. It is preferred, however, that the sequence of the second primer is located within 500 nucleotides from that of the first, such that in the end the entire amplified region of DNA bounded by the primers can be easily sequenced. PCR amplification using a primer pair like the one just described results in a population of DNA fragments that differ at the position of the mutation specified by the primer, and possibly at other positions, as template copying is somewhat error-prone.

[0036] If the ratio of template to product material is extremely low, the vast majority of product DNA fragments incorporate the desired mutation(s). This product can be used to replace the corresponding region in the plasmid that served as PCR template using standard DNA technology. Mutations at separate positions can be introduced simultaneously by either using a mutant second primer or performing a second PCR with different mutant primers and ligating the two resulting PCR fragments simultaneously to the vector fragment in a three (or more) part ligation.

[0037] Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al. Gene 34, 315 (1985). The starting material can be the plasmid (or vector) comprising the CHK DNA to be mutated. The codon(s) within the CHK to be mutated are identified. There must be unique restriction endonuclease sites on each side of the identified mutation site(s). If such restriction sites do not exist, they can be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the CHK DNA. After the restriction sites have been introduced into the plasmid, the plasmid is cut at these sites to linearize it. A double stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 3' and 5' ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated CHK DNA sequence, that can be expressed to produce CHK with altered binding activity.

[0038] Specifically encompassed by the present invention are methods of detecting the presence or absence of CHK protein in mammalian cells wherein detection of the presence of (e.g., the expression of) CHK is indicative of breast cancer. A biological sample to be tested for the presence or absence of CHK protein is obtained from the mammal. Typically, the sample is breast tissue or tissue adjacent to the breast. The tissue sample can include lymph nodes. The sample is typically obtained by biopsy techniques well known to those of skill in the art.

[0039] CHK protein expression can be detected in a tissue sample by immunohistochemical techniques as described herein. For example, the tissue can be imbedded in paraffin or frozen and sectioned in to thin slices, typically mounted on microscope slides. The tissue is contacted with an anti-CHK antibody under conditions suitable for the anti-CHK antibody to specifically bind to CHK present in the tissue sample as described herein. The anti-CHK antibodies can be monoclonal or polyclonal. The antibody can be detectably labeled, for example, with a flourescent dye. Alternatively, a second antibody that is detectably labeled can be used. For example, if the first antibody is a mouse anti-CHK antibody, a second antibody can be detectably labeled rabbit anti-mouse. Techniques for producing, purifying and labeling antibodies are well-known to those of skill in the art.

[0040] Expression of CHK protein can also be detected by Western blot (immunoblot) analysis using anti-CHK antibodies as described herein. Additionally, the expression of CHK protein can be detected by immunoprecipitation using anti-CHK antibodies, also as described herein. Additional techniques suitable for use to detect the presence of CHK protein includes e.g., immunofluorescence staining, confocal staining and ELISA when using soluble lysates. Such techniques are also well known to those of skill in the art.

[0041] Detection of the presence or absence of CHK can also be accomplished by the detection of the presence or absence of nucleic acids, either DNA or RNA, encoding the CHK protein in a biological sample. The biological sample, e.g., breast tissue, can be prepared in a manner that renders the nucleic acid encoding CHK available for hybridization with a nucleic acid probe that specifically hybridizes with a nucleic acid sequence that encodes all, or a portion, of CHK. For example, Northern blot analysis, or Southern blot analysis can be used to detect the presence of CHK RNA or DNA in a biological sample. These techniques are well-known to those of skill in the art. (See e.g., Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989) or Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, J. Wiley and Sons, New York, N.Y. (1992)). For example, the standard Southern blot methods includes extracting genomic DNA from the sample, and digesting the genomic DNA with suitable restriction enzymes to obtain DNA fragments. The DNA fragments are then separated by electrophoretic means on e.g., agarose gels and transferred to nylon membranes which are exposed to detectably-labeled probes under conditions sufficient for the probes to specifically hybridize to nucleic acids encoding CHK. Detection can be accomplished by, e.g., autoradiography, spectrometry or fluorometry.

[0042] Nucleic acid probes useful in the present invention comprise at least about 15 nucleotides, typically about 21 to 45 nucleotides and most typically about 100 nucleotides. This number of nucleotides typically provides the minimal length required of a probe that would specifically hybridize to a CHK-encoding sequence. The probes are of a specificity and sufficient length to form stable hybrid duplexes with the target sequence under stringent conditions. As used herein, stringent conditions are defined as conditions under which specific hybrid duplexes will be stable and maintained and under which non-specific hybrid duplexes will be not be stable (e.g., stable during wash conditions while non-specific hybrid duplexes will be (eluted during wash conditions). Probes and conditions useful in the present invention are described in WO 93/15201, entitled "Novel Protein Tyrosine Kinases", the teachings of which are herein incorporated in their entirety by reference (Also see FIG. 3, SEQ ID NO: 3, which is a nucleic acid sequence encoding a CHK peptide). Techniques for identifying probes and conditions of stringency (e.g., moderate or high) are also well-known to those of skill in the art and e.g., are described in Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989) or Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, J. Wiley and Sons, New York, N.Y. (1992).

[0043] It is important to note that the nucleotide sequences of probes that are useful in the present invention need not be fully complementary to the target sequence. Probes need only be substantially complementary. As defined herein, substantially complementary means that the probe sequence is sufficiently similar in sequence identity to the target sequence that the probe specifically hybridizes with the target sequence under specified conditions. For example, non-complementary bases can be interspersed within the probe sequence, or the probe can be longer or shorter than the target sequence, provided that the probe still specifically hybridizes with the target sequence.

[0044] Detection of hybrid duplexes is typically accomplished by the use of detectably labeled probes. Such labels and methods of labeling probes are well-known to those of skill in the art. For example, labels can be radiolabels, chemiluminescent labels, fluorescent labels, biotin, enzymes or other labels known to those of skill in the art. Alternatively, the probe can be unlabeled but detectable by subsequent binding or hybridization to a second, detectably labeled molecule.

[0045] Detection of nucleic acids encoding CHK can also be accomplished by amplification techniques which directly amplify the target nucleic acid present in a sample, for example, by polymerase chain reaction (PCR) (See e.g., Saiki, et al., Science, 230:1350-1353 (1986) or ligase chain reaction (LCR) (See e.g., Weiss, R., Science, 254:1292-1293 (1991)). Such amplification techniques can also be used a preliminary steps for detection techniques described above. For example, conditions and specific primers suitable for use in a PCR method to amplify nucleic acids encoding CHK are described herein.

[0046] In situ hybridization analysis on tissue samples can also be used to detect the presence or absence of CHK in a biological sample. Such techniques are also well-known to those of skill in the art. (See for example, Sure Site II System.TM. hybridization kit by NoVagen.)

[0047] The present invention also encompasses the use of the CHK proteins and nucleic acids encoding these proteins as a basis of rational drug design to produce biologically active CHK analogs that have substantially comparable, or lesser or greater biological activity of CHK. Also encompassed are the use of the CHK proteins to identify small molecules which interact with CHK and, thus, can act as agonists, antagonists or inhibitors of CHK activity.

[0048] A further embodiment encompassed by the present invention includes methods of inhibiting neoplastic (tumor) cell growth by supplying CHK to cells. Specifically encompassed by the present invention are methods that inhibit ErbB-2 mediated-breast cancer cell growth. Cells that are in need of CHK and are supplied with, or receive CHK protein, are referred to herein as target or recipient cells. The recipient cells are either substantially deficient in CHK (e.g., fail to produce an amount of CHK sufficient to suppress neoplastic growth, or hyperplasia, which is abnormal growth) or produce adequate amounts of CHK, but the CHK produced is functionally abnormal (e.g., the CHK lacks biological activity to suppress neoplastic growth). As defined herein, the term "inhibit" means either to completely suppress or prevent neoplastic cell growth or to substantially, or significantly decrease neoplastic or hypeplastic cell growth. Inhibition or decrease of cancer cell growth, or hyperplasia, can be measured as described herein, e.g., by comparing growth of breast tumor cells that have been supplied with CHK to breast tumor cells that have not been supplied with CHK, and by other methods well-known to those of skill in the art.

[0049] CHK protein, peptide or a biologically active fragment thereof, or a CHK analog or derivative, can be supplied to mammalian breast tissue that manifests neoplastic cell growth, or is at risk of producing neoplastic cell growth e.g., hyperplastic tissue. CHK can be supplied to (e.g., introduced into) the target recipient cells by methods well-known to those of skill in the art. For example, CHK can be introduced into recipient cells by injection of a pharmaceutical composition that contains an effective amount of CHK in a physiologically compatible solution, or by a liposome preparation that contains an effective amount of CHK. Specifically encompassed by this invention is the topical application of liposomes in a cream or ointment which contain an effective amount of CHK. An effective amount of CHK is defined herein as an amount of CHK which inhibits neoplastic or hyperplastic cell growth, specifically ErbB-2 mediated breast cancer cell growth.

[0050] Suitable physiologically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oil, benzyl alcohols, polyethylene glycols, gelatine, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid. Viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, etc. The pharmaceutical preparations can be sterilized and if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers and the like which do not deleteriously react with the active compounds. They can also be combined, where desired, with other active agents, e.g., enzyme inhibitors, to further reduce metabolic degradation.

[0051] For topical application, there are employed as nonsprayable forms, viscous semi-solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water. Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, foams, ointments, powders, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc. For topical application, also suitable are sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., pressurized air. Also included are transdermal patches as discussed, for example, in WO 93/07870, the teachings of which are incorporated by reference.

[0052] Alternatively, CHK can be supplied to the recipient cells by introducing a nucleic acid sequence encoding CHK, or a biologically active fragment, analog, or derivative of CHK which is then expressed in the recipient cells. Methods of introducing nucleic acids encoding specific proteins such as CHk are well-known to those of skill in the art. For example, expression vectors can be designed and produced that contain a nucleic acid insert which encodes CHK or a biologically active fragment of CHK. Methods to construct these expression vectors are well-known to those of skill in the art. For example, described herein is an expression vector comprising vaccinia virus useful for expressing a DNA insert encoding CHK. In addition to vaccinia virus, other virus or plasmid vectors, such as retroviruses, or plasmid vectors can be used to introduce nucleic acids encoding CHK into recipient cells. Additionally naked DNA can be injected into recipient cells, or methods such as elctroporation, co-precipitation or a "gene gun" can be used to deliver the DNA to the recipient cells.

[0053] Other techniques using naked plasmids or DNA, and cloned genes encapsidated in targets liposomes or in erythrocyte ghosts, can be used to introduce the receptor into the host (Friedman, T., Science, 244:1275-1281 (1990); Rabinovich, N. R., et al., Science, 265:1401-1404 (1994)). The construction of expression vectors and the transfer of vectors and nucleic acids into various host cells can be accomplished using genetic engineering techniques, or by using commercially available kits as described in Sambrook, J., et al. Molec. Cloning, Cold Spring Harbor Press (1989) or Ausubel, F. M., et al. Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1989) the teachings of which are hereby incorporated, in their entirety, by reference.

[0054] Cells from a patient's tumor can be analyzed by the diagnostic methods described above to determine the presence of CHK or to determine the biological activity of CHK present in their cells. A vector as described herein, containing a nucleic acid encoding CHK and operably linked to expression control elements required for the expression of a protein in the recipient cells, is then introduced into the patient, either at the site of the tumor or by intravenous or other parenteral injection in order to reach any tumor cells that may have metastasized to other sites. The introduction may be repeated as necessary in order to achieve the desired effect of inhibiting neoplastic growth. A description of techniques that may be used to specifically target breast cells is described in EP 0 699 754 A1, the teachings of which are herein incorporated by reference.

[0055] Thus, as a result of the work described herein, novel methods of detecting and inhibiting breast cancer, or hyperplastic growth that may result in cancer, are now available.

[0056] The following examples more specifically illustrate the invention and are not intended to be limiting in any way.

EXAMPLE 1

Expression of CHK in Human Breast Cancer Tissue

[0057] Materials:

[0058] Recombinant heregulin (rHRG(1, 177-244), rabbit polyclonal anti-ErbB-2 antibodies, and 3E8-monoclonal anti-ErbB-2 antibodies, were obtained from Genentech, Inc. (San Francisco, Calif.), Levi, A. D., Bunge, R. P., Lofgren, J. A., Meima, L., Hefti, F., Nikolics, K., and Sliwkowski, M. X., J. Neurosci., 15, 1329-1340 (1995)). EGF and IL-6 were purchased from Collaborative Biomedical Products (Bedford, Mass.) and from R & D Systems (Minneapolis, Minn.) respectively. Monoclonal anti-phosphotyrosine antibody (PY20) conjugated to horse radish peroxidase (HRP) was obtained from Zymed, Inc. (San Francisco, Calif.). Polyclonal antibodies for EGF-R, ErbB-3, ErbB-4 and polyclonal anti-CHK (anti-LSK) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.). Anti-GST monoclonal antibodies were purchased from Pharmacia Biotech, Inc. (Piscataway, N.J.). GST fusion proteins containing the NH2-SH2 domain of p85 P13 kinase and SH2-SH2-SH3 domains of PLC-1 were obtained from Santa Cruz Biotechnology. The primers for the polymerase chain reaction (PCR) were synthesized by an automated DNA Synthesizer (Applied Biosystems, Model 394). Reagents for electrophoresis were obtained from BioRad (Hercules, Calif.). ECL reagents were purchased from Amersham Corp. (Arlington Heights, Ill.). All other reagents were purchased from Sigma (St. Louis, Mo.).

[0059] Experimental Procedures

[0060] Immunohistochemical staining was performed on paraffin-embedded 5 mm-thick tissue sections of human breast cancer. Sections were deparaffinized in xylene and then incubated in decreasing concentrations of ethyl alcohol. After several rinses in water, the slides were incubated in methanol/hydrogen peroxide (1:4), briefly rinsed in water and then in PBS (pH 7.6). Subsequent immunohistochemical staining was performed using a 1:100 dilution in PBS of rabbit anti-CHK antisera (1 hr incubation) followed by the addition of the secondary antibodies, peroxidase-conjugated rabbit anti-mouse IgG (Sigma) at 50 .mu.g/ml in PBS.

[0061] Analyses of CHK expression in human breast cancer tissues at different stages were performed using immunohistochemistry on paraffin sections. Results (Table 1) revealed that CHK is expressed in the majority of breast cancers, but was not detected in normal adjacent tissue.

1TABLE 1 CHK Expression in Primary Breast Cancer Tissues BREAST CANCER PATIENTS NO. PATIENTS (+) FOR CHK Stage I 32/41 Stage II 29/35 Stage III 4/4 Unknown Stage 5/6 Normal Breast 0/3 Fibroadenoma 0/6

[0062] Immunohistochemical staining was performed on paraffin embedded sections of infiltrating ductal carcinoma using anti-CHK antibodies.

EXAMPLE 2

CHK is Associated With Activated ErbB-2 Upon Stimulation With HRG

[0063] Experiments were performed using the T47D breast cancer cell line and the GST-fusion protein containing the SH2 domain of CHK (CHK-SH2). T47D cells express the ErbB family receptors and the CHK protein as observed by immunohistochemistry. The T47D human breast cancer cell line was obtained from ATCC (American Type Culture Collection, Rockville, Md.). T47D cells were grown in RPMI-1640 medium (GIBCO/BRL, Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal bovine serum (FBS) and 3.5 (g/ml insulin (Sigma). Prior to stimulation with HRG, cells were starved overnight in media containing 1% FBS and then for 4 hr in serum-free medium. The starved cells were then stimulated with HRG (10 nM) for the indicated time points (FIGS. 1A-D). Cells were lysed, and the supernatants were incubated with the purified CHK-SH2 fusion protein (FIGS. 1A, 1B) or with the 3E8 monoclonal antibody to ErbB-2 (FIGS. 1C, 1D). The co-precipitated proteins were analyzed on 7% SDS-PAGE, and immunoblotted with PY20 (FIGS. 1A, 1C).

[0064] As shown in FIG. 1A, a tyrosine-phosphorylated 185 kD protein was associated with CHK-SH2 within 2 min of the HRG stimulation. The association of the 185 KD with CHK-SH2 was maximal at 2-8 min after HRG stimulation and then gradually decreased. In order to determine whether the 185 kD protein was ErbB-2, the blot was deprobed and reblotted with polyclonal anti-ErbB-2 antibody. As shown in FIG. 1B, the 185 kD protein was confirmed to be the ErbB-2 protein. These results indicated that the CHK protein can interact with the HRG-activated ErbB-2 receptor.

[0065] When lysates from HRG-treated cells were immunoprecipitated with the 3E8 monoclonal anti-ErbB-2 antibody, the pattern of the phosphorylated ErbB-2 was different from that of the ErbB-2 precipitated with the SH2 domain of CHK (compare FIG. 1C with FIG. 1A). Blotting of the same samples with the polyclonal anti-ErbB-2 antibody (FIG. 1D) confirmed these observations.

[0066] CHK-SH2 fusion proteins also precipitated other as of yet unidentified tyrosine-phosphorylated proteins as shown in FIG. 1A. However, these phosphorylated proteins were also precipitated from the unstimulated cells and their phosphorylation pattern did not appear to change over the time course of these studies.

EXAMPLE 3

The Association of CHK With ErbB-2 is Specific for HRG Stimulation

[0067] In order to determine whether the observed association of CHK with ErbB-2 was receptor-specific and stimulus-specific, experiments were performed to analyze whether CHK could associate with either the EGF-R or IL-6 receptors which are both known to be expressed in T47D cells. The association of CHK-SH2 with ErbB-2 in lysates from HRG, EGF and IL-6 stimulated cells was compared. T47D cells were serum starved as described above and then activated either with HRG (10(nM) for 8 min or with (EGF (100 ng/ml) or IL-6 (100 ng/ml) for 5 min. The experimental time points and the concentrations of EGF and IL-6 were optimized in initial kinetic studies. The stimulated cells were lysed and precipitated with the CHK-SH2 fusion protein as described above. The precipitates were then analyzed on SDS-PAGE and immunoblotted with PY20 antibodies or with polyclonal anti-ErbB-2 antibodies. Only HRG stimulation induced the association of ErbB-2 with the purified CHK-SH2 fusion protein. EGF or IL-6 stimulation failed to induce CHK-SH2 association either to ErbB-2, or to the EGF-receptor or the IL-6 receptor.

[0068] The association of ErbB-2 with other SH2 domain-containing signaling molecules such as p85 of P13-kinase, PLC-1 or Shc was also examined. The SH2-SH2-SH3 domain of PLC-1 was found to be associated with the HRG-activated ErbB-2 as well as with Shc. The SH2 domain of P13-kinase precipitated ErbB-2, probably as a result of the ErbB-2 heterodimerization with ErbB-3. Taken together, these results indicate that ErbB-2 associates with all three signaling molecules in HRG-activated T47D cells.

[0069] Experiments were also performed to show that the SH3 domain of CHK is not involved in the interaction between CHK and ErbB-2.

[0070] The potential involvement of other domains of CHK in the interaction with ErbB-2 was also examined. GST-fusion proteins containing the SH3 domain of CHK (CHK-SH3), the N-terminal domain plus SH3 domain (NH2-SH3), the SH3 and SH2 domains of CHK (SH3-SH2), the SH2 domain of CHK as well as the GST protein alone were prepared as follows.

[0071] Generation of Flag-CHK Construct in pCDNA3 Vector:

[0072] The CHK cDNA (1.6 kb, (SEQ ID NO: 1) was cloned into EcoRI sites in the pCDNA3 neo vector. The nucleotide sequence for the Flag epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) (SEQ ID NO: 5) was introduced to the 5' end of the ORF (open reading frame) of the CHK cDNA sequence by PCR, using 1.6 kb CHK cDNA as a template. The 5' sense primer included a BamH I restriction site, ATG initiation codon, the Flag sequence and CHK sequences from nucleotides #269 to #295 of SEQ ID NO: 1, FIG. 2 (Bennett, B. D., et al. (1994) J. Biol. Chem. 269, 1068-1074), the teachings of which are herein incorporated, in their entirety, by reference). The 3'-antisense primer was composed of CHK sequences from nucleotides #510 to #481 of SEQ ID NO: 1. The PCR product was double digested with BamHI and BstEII (New England Bio Labs, Beverly, Mass.), gel-purified and then cloned into BamHI, and BstEII sites in the pDNA3 neo-Flag-CHK. The construct was analyzed by restriction mapping and nucleotide sequencing.

[0073] Transfection:

[0074] Transfection of MCF-7 cells was performed using the Lipofectamine.TM. (Gibco/BRL) according to the manufacturer's protocol. The transfected cells were selected in 1.2 mg/ml G418 (Sigma). Positive transfectants were chosen based on their immunoreactivity on Western blots probed with polyclonal anti-CHK and monoclonal anti-Flag (M5) antibodies (Eastman Kodak Company, New Haven, Conn.).

[0075] Construction and Purification of GST-Fusion Proteins of CHK:

[0076] To express the NH2-SH3 and SH3-SH2 domains of CHK as GST-fusion proteins, the corresponding DNA sequences were amplified by PCR with sense and antisense primers of CHK cDNA which contained BamHI and EcoRI restriction sites. For the NH2-SH3 construct, we used the sense primer from nucleotides #4 to #27 SEQ ID NO: 1 and the antisense primer from nucleotides #343 to #321 of SEQ ID NO: 1. For the SH3-SH2 construct, we the sequence from nucleotides #127 to #150 of SEQ ID NO: 1 was used as the sense primer and from nucleotides #657 to #634 SEQ ID NO: 1 as the antisense primer. The DNA fragments obtained from PCR were restriction digested with BamHI and EcoRI and ligated into the pGEX-2T vector (Pharmacia). The sequence and orientation were confirmed by sequencing both strands. Construction of the GST-fusion proteins of CHK-SH2 and CHK-SH3 were performed as described in Jhun, B. H., Rivnay, B., Price, D., and Avraham, H. (1995) J. Biol. Chem. 270, 9661-9666.

[0077] GST-fusion proteins were produced by the induction of transformed bacteria using 10 mM isopropyl-(-thiogalactopyranoside (IPTG), and purified on a large scale by affinity chromatography on glutathione-sepharose beads (Pharmacia) according to the manufacturer's protocol.

[0078] HRG-stimulated T47D cell lysates were incubated with the different GST-fusion proteins, analyzed by SDS-PAGE, and immunoblotted either with PY20, rabbit anti-ErbB-2 antibody or with anti-GST antibody. Neither the SH3 domain of the CHK protein nor the NH2-SH3 domain precipitated ErbB-2. Binding to ErbB-2 was detected only in the presence of the CHK-SH2 and CHK-SH3-SH2 fusion proteins. As expected, no binding was detected when the same lysates were incubated with the GST protein alone. The amounts of the different fusion proteins loaded on the gel were comparable. These results confirm that CHK can interact with the HRG-stimulated ErbB-2 in a specific manner via its SH2 domain.

EXAMPLE 4

In Vivo Association of Intact CHK With ErbB-2

[0079] The MCF-7 human breast cancer cell line was obtained from ATCC (American Type Culture Collection, Rockville, Md.). The MCF-7 cells were grown in MEM (GIBCO) supplemented with 10% FBS, 5 .mu.g/ml insulin (Sigma), 1 mM non-essential amino acids and 1 mM sodium pyruvate. Prior to stimulation, cells were starved overnight in media containing 1% FBS and then for 4 hr in serum-free medium.

[0080] To further confirm the association of ErbB-2 with CHK, the CHK protein was overexpressed in MCF-7 breast cancer cells. CHK expression in MCF-7 cells was detected only by PCR analysis. Expression of the ErbB receptor family in MCF-7 cells was similar to that observed in T47D cells. Stable transfections were performed using the Flag-CHK pCDNA3 neo construct as described above. The transfected cells were analyzed for CHK expression by Western blot using anti-Flag and anti-CHK antibodies and also by immunofluorescence using confocal microscopy. MCF-7 cells transfected with Flag-CHK pCDNA3 neo (Flag-CHK), MCF-7 cells transfected with the pCDNA3 neo vector alone, or untransfected MCF-7 control cells, were stimulated with HRG and then lysed.

[0081] Immunoprecipitation studies were performed as follows. Approximately 5.times.10.sup.6 cells/plate were starved overnight in media containing 1% FBS, followed by additional starvation in serum-free medium for 4 hr at 37.degree. C. The starved cells were then stimulated with 10 nM HRG for 8 min or with 100 ng/ml EGF or 100 ng/ml IL-6 for 5 min at room temperature. The stimulation was terminated by the addition of an ice-cold lysis buffer (0.1% SDS, 1% Triton X-100, in Tris-buffered saline containing 10% glycerol, 1 mM EDTA, 0.5 mM Na3VO4, 0.2 mM phenylmethylsulfonyl fluoride, 1 .mu.g/ml aprotinin, and 10 mM leupeptin). Lysates were pre-cleared by centrifugation (14,000 rpm, 15 min) and then incubated for 90 min at 4.degree. C. with 10 .mu.g of GST-fusion proteins coupled to glutathione-sepharose beads. The beads were washed three times with the lysis buffer. For the immunoprecipitation experiments, polyclonal anti-CHK antibody, monoclonal anti-ErbB-2 antibody, 3E8 (10 .mu.g/ml), polyclonal anti-ErbB-3 antibody (10 .mu.g/ml) or polyclonal anti-ErbB-4 antibody (10 .mu.g/ml) were used. SDS-sample buffer was added to the samples and analyzed on 7% polyacrylamide SDS-PAGE. Proteins were transferred onto nitrocellulose or Immobilon-PTM (Millipore, Inc., Bedford, Mass.) membranes. Bound proteins were immunoblotted with anti-phosphotyrosine antibody (PY20), polyclonal anti-ErbB-2 antibody, or polyclonal anti-CHK, EGF-R, ErbB-3 or ErbB-4 antibodies. The blots were developed using the enhanced chemiluminescence (ECL) system (Amersham). Blots were stripped for 30 min at 55.degree. C. in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7), according to the manufacturer's protocol (Amersham).

[0082] The 185 KD tyrosine-phosphorylated protein was immunoprecipitated with anti-Flag antibodies or anti-CHK antibodies only in HRG-stimulated Flag-CHK transfected cell lysates, but not in the untransfected MCF-7 cell lysates or the MCF-7 cell lysates transfected with the pcDNA3-neo Flag vector alone. Blotting with the anti-ErbB-2 antibody confirmed that the co-precipitated 185 KD protein was indeed the ErbB-2. Analysis of the total lysates from the same experiment revealed that the ErbB-2 was tyrosine-phosphorylated as a result of the HRG stimulation in the Flag-CHK cells as well as in the MCF-7 untransfected cells. The expression of ErbB-2 appeared to be equal in both the Flag-CHK and MCF-7 cells. Taken together, these in vitro and in vivo data indicate that the HRG-stimulated ErbB-2 associates with CHK through the SH2 domain.

EXAMPLE 5

Involvement of Other ErbB-2 Family Members in the Interaction With CHK

[0083] To further investigate the possible involvement of other members of the ErbB family in the observed interaction between CHK and ErbB-2, co-immunoprecipitation experiments using MCF-7 cells transfected with Flag-CHK were performed. Flag-CHK transfected cells were stimulated with HRG, and then lysed and immunoprecipitated with anti-CHK antibody as described above. The immunocomplexes were separated by SDS-PAGE and immunoblotted with anti-ErbB-2 antibody or with anti-ErbB-3 antibody. The results indicated that anti-CHK antibody immunoprecipitated the HRG-activated ErbB-2. In contrast, no detectable ErbB-3 was found. However, the possibility that very low amounts of ErbB-3 were present in the precipitates as a result of the heterodimerization with the ErbB-2 receptor upon HRG stimulation cannot be excluded.

[0084] It was also investigated whether ErbB-4 interacted with CHK under these conditions, however, findings indicated that ErbB-4 was not involved in the ErbB-2-CHK association.

[0085] In order to confirm the presence and phosphorylation of the ErbB-3 as well as the heterodimerization of ErbB-3 with ErbB-2 in the Flag-CHK transfected cells, lysates from HRG-stimulated Flag-CHK cells were immunoprecipitated with anti-ErbB-3 antibodies or with anti-ErbB-2 antibodies. Both ErbB-3 and ErbB-2 were tyrosine-phosphorylated upon HRG stimulation and the formation of ErbB-2-ErbB-3 heterodimers was demonstrated by the presence of ErbB-2 in the precipitates of the anti-ErbB-3 antibodies. However, under these conditions, ErbB-3 was not detected in the samples immunoprecipitated with anti-ErbB-2 antibody. Taken together, these observations indicate that upon HRG stimulation, heterodimerization of ErbB-3 with ErbB-2 receptors occurred in the transfected cells, suggesting that the ErbB signaling in these cells is not altered.

[0086] To determine whether EGF-R (ErbB-1) might be involved in ErbB-2-CHK interactions, Flag-CHK MCF-7 transfected cells were serum-starved and then stimulated with HRG (10 nM) or with EGF (100 ng/ml). The lysates were immunoprecipitated with anti-CHK antibodies and analyzed by SDS-PAGE. Only the tyrosine-phosphorylated ErbB-2 protein was immunoprecipitated with anti-CHK-antibodies in the HRG-stimulated lysates. No tyrosine-phosphorylated proteins were detected in the immunoprecipitates with anti-CHK antibodies from the EGF-stimulated cells. Reprobing of this blot with anti-ErbB-2 or with anti-EGF-R antibodies confirmed that neither of these receptors were present in the CHK immunoprecipitates.

[0087] As a control, immunoprecipitations with anti-EGF-R antibodies of the EGF-stimulated Flag-CHK cell lysates as well as of lysates from untransfected MCF-7 cells were performed. The EGF-R and the ErbB-2 proteins were present in the immunoprecipitates from the EGF-stimulated cells as a result of the EGF-ErbB-2 heterodimerization. Probing of the same blot with anti-ErbB-2 or anti-EGF-R antibodies confirm this observation.

[0088] These analyses indicate that CHK associates via its SH2 domain with HRG-stimulated ErbB-2. This association is specific to HRG-stimulated ErbB-2 and does not appear to prominently involve other ErbB family members.

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