The Src tyrosine kinase Hck is required for Tel-Abl- but not for Tel-Jak2-induced cell transformation

Tel-Abl and Tel-Jak2 are fusion proteins associated with human haematologic neoplasms. They possess constitutive tyrosine kinase activity and activate common downstream signalling pathways like Stat-5, PI3-K/Akt, Ras/MapK and NF-kappaB. In this study, we showed the specific requirement of Src family members for the Tel-Abl-mediated cell growth, activation of Stat5, PI3-K/Akt and Ras/MapK while dispensable for Tel-Jak2. Hck was found strongly phosphorylated in Tel-Abl-expressing Ba/F3 cells and sensitive to imatinib mesylate treatment, providing evidence that Hck is a target of Tel-Abl tyrosine kinase activity. Overexpression of a kinase dead form of Hck inhibits the proliferation of Ba/F3 cells expressing Tel-Abl as the phosphorylation of Akt and Erk1/2. These results argue for an important role of Hck in Tel-Abl oncogenic signalling.     

Antibody-induced growth inhibition is mediated through immunochemically and functionally distinct epitopes on the extracellular domain of the c-erbb-2 (her-2/neu) gene product p185

Over-expression of the c-erbB-2 (HER-2/neu) gene product p185 occurs in 30% of breast and ovarian cancers. The p185 protein might serve as a target for serotherapy in that antibodies against different epitopes on the extracellular domain of p185 can inhibit growth of tumor cells in the absence of cellular or humoral effector mechanisms. To define epitopes of functional relevance, II monoclonal antibodies (MAbs) were evaluated for their ability to bind to the extracellular domain of p185. Results of competition studies with ¹²⁵1-labeled and non-labeled antibodies indicated that 10 of 11 epitopes were grouped in a linear array. Antibodies against 7 epitopes inhibited anchorageindependent growth and antibodies against 2 of these epitopes also inhibited anchorage-dependent growth of SKBr3 breast-cancer cells that over-expressed p185. Treatment with antibodies exerted cytotoxic rather than cytostatic effects. When antibodies were used in combination, additive or supra-additive inhibition of anchorage-independent and anchorage-dependent growth was observed between pairs of antibodies. Growth inhibition did not relate to the affinity of the antibody or its isotype. Two antibodies that inhibited both anchorage-dependent and anchorage-independent growth also blocked binding of the HER-2/neu ligand, whereas 5 antibodies that inhibited only anchorage-independent growth had no effect on ligand binding. Inhibition of cell growth did not correlate with internalization of p185 or down-regulation of p185 on the cell surface. Fab fragments of active antibodies could also inhibit anchorage-independent growth of SKBr3. Thus, murine MAbs and their fragments recognized both immunochemically distinct and functionally distinct epitopes on the p185 molecule. Whereas inhibition of anchorage-dependent growth correlated with the ability of antibodies to block ligand binding, inhibition of anchorage-independent growth did not correlate with effects on ligand binding, internalization, cell-surface expression or cross-linking of pl85.     

p185(neu) protein is required for tumor and anchorage-independent growth, not for cell proliferation of transgenic mammary carcinoma

Transgenic FVB-NeuN mice (N202) bearing the rat neu protooncogene driven by the mouse mammary tumor virus promoter/enhancer develop focal mammary carcinomas overexpressing the neu-encoded p185(neu) protein. In vitro expression of p185(neu) among mammary carcinoma cultures was heterogeneous, and we could establish some cell lines and clones displaying a complete loss of p185(neu) expression, along with others with very high p185(neu) protein level. Upon in vivo injection, p185(neu)-positive cells gave rise to fast-growing tumors with a short latency, while p185(neu)-negative cells required a very long latency time, and the resulting tumors were invariably p185(neu)-positive. The lower growth ability of p185(neu)-negative cells in vivo was also confirmed in athymic nude mice. In vitro, analysis of anchorage-independent growth in soft agar revealed colony formation from p185(neu)-positive but not p185(neu)-negative cells. The direct involvement of p185(neu) in clonogenicity was demonstrated by the inhibition of p185(neu)-positive colony growth in soft agar in the presence of an anti-p185(neu) monoclonal antibody. By contrast, a higher level of anchorage-dependent clonogenic growth and proliferation was observed in p185(neu)-negative cells as compared to p185(neu)-positive cells, thus explaining the relative ease with which p185(neu)-negative cell lines and clones were established in vitro. Together, the results indicate that p185(neu) expression can lead to tumor formation and metastasis through the modification of intrinsic properties of cells related to anchorage-independent growth ability rather than to proliferation or host-dependent mechanisms.     

Expression of the c-erbB-2 gene product (p185) at different stages of neoplastic progression in the colon

The c-erbB-2 gene has been found amplified in a number of human adenocarcinomas leading to elevated levels of expression of the p185 protein product. Increased expression of this putative growth factor receptor has been reported to occur by molecular mechanism other than gene amplification and for this reason we have studied the expression of the p185 protein in normal colon and in lesions representing different stages of neoplastic progression. We report amplification of the c-erbB-2 gene in 3 of 44 colon carcinomas and 1 of 5 preneoplastic polyps studied. Confirmation of expression of the p185 protein product was established in Western blot analysis and by immunocytochemical staining of tissue sections. An extended study, involving adenomatous polyps and carcinomatous material in immunostaining, revealed detectable presence of the p185 protein in 20% of carcinomas, consistent with immunoprecipitation data derived using established cell lines. In contrast, a high percentage of polyps showed strong staining with both p185 antibodies used, indicating elevated levels of expression of the c-erbB-2 protein associated with preneoplastic lesions. Staining of normal human colon revealed a restricted localization of this putative receptor to cells on the luminal colonic surface, with no expression in cells of the crypt. Histologically normal mucosa, adjacent to the tumor, showed a more extensive distribution involving the crypt suggestive of a disturbance in the normal expression of c-erbB-2. These results indicate that elevated expression of the c-erbB-2 protein is associated with early stages of colonic neoplasia but do not establish it as a primary factor in these events. The occurrence of multiple copies of the c-erbB-2 in a percentage of colon lesions, however, suggests a possible role for this gene in some colon malignancies.     

Engineering high affinity humanized anti-p185HER2/anti-CD3 bispecific F(ab′)2 for efficient lysis of p185HER2 overexpressing tumor cells

We previously constructed a humanized anti-p185^HER2/anti-CD3 bispecific antibody variant, BsF(ab′)₂ v1 which retargets the cytotoxic activity of human T cells in vitro against human breast tumor cells which overexpress the p185^HER2 product of the HER2/neu (c-erbB-2) protooncogene. Subsequently we identified an improved anti-CD3 variant, v9, which binds to T cells with approx. 100-fold higher affinity than the original variant, v1. Here we demonstrate that BsF(ab′)₂ v9 is more potent than BsF(ab′)₂ v1 in stimulating the proliferation of both resting peripheral blood lymphocytes (PBL) and IL-2-activated, long-term cultured T lymphocytes (ATL). In addition, at low concentrations (0.01-1 ng/ml) BsF(ab′)₂ v9 is much more efficient than BsF(ab)₂ v1 in directing lysis of p185^HER2-overexpressing tumor cells by IL-2 activated PBL. In contrast, at higher concentration BsF(ab′)₂ v9 and BsF(ab′)₂ v1 have similar potency in retargeted cytotoxicity. At BsF(ab′)₂ v9 concentrations of ? 1 ng/ml the susceptibility of p185^HER2-expressing tumor cells to lysis is apparently independent of the level of p185^HER2 expression. At lower concentrations of BsF(ab′)₂ v9 and/or lower ratios of effector to target cells the extent of lysis is reduced, in some cases improving the selectivity of lysis of high p185^HER2 expressors over low expressors. Thus selection of a high affinity anti-CD3 arm is likely important in the design of BsF(ab′)₂ for retargeting the cytotoxicity of T cells to tumors. The dose of BsF(ab′)₂ v9 in any future clinical evaluation will require optimization to maximize anti-tumor efficacy whilst minimizing potential toxicity against normal tissue expressing p185^HER2. © 1995 Wiley-Liss Inc.     

Relative cytotoxic activity of immunotoxins reactive with different epitopes on the extracellular domain of the c-erbB-2 (HER-2/neu) gene product p185.

Different epitopes on the extracellular domain of the HER-2 receptor can serve as distinct targets for immunotoxins. To determine the optimal epitope target for immunotoxin therapy, 7 anti-HER-2 ricin A chain murine monoclonal immunotoxins, each reactive with different epitopes of HER-2 receptor, were tested for cytotoxic activity. The immunotoxins produced 1.2-4.6 logs of cytotoxicity in limiting dilution clonogenic assays with 2 breast cancer cell lines that overexpressed HER-2. Cytotoxicity did not correlate with immunoglobulin isotype, binding affinity, relative position of epitopes or internalization of the anti-HER-2 immunotoxins. Interestingly, the most and least effective immunotoxins bound to epitopes in very close proximity. Competitive binding assays with unconjugated antibodies have previously indicated that our antibodies recognized epitopes that are arranged in a linear array. To orient this relative epitope map, deletions were prepared in the HER-2/neu gene and these mutant constructs were expressed in NIH3T3 cells. Epitope expression was determined by antibody binding and radioimmunoassay. Epitopes targeted by the PB3, 454C11 and NB3 antibodies are localized N-terminal to the epitopes recognized by ID5, BD5, 741F8 and 520C9 antibodies. The 2 non-conformational epitopes PB3 and NB3 were localized to regions corresponding to amino acides 78-242 of the p185(HER-2) protein.     

Selection of monoclonal antibodies which induce internalization and phosphorylation of p185HER2 and growth inhibition of cells with HER2/NEU gene amplification.

In order to obtain further information on the biological role of the HER2/neu oncoprotein monoclonal antibodies (MAbs) were produced against the p185 extracellular domain. To immunize the mice and screen the hybridoma supernatants we selected a lung adenocarcinoma cell line (Calu-3), which demonstrated an over-expression of p185HER2 measured as the reactivity with polyclonal rabbit serum to the 14-amino-acid carboxy-terminal-HER2/neu. Two MAbs, designated MGR2 (IgG1) and MGR3 (IgG2), selected for reactivity on Calu-3 and negativity on A43I live cells, the reference target cell for EGF receptor expression, were found to immunoprecipitate a 185-kDa molecule. Immunodepletion experiments with the polyclonal antiserum and cross-competition experiments indicated that the 2 reagents recognized 2 different epitopes located on the p185HER2 molecule. One of the 2 MAbs, MGR3, was found to internalize, induce p185HER2 phosphorylation and inhibit tumor cell growth in vitro. These results indicate that MGR3 is directed against a determinant located in the p185HER2 ligand binding site and may compete with the p185HER2 ligand, but is incapable of inducing a complete mitotic signal.     

Alteration of the CDKN2/p16 gene is not required for HPV-positive uterine cervical cancer cell lines

To determine whether alterations of the CDKN2/p16 might be involved in HPV-positive cervical cancers, we examined for alterations of this gene and function of the protein p16 to interact with CDK4 in 5 cervical cancer cell lines. No alteration of this gene was detected. Proteins for p16 and CDK4 were normally expressed and function of p16 to interact with CDK4 was not abrogated in these cell lines. These cell lines were human papillomavirus (HPV)-positive and carried wild-type p53. These findings suggest that phosphorylation of pRb by CDK4 is not critical in the carcinogenesis or in the establishment of HPV-positive cervical cancer cell lines, since HPV E6 or E7 viral-transforming proteins inactivate p53 and pRb tumor suppressor protein function, resulting in deregulated progression of the cell cycle.     

Inactivation of the p53 gene is not required for tumorigenesis of medullary thyroid carcinoma or pheochromocytoma.

A polymerase chain reaction (PCR)-mediated RNase protection analysis was performed to detect subtle genetic alterations of p53 in medullary thyroid carcinoma (MTC) and pheochromocytoma. None of the 30 pheochromocytomas showed abnormal RNase protection patterns. Only one of 32 MTCs showed an abnormal pattern, and subsequent DNA sequencing of the PCR product revealed that it had a G to C transversion in codon 49 that resulted in a change from aspartic acid to histidine. However, this was a sporadic MTC with no specific clinicopathological characteristics. On the basis of a previous report that genes on chromosome 17p were not deleted in MTCs and were relatively infrequently deleted in pheochromocytomas, our results suggest that the p53 gene is not involved in tumorigenesis of MTC or pheochromocytoma.     

Ribosomal protein L7a gene is up-regulated but not fused to the tyrosine kinase receptor as chimeric trk oncogene in human colorectal carcinoma

Ribosomal protein L7a (rp L7a) was identified in a subtractive hybridization screen as a gene up-regulated in human colorectal cancer. Expression of rp L7a was greater than 2-fold higher in tumors compared to adjacent normal mucosa in 72% of the patients studied (n=36). rp L7a was also up-regulated in concomitant polyps. The number of patients with rp L7a T/N ratio of >2 was significantly higher in the female (16/18) than in the male (10/18). rp L7a expression was also significantly higher in females with lymph node involvement compared to males. These results indicate that rp L7a expression is related to tumor growth in colorectal cancer especially in females, where it may also be related to tumor spread. There was no correlation of rp L7a expression with tumor cell differentiation. We also show that rp L7a does not exist as a fusion oncogene (trk-2h) in colorectal cancer.     

Cell cycle inhibition mediated by the outer surface of the C/EBPalpha basic region is required but not sufficient for granulopoiesis

CCAAT/enhancer binding protein alpha (C/EBPalpha) transactivates target genes dependent upon DNA binding via its basic region-leucine zipper domain and slows G1 progression by interaction with E2F, cdk2, or cdk4. E2F interacts with the non-DNA-binding surface of the C/EBPalpha basic region and C/EBPalpha residues 1-70 are required for repressing E2F targets, while cdk2 and cdk4 bind residues 177-191. C/EBPalpha-ER induces the 32D cl3 myeloblast cell line to differentiate to granulocytes. C/EBPalpha-ER variants incapable of binding DNA slowed G1, but did not induce early or late granulopoiesis, indicating that cell cycle inhibition as mediated by C/EBPalpha is not sufficient for differentiation. C/EBPalpha-ER variants lacking residues 11-70 or residues 11-70 and 178-200 both slowed the G1 to S transition. C/EBPalpha(GZ)-ER, containing the GCN4 rather than the C/EBPalpha leucine zipper, also slowed G1. In contrast, C/EBPalpha(BRM2)-ER, carrying mutations in the outer surface of the basic region required for interaction with E2F, did not slow G1. C/EBPalpha(BRM2)-ER induced early markers of granulopoiesis much less efficiently than C/EBPalpha-ER and did not direct terminal maturation. Inhibition of G1 progression using mimosine increased induction of late markers by G-CSF. Thus, both DNA binding and cell cycle arrest, mediated by opposite surfaces of the C/EBPalpha basic region, are required for granulopoiesis.     

Evidence that reduction of hepatocyte growth factor (HGF) is not required for peroxisome proliferator-induced hepatocyte proliferation

The mechanisms underlying peroxisome proliferator-induced hepatocarcinogenesis are not understood. Because of the uncertainty of human cancer risk associated with peroxisome proliferators, delineating the mechanisms of carcinogenesis by these agents is of great interest. Alterations in liver growth factors were postulated to contribute to the carcinogenic effect of peroxisome proliferators. Administration of these compounds to rodents results in down-regulation of hepatocyte growth factor (HGF) and supplementing culture medium with HGF is reported to suppress cell proliferation of preneoplastic and neoplastic cells from WY-14,643-treated livers. Combined, these observations suggest that reduced levels of hepatic HGF contribute to the mechanisms underlying peroxisome proliferator-induced hepatocarcinogenesis. To determine if HGF can prevent the effects of peroxisome proliferators in liver, the short-term influence of WY-14,643 in two different lines of HGF transgenic mice was examined. Mice were fed either a control diet or one containing 0.1% WY-14-643 for one week. Hepatomegaly was found in both HGF transgenic mouse lines fed WY-14,643 compared with controls. Additionally, hepatic expression of typical mRNA markers of peroxisome proliferation including those encoding peroxisomal fatty acid metabolizing enzymes and cell cycle control proteins were all significantly elevated in HGF transgenic mice fed WY-14,643 compared with controls. Down-regulation of HGF was found to be dependent on PPARalpha since lower levels of HGF mRNA and protein were observed in wild-type mice fed WY-14,643 for 1 week and not in similarly treated PPARalpha-null mice. These results demonstrate that the early increase in hepatic mRNAs associated with peroxisome and cell proliferation induced by WY-14,643 treatment can not be prevented by overexpression of HGF in vivo.     

Hepatocyte growth factor (HGF) stimulates the tyrosine kinase activity of the receptor encoded by the proto-oncogene c-MET.

The human proto-oncogene c-MET encodes a heterodimeric 190 kDa transmembrane protein (p190c-met) with structural features of a tyrosine kinase receptor. The ligand for this putative receptor has not yet been identified. By Northern blot hybridization we found that, among a restricted number of human tissues, c-MET is highly expressed in the liver. This prompted us to test the hypothesis of a functional interaction between the c-MET receptor and Hepatocyte Growth Factor (HGF), a heparin-binding polypeptide consisting of heavy and light chains of 65 and 35 kDa. Nanomolar concentrations of highly purified HGF added to GTL-16 cells, which overexpress the c-MET receptor, enhanced the phosphorylation on tyrosine of the p190c-met kinase. Addition of other known growth factors or serum was ineffective. The kinase activity of the c-MET receptor was also stimulated by HGF in an in vitro assay, after detergent solubilization and partial purification of p190c-met. Moreover, elution of immunoprecipitates obtained with anti-MET antibodies from GTL-16 cell lysates yielded an HGF-responsive kinase activity. These results suggest that HGF, or a growth factor structurally related to HGF, is a candidate ligand for the receptor encoded by c-MET.     

Development of hygromas or severe edema during treatment with the tyrosine kinase inhibitor STI571 is not associated with platelet-derived growth factor receptor (PDGFR) gene polymorphisms

STI571 is active against Bcr/Abl-, c-kit- and platelet-derived growth factor receptor (PDGFR)-driven malignancies. Mild to moderate edema is common, whereas severe edema, body cavity effusions and subdural hygromas are rarely observed. These effects have been suggested to involve inhibition of PDGFR signaling, but predisposing factors are unknown. We examined SNPs in the PDGFR alpha and beta gene regions in STI571-treated patients with and without life-threatening edema or cerebral hygromas, and in healthy volunteers. By RFLP analysis of 15 SNPs, the frequencies of genotypes did not differ between the three groups. SNPs of PDGFR genes do not appear to play a role in patient's susceptibility to clinically severe edema formation during treatment with STI571.