c-Met autocrine activation induces development of malignant melanoma and acquisition of the metastatic phenotype.

The molecular and genetic events that contribute to the genesis and progression of cutaneous malignant melanoma, a complex and aggressive disease with a high propensity for metastasis, are poorly understood due in large part to the dearth of relevant experimental animal models. Here we used transgenic mice ectopically expressing hepatocyte growth factor/scatter factor (HGF/SF) to show that the Met signaling pathway is an important in vivo regulator of melanocyte function, whose subversion induces malignant melanoma. Tumorigenesis occurred in stages, beginning with the abnormal accumulation of melanocytes in the epidermis and dermis and culminating in the development of metastatic melanoma. Oncogenesis in this model was driven by creation of HGF/SF-Met autocrine loops through forced expression of the transgenic ligand and apparent selection of melanocytes overexpressing endogenous receptor, rather than paracrine stimulation or mutational activation of c-met. Preference for liver as a metastatic target correlated with high HGF/SF-Met autocrine activity, consistent with the notion that such activity may influence colonization. Although basic fibroblast growth factor and its receptor were both weakly expressed in the majority of melanomas examined, high levels were found only in those rare neoplasms with low or undetectable HGF/SF and Met expression, suggesting that these two tyrosine kinase receptor autocrine loops serve a critical overlapping function in melanocytic tumorigenesis. Our data support a causal role for HGF/SF-Met signaling in the development of melanoma and acquisition of the metastatic phenotype. Moreover, this transgenic mouse should serve as a highly useful model, facilitating our understanding of mechanisms by which human melanoma progresses to malignancy and expediting the development of efficacious therapeutic modalities designed to constrain metastasis.

ErbB2 expression increases the spectrum and potency of ligand-mediated signal transduction through ErbB4.

Interleukin 3-dependent murine 32D cells do not detectably express members of the ErbB receptor family and do not proliferate in response to known ligands for these receptors. 32D transfectants were generated expressing human ErbB4 alone (32D.E4) or with ErbB2 (32D.E2/E4). Epidermal growth factor (EGF), neuregulin 1-beta (NRG1-beta), betacellulin (BTC), transforming growth factor-alpha (TGF-alpha), heparin binding-EGF (HB-EGF), and amphiregulin were analyzed for their ability to mediate mitogenesis in these transfectants. 32D.E4 responded mitogenically to NRG1-beta and BTC. Surprisingly, EGF also induced significant DNA synthesis and TGF-alpha was negligibly mitogenic on 32D.E4 cells, whereas HB-EGF and amphiregulin were inactive. Although coexpression of ErbB2 with ErbB4 in 32D.E2/E4 cells did not significantly alter DNA synthesis in response to NRG1-beta or BTC, it greatly enhanced mitogenesis elicited by EGF and TGF-alpha and unmasked the ability of HB-EGF to induce proliferation. EGF-related ligands that exhibited potent mitogenic activity on 32D.E2/E4 cells at low concentrations induced adherence, morphological alterations, and up-regulation of the Mac-1 integrin and FcgammaRII/III at higher concentrations. While 125I-EGF could be specifically crosslinked to both 32D.E4 and 32D.E2/E4 cells, its crosslinking capacity was greatly enhanced in the cotransfected cells. The ability of the various ligands to mediate proliferation and/or adhesion in the two transfectants correlated with their capacity to induce substrate tyrosine phosphorylation and to initiate and sustain activation of mitogen-activated protein kinase. We conclude that the ability of ErbB4 to mediate signal transduction through EGF-like ligands is broader than previously assumed and can be profoundly altered by the concomitant expression of ErbB2.

PLC-gamma activation is required for PDGF-betaR-mediated mitogenesis and monocytic differentiation of myeloid progenitor cells.

To investigate the molecular mechanisms mediating hematopoietic cell differentiation and mitogenesis by activation of the platelet-derived growth factor beta receptor (PDGF-betaR), the wild type PDGF-betaR (PDGF-betaRWT) and tyrosine to phenylalanine mutants of the PDGF-betaR, including F751, F966, F970, F1009, F1021 and F1009/F1021 were overexpressed in FDC-P2 myeloid progenitor cells by retroviral-mediated gene transfer. Stimulation of PDGF-betaRWT and F966, F970 and F1009 infectants with PDGF-BB led to the increased expression of monocytic differentiation markers. In contrast, activation of PDGF-betaR in the parental line or the F1021 or F1009/F1021 mutant infectants failed to induce monocytic differentiation. PDGF-BB stimulation of PDGF-betaRWT, F751, F966, F970 and F1009 infectants led to pronounced DNA synthesis, whereas F1021 and F1009/F1021 infectants did not reveal any increase in mitogenesis when compared to that of the FDC-P2 line. While PDGF stimulation of FDC-P2 cells overexpressing PDGF-betaRWT led to a pronounced increase in inositol phosphate formation due to phospholipase C-gamma (PLC-gamma) activation, PDGF-BB induced phosphoinositol hydrolysis was completely abolished in the F1021 and F1009/F1021 infectants. GF 109203X, a specific inhibitor of protein kinase C (PKC) activation, fully blocked PDGF-betaR-mediated monocytic differentiation and mitogenesis. Taken together, these results suggest that stimulation of the PDGF-betaR signaling pathway can mediate monocytic differentiation when PDGF-betaR is expressed at sufficient levels and that activation of PLC-gamma and PKC plays a pivotal role in PDGF-betaR-mediated differentiation and mitogenesis in FDC-P2 cell system.

Jak1 expression is required for mediating interleukin-4-induced tyrosine phosphorylation of insulin receptor substrate and Stat6 signaling molecules.

The Jak1, Jak2, Jak3, and Fes tyrosine kinases have been demonstrated to undergo tyrosine phosphorylation in response to interleukin (IL)-4 stimulation in different cell systems. However, it is not clear which, if any, of these kinases are responsible for initiating IL-4-induced tyrosine phosphorylation of intracellular substrates in vivo. In the present study, we have utilized a mutant Jak1-deficient HeLa cell line, E1C3, and its parental Jak1-expressing counterpart, 1D4, to analyze the role of Jak1 in mediating IL-4-induced tyrosine phosphorylation events. IL-4 treatment rapidly induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 in 1D4 but not in E1C3 cells. IL-4-mediated tyrosine phosphorylation of Stat6 was pronounced in 1D4 cells, while no IL-4-induced Stat6 phosphorylation was detected in E1C3 cells. IL-4 also induced Stat6 DNA binding activity from lysates of 1D4 but not E1C3 cells utilizing a radiolabeled immunoglobulin heavy chain germline epsilon promotor sequence (Iepsilon) in an electrophoretic mobility shift assay. Reconstitution of Jak1 expression in E1C3 cells restored the ability of IL-4 to induce IRS and Stat6 tyrosine phosphorylation. These results provide evidence that Jak1 expression is required for mediating tyrosine phosphorylation and activation of crucial molecules involved in IL-4 signal transduction.

Stimulation of the platelet-derived growth factor beta receptor signaling pathway activates protein kinase C-delta.

The murine myeloid progenitor cell line 32D was recently shown to undergo monocytic differentiation when protein kinase C-delta (PKC-delta) was overexpressed and activated by 12-O-tetradecanoylphorbol-13-acetate (TPA) (H. Mischak, J.H. Pierce, J. Goodnight, M.G. Kazanietz, P.M. Blumberg, and J.F. Mushinski, J. Biol. Chem. 268:20110-20115, 1993). Tyrosine phosphorylation of PKC-delta occurred when PKC-delta-transfected 32D cells were stimulated by TPA (W. Li, H. Mischak, J.-C. Yu, L.-M. Wang, J.F. Mushinski, M.A. Heidaran, and J.H. Pierce, J. Biol. Chem. 269:2349-2352, 1994). In order to elucidate the role played by PKC-delta in response to activation of a receptor tyrosine kinase, we transfected platelet-derived growth factor beta receptor (PDGF-beta R) alone (32D/PDGF-beta R) or together with PKC-delta (32D/PDGF-beta R/PKC-delta) into 32D cells. NIH 3T3 cells which endogenously express both PDGF-alpha R and PDGF-beta R were also transfected with PKC-delta (NIH 3T3/PKC-delta). Like TPA treatment, PDGF-BB stimulation caused striking phosphorylation of PKC-delta in vivo and translocation of some PKC-delta from the cytosol fraction to the membrane fraction in both cell systems. Some of the phosphorylation induced by PDGF-BB treatment was found to be on a tyrosine residue(s). Tyrosine-phosphorylated PKC-delta was observed only for the membrane fraction after stimulation with PDGF-BB or TPA. The enzymatic activity of PKC-delta in the membrane fraction also increased after stimulation with TPA or PDGF, providing a positive correlation between PKC-delta tyrosine phosphorylation and its activation. Overnight treatment of 32D/PDGF-beta R/PKC-delta cells with PDGF-BB induced monocytic differentiation as judged by an increase in expression of cell surface macrophage differentiation markers. PDGF-BB had much weaker effects on 32D/PDGF-beta R cell differentiation, suggesting that increased PKC-delta expression enhanced monocytic differentiation. These results indicate that PKC-delta is a downstream molecule in the PDGFR signaling pathway and may play a pivotal role in PDGF-beta R-mediated cell differentiation.

Functional evidence for a second tumor suppressor gene on human chromosome 17.

The development and progression of human tumors often involves inactivation of tumor suppressor gene function. Observations that specific chromosome deletions correlate with distinct groups of cancer suggest that some types of tumors may share common defective tumor suppressor genes. In support of this notion, our initial studies showed that four human carcinoma cell lines belong to the same complementation group for tumorigenic potential. In this investigation, we have extended these studies to six human soft tissue sarcoma cell lines. Our data showed that hybrid cells between a peripheral neuroepithelioma (PNET) cell line and normal human fibroblasts or HeLa cells were nontumorigenic. However, hybrid cells between the PNET cell line and five other soft tissue sarcoma cell lines remained highly tumorigenic, suggesting at least one common genetic defect in the control of tumorigenic potential in these cells. To determine the location of this common tumor suppressor gene, we examined biochemical and molecular polymorphic markers in matched pairs of tumorigenic and nontumorigenic hybrid cells between the PNET cell line and a normal human fibroblast. The data showed that loss of the fibroblast-derived chromosome 17 correlated with the conversion from nontumorigenic to tumorigenic cells. Transfer of two different chromosome 17s containing a mutant form of the p53 gene into the PNET cell line caused suppression of tumorigenic potential, implying the presence of a second tumor suppressor gene on chromosome 17.

Production of placental protein 5 (PP5) by non-malignant human fibroblasts in culture. Comparison with 'pregnancy-specific' glycoprotein (SP1).

Placental protein 5 (PP5) was detected by radioimmunoassay in the culture media from 11/19 (58%) of cell lines derived from non-malignant human fibroblasts. One line was studied in detail. Its rate of PP5 secretion was polyphasic, with maxima of 3.1 pmol/10(6) cells per 24 h on day 1, 1.7 on day 8 and 2.0 on day 14, and minima of 0.9 on day 4, 1.0 on day 10 and 0.7 on day 15 of culture. A discordance with the secretion of pregnancy-specific glycoprotein (SP1) was demonstrated; the SP1 concentration was constant initially at 2.9 pmol/10(6) cells per 24 h, followed by a steady decrease to 0.5 on day 15. A small amount of PP5 was retained in the cells, but usually less than 5% of that secreted. When the culture medium was changed daily, more PP5 and SP1 were produced than when the cells were grown in conditioned media. A cell line derived from a human fibrosarcoma produced SP1 at rates exceeding that of the normal fibroblasts, but PP5 production was not detected in the fibrosarcoma.

Cellular genes analogous to retroviral onc genes are transcribed in human tumour cells.

Polyadenylated RNAs of certain human tumour cell lines are shown to contain transcripts related to the cell-derived transforming onc genes of molecularly cloned primate, murine or avian transforming retrovirus genomes. Thus, analogues of retroviral transforming genes are both present and frequently expressed in human neoplastic cells.

Ectopic production of "pregnancy-specific" beta-1 glycoprotein in vitro: discordance with three other placental proteins.

"Pregnancy-specific" beta-1 glycoprotein (SP1) was produced in vitro by nine of 32 established cell lines derived from human malignant neoplasms. The nine positive lines comprised a variety of cell types, and SP1 production ranged from 0.36 to 35.5 pmol/mg cell protein at confluence. The highest production was by a fibrosarcoma line. SP1 in medium and cells from this line was indistinguishable immunochemically and gel chromatographically from purified (Bohn) placental SP1. None of the SP1-positive lines produced placental lactogen, only three produced the alpha subunit of chorionic gonadotropin (CG), and only four produced the beta subunit of CG. SP1-positive lines that produced cg-alpha did not produce CG-beta. Secretion of SP1 and of CG-beta by the fibrosarcoma line followed different time courses.

Ultrastructural studies of surface features of human normal and tumor cells in tissue culture by scanning and transmission electron microscopy.

Human tumors of a variety of histopathologic types have been established in tissue culture. The surface features of these cell lines were investigated by scanning electron microscopy (SEM) with the use of new techniques for specimen preparation. Tumor cells demonstrated striking degrees of surface activity with numerous microvilli, filopodia, blebs, and ruffles. Intercellular contacts were also prominent in cultures of most solid tumors observed by SEM. At low cell density, normal human fibroblasts exhibited some surface features such as microvilli and blebs, but at higher cell density they lacked extensive surface modifications. By transmission electron microscopy (TEM), the cytoskeleton of normal fibroblasts was shown to be well organized, with parallel orientation of microfilaments, filaments, and microtubules. These structures were also in tumor cells, but they lacked the degree of organization of fibroblasts. Desmosomes were readily demonstrated in normal fibroblasts and carcinoma cells in culture but not in sarcomas, melanomas, or tumors of neural origin. These studies have provided the first correlative SEM and TEM analyses of solid human tumor cells of diverse pathologic types in vitro.

Effect of cytosine arabinoside, adenine arabinoside, tilorone, and rifamycin SV on multiplication of Herpesvirus saimiri in vitro.

The multiplication of Herpesvirus saimiri was inhibited in vitro by cytosine arabinoside, adenine arabinoside, and tilorone, but not by rifamycin SV.