transforming-growth-factor-alpha and Cell-Transformation--Viral

Normal human keratinocytes grown under serum-free conditions can be triggered to differentiate by exposure to serum or to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). We found that TPA treatment of human papillomavirus (HPV) type 16-immortalized cells in culture induced formation of cornified envelopes indicative of squamous differentiation. Concurrent with differentiation, TPA inhibited the expression of HPV 16 E6 and E7 mRNA transcripts. Adaptation of the immortalized cells to growth in serum-containing medium led to the selection of a subpopulation of HPV-transformed cells that was resistant to TPA-induced differentiation. In this cell line, a transient suppression of HPV transcripts was observed at 5 h, whereas in differentiation-resistant, carcinoma-derived lines, TPA had little effect on HPV oncogene expression. c-myc transcripts were suppressed for the duration of exposure to TPA in only the differentiation-competent cells; c-fos and c-jun were transiently induced in all cell lines. Transforming growth factor-alpha mRNAs were also increased approximately eightfold as HPV 16-immortalized cells were induced to differentiate. These results demonstrate that, in HPV 16-immortalized keratinocytes, acquisition of resistance to inducers of squamous differentiation is accompanied by altered regulation of cell growth and gene expression.

Topics: Cell Differentiation; Cell Division; Cell Transformation, Viral; Gene Expression Regulation, Viral; Humans; In Vitro Techniques; Keratinocytes; Oncogenes; Papillomaviridae; Proto-Oncogenes; RNA, Messenger; RNA, Viral; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha; Virus Replication

The secretion of transforming growth factor alpha (TGF alpha) and the expression of cell-surface receptors for epidermal growth factor (EGF) were measured in a series of human mammary cell lines. The amount of TGF alpha secreted by the cells did not correlate with the phenotype of the cells (epithelial or myoepithelial), the mechanism of immortalization of the cells (SV40 or spontaneous) or the source of the cells (normal mammary gland, benign hyperplastic lesion, malignant tumour). The level of expression of cell-surface receptors for EGF was markedly increased as a consequence of SV40-immortalization of mammary cells, but otherwise did not correlate with the phenotype of the cells or the source of the cells. Much of the increase was accounted for by the appearance of a large number of low-affinity receptors for EGF in the SV40-immortalized cells. It is suggested that one of the mechanisms whereby SV40-immortalization suppresses the senescence of primary cultures of human mammary epithelial cells involves increasing the level of expression of receptors for EGF. In contrast the level of secretion of TGF alpha by cells in culture is probably a consequence of the mechanisms of adaptation of each cell line to culture conditions, and does not reflect the level of secretion of TGF alpha by cells in vivo.

Topics: Breast; Cell Line; Cell Transformation, Viral; Epithelial Cells; Epithelium; ErbB Receptors; Female; Humans; Phenotype; Simian virus 40; Transforming Growth Factor alpha

The epidermal growth factor receptor, EGFR, has been implicated in cell transformation in both mammalian and avian species. The v-ErbB oncoprotein is an oncogenic form of the chicken EGFR. The tyrosine kinase activity of this oncoprotein is required for transformation, but no transformation-specific cellular substrates have been described to date. Recently activation of the ras signal transduction pathway by the EGFR has been shown to involve the Shc and Grb2 proteins. In this communication, we demonstrate that the Shc proteins are phosphorylated on tyrosine residues and are complexed with Grb2 and the chicken EGFR following ligand activation of this receptor. In fibroblasts and erythroid cells transformed by the avian erythroblastosis virus (AEV) strains H and ES4, the Shc proteins are found to be constitutively phosphorylated on tyrosine residues. The tyrosine-phosphorylated forms of the AEV strain H v-ErbB protein are found in a complex with Shc and Grb2, but the Shc proteins do not bind to the AEV strain ES4 v-ErbB protein. Mutant forms of the v-ErbB protein (in which several of the tyrosines that become autophosphorylated have been deleted by truncation) are unable to transform erythroid cells but can still transform fibroblasts. Analysis of cells transformed by one of these mutants revealed that the truncated v-ErbB protein could no longer bind to either Shc or Grb2, but this oncoprotein still gave rise to tyrosine-phosphorylated Shc proteins that complexed with Grb2 and led to activation of mitogen-activated protein (MAP) kinase. The results suggest that stable binding of Grb2 and Shc to the v-ErbB protein is not necessary to activate this signal transduction pathway and assuming that the mutant activate MAP kinase in erythroid cells in a manner similar to that of fibroblasts, that activation of this pathway is not sufficient to transform erythroid cells.

Topics: Alpharetrovirus; Animals; Blotting, Western; Calcium-Calmodulin-Dependent Protein Kinases; Cell Division; Cell Transformation, Viral; Cells, Cultured; Chick Embryo; Enzyme Activation; ErbB Receptors; Erythroblasts; Fibroblasts; Humans; Oncogene Proteins v-erbB; Oncogene Proteins, Viral; Oncogenes; Phosphorylation; Proteins; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Receptor, ErbB-2; Retroviridae Proteins, Oncogenic; Signal Transduction; Transforming Growth Factor alpha

We immortalized oral keratinocytes by transfecting them with recombinant human papillomavirus (HPV) type 18 DNA and established three cell lines. These lines were morphologically different from their normal counterpart, contained integrated entire HPV-18 DNA, and expressed the viral E6/E7 genes. The cells contained less p53 protein and more c-myc mRNA than normal cells. However, they proliferated only in keratinocyte growth medium (KGM) containing low calcium and were not tumorigenic in nude mice. To test the hypothesis that tumors result from the combined effect of a "high-risk" HPV and chemical carcinogens in the human oral cavity, we exposed the immortalized cells to the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine. Three chemically transformed cell colonies were isolated. These cells (a) proliferated well in both KGM and Dulbecco's modified minimum essential medium containing physiological levels of calcium; (b) were capable of proliferating in nude mice; (c) contained intact, integrated HPV-18 sequences; (d) transcribed substantially more HPV-18 E6/E7, transforming growth factor-alpha, and c-myc than the immortalized counterpart; and (e) contained, like the immortalized counterpart, less wild-type p53 protein and DCC message. These data indicate that human oral keratinocytes can be transformed by sequential exposure of normal keratinocytes to a "high-risk" HPV and chemical carcinogens.

Topics: Animals; Base Sequence; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA, Viral; ErbB Receptors; Gene Expression; Genes, myc; Genes, p53; Humans; Keratinocytes; Methylnitronitrosoguanidine; Mice; Mice, Nude; Molecular Sequence Data; Mouth Neoplasms; Papillomaviridae; RNA, Messenger; RNA, Viral; Transcription, Genetic; Transfection; Transforming Growth Factor alpha

We immortalized oral keratinocytes by transfection with recombinant human papillomavirus type 16 (HPV-16) DNA and established two cell lines, human oral keratinocytes-16A (HOK-16A) and -16B (HOK-16B). These cell lines were morphologically different from the normal counterpart, contained HPV-16 DNA as integrated form and expressed numerous viral genes. However, these cells proliferated only in culture medium containing low calcium (0.15 mM) and are not tumorigenic in nude mice. To test the hypothesis that tumors can be developed by sequential combined effect of human papillomavirus and chemical carcinogens in the oral cavity, these immortalized cell lines were chemically transformed by exposure to either benzo[a]pyrene or methanesulfonic acid ethyl ester. Such transformants proliferated in medium containing physiological calcium levels (1.5 mM) and demonstrated enhanced growth potential in nude mice, whereas primary human oral keratinocytes treated with these chemical carcinogens failed to show any evidence of transformation. Chemically transformed cells contained integrated, intact HPV-16 sequences and transcribed significantly higher amount of HPV-16 E6/E7 messages and transforming growth factor-alpha (TGF-alpha) compared with the immortalized oral keratinocytes. Like the HPV-immortalized cell lines, the chemically transformed oral keratinocytes contained lower levels of newly synthesized, wild-type p53 proteins compared to normal cells, and expressed wild-type c-Ha-ras. These results indicate that this in vitro system is useful for investigating the mechanisms of multistep oral carcinogenesis.

Topics: Base Sequence; Blotting, Southern; Carcinogens; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cocarcinogenesis; DNA, Viral; Humans; Keratinocytes; Molecular Sequence Data; Papillomaviridae; Polymerase Chain Reaction; Proto-Oncogene Proteins p21(ras); RNA, Messenger; RNA, Viral; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

c-erbB, encoding the EGF receptor (EGF-R), was originally identified as the cellular homolog of a chicken leukemia oncogene. In humans, EGF-R is distributed widely except in hemopoietic tissues, and its amplification is associated with epidermal and glial malignancies. Here we show that c-erbB is present in normal chicken erythrocytic progenitors and transmits the mitogenic signal induced by TGF alpha. Cells that contain high affinity EGF-R are at approximately the BFU-E stage, and their long-term renewal can be induced by TGF alpha. Upon addition of insulin and erythropoietin, they can be induced to terminally differentiate into red cells. We previously demonstrated that v-erbA blocks differentiation of chicken erythrocytic progenitors but does not abrogate their growth factor dependence for proliferation. These data indicate that proliferation and differentiation are not necessarily coupled in these cells. They also demonstrate a direct role of c-erbB in the control of self-renewal of normal chicken erythrocytic progenitors and could account for the predominant leukemogenic potential of the chicken erbB gene.

Topics: Animals; Bone Marrow Cells; Cell Differentiation; Cell Division; Cell Transformation, Viral; Cells, Cultured; Chickens; ErbB Receptors; Erythroid Precursor Cells; Erythropoietin; Insulin; Oncogene Proteins v-erbA; Oncogenes; Proto-Oncogene Proteins; Receptors, Colony-Stimulating Factor; Retroviridae Proteins, Oncogenic; Transforming Growth Factor alpha

The middle tumor antigen (mT) of polyomavirus is unable to transform a clone of NIH 3T3 cells to anchorage independence (L. Raptis and J.B. Bolen, J. Virol. 63:753-758, 1989). However, this oncogene increased the responsiveness of these cells to the growth factors (alpha-like and beta-type transforming growth factors) produced by cells possessing the whole transforming region of polyomavirus. This resulted in the growth of NIH 3T3 cells, expressing mT under control of the dexamethasone-regulatable mouse mammary tumor virus promoter, in agar medium supplemented with these growth factors upon addition of the inducer. Therefore, mT, a transforming oncogene, is able to enhance the responsiveness of established cells to growth factors, a property previously attributed primarily to myc and other establishment type oncogenes.

Topics: Animals; Antigens, Polyomavirus Transforming; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Mice; Oncogenes; Phosphorylation; Promoter Regions, Genetic; Transforming Growth Factor alpha; Transforming Growth Factor beta