involucrin and Cell-Transformation--Viral

Calcium is an important regulator of normal human cervical cell differentiation; however, it is not known whether the calcium responsiveness of the cells would be altered by immortalization with human papillomavirus. In the present study we examine the effects of extracellular calcium on morphology, expression of biochemical markers of cervical cell differentiation, and HPV16 oncogene transcription in an HPV16-immortalized human cervical cell line, ECE16-1. ECE16-1 cells respond to increased calcium with increased levels of mRNA encoding cytokeratin K13, transglutaminase, and involucrin. Dose-response studies indicate that these markers are coordinately regulated and that maximal levels are present at calcium concentrations of > or = 0.4 mM. RNA levels begin to increase within 24 h after cells are shifted to medium containing high calcium and are maximal by 3 days. Protein levels change directly with the change in mRNA, suggesting that mRNA level determines protein concentration; however, transcription runoff experiments indicate that the increased mRNA does not result from new synthesis. Parallel studies indicate that expression of the HPV16 oncogenes, E6 and E7, are not regulated by extracellular calcium. These results are consistent with in vivo experiments showing that in advanced cervical intraepithelial neoplasia, where HPV16 DNA is integrated in host cell DNA, E6/E7 transcript production is not regulated in a differentiation-dependent manner.

Topics: Calcium; Cell Differentiation; Cell Transformation, Viral; Cervix Uteri; Epithelial Cells; Epithelium; Female; Gene Expression Regulation, Viral; Humans; In Vitro Techniques; Keratins; Oncogene Proteins, Viral; Oncogenes; Papillomaviridae; Papillomavirus E7 Proteins; Protein Precursors; Repressor Proteins; RNA, Messenger; Transglutaminases; Tumor Virus Infections

Human papillomavirus (HPV) DNAs are detected in approximately 90% of anogenital carcinomas. To assess directly the effect of HPV on squamous differentiation, normal human cervical and foreskin epithelial cells and cells immortalized by recombinant HPV DNAs were transplanted beneath a skin-muscle flap in athymic mice. Xenografts containing normal cells formed well-differentiated stratified squamous epithelia 2 to 3 weeks after transplantation, but cell lines immortalized by four HPV types (HPV16, HPV18, HPV31, and HPV33) detected in anogenital cancer exhibited dysplastic morphology and molecular alterations in gene expression characteristic of intraepithelial neoplasia. Morphological alterations were accompanied by delayed commitment to terminal differentiation, alterations in the pattern of involucrin expression, and reductions in levels of involucrin and keratin 1 RNAs. HPV18-immortalized cells developed dysplastic changes more rapidly than cells immortalized by HPV16 DNA. These results show that human genital epithelial cells immortalized by HPV DNAs detected in genital cancers undergo dysplastic differentiation in vivo.

Topics: Animals; Cell Adhesion; Cell Differentiation; Cell Line; Cell Transformation, Viral; Cervix Uteri; Epithelium; Female; Gene Expression Regulation, Viral; Genes, Viral; Humans; Keratins; Laminin; Male; Mice; Mice, Nude; Papillomaviridae; Penis; Protein Precursors; RNA; Transfection

Immortalized, but nontumorigenic, human keratinocyte cell lines have many potential therapeutic and experimental uses. We have utilized a recombinant retrovirus, encoding the simian virus 40 large T-antigen, to immortalize normal human epidermal keratinocytes. The KER-1 cells derived from the immortalization process grow without feeder layer support, but do not form colonies in soft agar. Morphologically, the KER-1 cells appear similar to nonimmortalized cells, except that stratification is somewhat reduced. The pattern of keratin gene expression in nonimmortalized and KER-1 cells is similar, except for the retinoid-dependent regulation of type I cytokeratin, K7, in the KER-1 cells. This keratin is not expressed in nonimmortalized keratinocytes, but is present at low levels in KER-1 cells. Incubation with trans-retinoic acid (20 or 200 nM) or retinol (200 or 2000 nM) results in a 40-fold increase in K7 expression in KER-1 cells. The cornified envelope precursor, involucrin, is expressed at normal levels in KER-1 cells. Moreover, as in nonimmortalized cells, KER-1 involucrin levels are not suppressed by retinoids. trans-Retinoic acid and retinol reduce envelope formation in both nonimmortalized keratinocytes and KER-1 cells. Surprisingly, the synthetic retinoid, Ro 13-6298 (p [(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphthyl)-propenyl]benzoic acid ethyl ester), a potent regulator of keratin gene expression, cornified envelope formation and morphological change in nonimmortalized cells, is completely inactive in KER-1 cells.

Topics: Antigens, Polyomavirus Transforming; Cell Transformation, Viral; Gene Expression Regulation; Humans; Keratinocytes; Keratins; Molecular Weight; Protein Precursors; Retinoids

Primary human epithelial cells were cotransfected with pHPV-18 and pSV2neo, and cell strains were generated by selecting in G418. One cell strain (FE-A), which exhibits an extended life span, is currently in its 30th passage. In comparison, control cultures can only be maintained up to the seventh passage. Southern blot analysis revealed the presence of at least one intact, integrated viral genome in these cells. FE-A cells showed altered growth properties, characterized by a change in morphology, and clonal density. Differentiation markers analyzed by Western blotting (immunoblotting), such as cytokeratins and involucrin, indicated that the cells resembled a partially differentiated epithelial population. Increased expression of the 40-kilodalton cytokeratin was observed in FE-A cells, similar to that observed in simian virus 40-immortalized human keratinocytes (M. Steinberg and V. Defendi, J. Cell Physiol. 123:117-125, 1985). FE-A cells were also found to be defective in their response to terminal differentiation stimuli. Calcium and 12-O-tetradecanoyl-phorbol-13-acetate treatment induced normal epithelial cells to differentiate, whereas the human papillomavirus 18 (HPV-18)-containing keratinocytes were resistant to these signals, indicating their partially transformed nature. These cells were not able to induce tumors in nude mice over a period of up to 8 months. A second cell strain, FE-H18L, also generated by transfecting HPV-18, also exhibited an extended life span and similar alterations in morphology. Viral RNA transcribed from the early region of HPV-18 was detected in both cell strains by Northern (RNA) blot analysis. These cell strains should provide a useful model for determining the role of HPV in carcinogenesis.

Topics: Animals; Calcium; Cell Differentiation; Cell Division; Cell Transformation, Viral; Cells, Cultured; DNA, Viral; Epithelial Cells; Epithelium; Gene Expression Regulation; Immunologic Techniques; Keratins; Male; Mice; Mice, Nude; Neoplasms, Experimental; Papillomaviridae; Protein Precursors; RNA, Messenger; RNA, Viral

SV-40 transformed human foreskin keratinocytes (line SV-K14) develop under conditions of serum starvation the competence to form cornified envelopes that are characteristic of terminally differentiating epidermal cells. In this cell line, the final assembly of the envelope does not occur spontaneously but must be induced using a calcium ionophore. Five potential precursor proteins with molecular weights of 140K, 90K, 61K, 53K, and 36K, respectively, could be detected in the extracts of envelope competent and noncompetent cells. The 61 kD and the 36 kD precursors were specifically decorated in immunoblots when using an antiserum directed against the purified cornified envelope of SV-K14 cells. The 140 kD protein was identified as involucrin by means of a commercial anti-involucrin antibody. Part of the 61 kD protein was found to be inserted into the plasma membrane after the cells gained envelope competence. The set of precursor proteins used by SV-K14 cells differed markedly from those described in the literature for epidermal cells in vivo and for normal human keratinocytes in vitro. Furthermore, cyanogen bromide cleavage of purified envelopes from transformed and normal keratinocytes revealed a completely different peptide pattern. This indicates that the exact molecular composition of the cornified envelope may not be strictly determined and may vary according to the availability of potential substrate proteins at the very moment when the cross-linking enzyme, the plasma membrane associated transglutaminase, becomes functional.

Topics: Cell Line; Cell Transformation, Viral; Electrophoresis; Humans; Keratins; Male; Protein Precursors; Simian virus 40; Skin; Subcellular Fractions; Tissue Distribution; Viral Envelope Proteins