RESUMO
We previously immortalized normal human oral keratinocytes (NHOK) by transfection with cloned human papillomavirus type 16 (HPV-16) genome and converted these immortalized cells to tumorigenic cells with chemical carcinogens. Since the tumorigenic cells expressed higher level of HPV-16 E6/E7 transcripts, we predicted that enhanced E6/E7 expression was induced by mutations at the long control region (LCR) of the viral genome integrated into cellular chromosome. To test this possibility, we sequenced the entire HPV-16 LCR from immortalized and tumorigenic cells, but no difference in the sequences in all of the tested cells was observed. However, it is possible that such differences in the expression of E6/E7 could have originated from different activities of cellular transcription factors in the different cells. To examine this prospect, we subcloned entire LCR into a reporter gene and determined the promoter activity of LCR in immortalized and tumorigenic cells. We found that the LCR promoter activity was significantly higher in tumorigenic cells when comparing to immortalized cells. We also observed that at least 477 nucleotides upstream of E6 open reading frame are needed for the maximum LCR promoter activity in tumorigenic cells.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Queratinócitos/metabolismo , Mucosa Bucal/citologia , Papillomaviridae/genética , Proteínas Repressoras , Fatores de Transcrição , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular Transformada , Transformação Celular Neoplásica , Cloranfenicol O-Acetiltransferase/genética , DNA Viral/genética , DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Expressão Gênica , Regulação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Reporter/genética , Humanos , Queratinócitos/citologia , Mutação , Fatores de Transcrição NFI , Proteínas Nucleares , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Sequências Reguladoras de Ácido Nucleico/genética , Proteína 1 de Ligação a Y-BoxRESUMO
Normal human epithelial cells cannot proliferate and undergo apoptosis in the presence of transforming growth factor-beta (TGF-beta) in vitro, but many human epidermoid cancer cells are resistant to TGF-beta. Resistance to TGF-beta may thus, in part, be responsible for uncontrolled proliferation of cancer cells. Though detailed mechanisms for the resistance of cancer cells to TGF-beta remain unknown, resistance may be due to decreased expression of TGF-beta receptors from cancer cells. To investigate this possibility, we determined the expression of TGF-beta and type II TGF-beta receptor in primary normal human oral keratinocytes (NHOK), human papillomavirus-immortalized human oral keratinocytes (HOK-16B) and two tumor cell lines derived from HOK-16B (CTHOK-16B-BaP and CTHOK-16B-DMBA). Our results show that (1) the cellular and secretory TGF-beta levels in immortalized and tumor cells were notably lower than in NHOK and (2) the level of type II TGF-beta receptor of the tested cells was similar to each other. Taken together, the conversion of NHOK to tumorigenic cells may, in part, be due to the acquisition of NHOK resistance to TGF-beta through underexpression of this cytokine.