Combined oral carcinogenicity of HPV-16 and benzo(a)pyrene: an in vitro multistep carcinogenesis model.

We previously immortalized normal human oral keratinocytes by transfection with recombinant HPV-16 DNA and subsequently exposed the cells to benzo(a)pyrene for 7 days. The exposure to benzo(a)pyrene modified the immortalized cells: the modified cells (HOK-16B-BaP) proliferated in an ordinary culture medium containing physiological calcium level (1.5 mM), but demonstrated only enhanced proliferation capacity without tumor formation in nude mice and failed to show in vitro anchorage-independency. In this study, we further modified the HOK-16B-BaP cells by subculturing the cells in a medium containing benzo(a)pyrene for 6 months. The cells were further modified with a chronic benzo(a)pyrene exposure and were termed HOK-16B-BaP-T cells (1) demonstrated a malignant phenotype in organotypic 'raft' culture, (2) showed in vitro anchorage-independency, (3) developed tumors in nude mice when injected subcutaneously, (4) contained a significantly higher copy number of intact and integrated HPV-16 DNA; (5) contained higher level of HPV-16 E6/E7 messages and E7 protein, (6) were more resistant to transforming growth factor-beta 1 and (7) secreted higher level of vascular endothelial growth factor with molecular weight of 56 kd than parental HOK-16B-BaP cells. However, the levels of p53 and ras proteins and the levels of p53, c-myc and c-fos transcripts in the HOK-16B-BaP-T cells were not different from those in the HOK-16B-BaP cells. The highly conserved coding regions of the p53, c-Ha-ras1, and c-Ki-ras2 genes of the tumor cells were not mutated. These data indicate that the HPV-immortalized human oral keratinocytes can convert to tumorigenic cells by chronic exposure to benzo(a)pyrene. The tumorigenic conversion seems to be associated with (1) the overexpression of viral oncogenes such as E6 and E7 genes, (2) the higher resistance of cells to transforming growth factor-beta 1 and (3) the high secretion of 56 kd vascular endothelial growth factor from the cells.

Abortive replication of influenza virus A/WSN/33 in HeLa229 cells: defective viral entry and budding processes.

Since influenza A virus replication is defective in HeLa229 cells but productive in Madin-Darby canine kidney (MDCK) cells, we have investigated the steps in the infectious cycle of A/WSN/33 virus defective in HeLa229 cells. We find that both the entry and exit processes of the infectious cycle were defective in HeLa229 cells. During entry, viral adsorption was apparently normal in HeLa229 cells but a subsequent step(s) involving one or more processes namely the fusion/uncoating and nuclear transport of viral ribonucleoprotein was inefficient and slow compared to those in MDCK cells. Fewer HeLa229 cells were infected at the same multiplicities of infection, resistance to ammonium chloride developed much more slowly and degradation of the incoming virus proteins was delayed when compared to those in MDCK cells. Subsequent to the entry process, there was no significant difference in either the synthesis of viral proteins or the transport, maturation, and membrane insertion of viral glycoproteins although the glycosylation pattern of hemagglutinin was different and the peak protein synthesis was albeit delayed in HeLa229 cells compared to that in MDCK cells. However, there was a major defect in the budding and release of viral particles. In HeLa229 cells, viral bud formation occurred but viral particles remained attached to the plasma membrane and were not released into the medium. This defect in virus release was not due to lack of neuraminidase activity but could be, at least partly, overcome by cytochalasin B treatment, suggesting a possible involvement of microfilaments in virus release. These results indicate that the abortive replication of influenza virus A/WSN/33 in HeLa229 cells appears to be due to multiple defects involving both the entry and release of viral particles and that host cell membrane and microfilaments may be important contributing factors in these processes.

Inactivation of the p53 gene by either mutation or HPV infection is extremely frequent in human oral squamous cell carcinoma cell lines.

The state of p53 tumour suppressor and the frequency of high-risk human papillomavirus (HPV) infections were studied in nine human oral cancer cell lines. Three cancer cell lines (SCC-4, Tu-177 and FaDu) had similar amounts of p53 transcripts to normal cells, but contained significantly higher levels of p53 protein than the normal control cells. Sequencing highly conserved open reading frames of the p53 gene of these cancer cells showed point mutations in the SCC-4 and Tu-177 cell lines, a base transition from CCC to TCC occurred at codon 151; and in the line FaDu, a mutation of CGG to CTG occurred at codon 248. The HEp-2 and 1483 cancer lines contained significantly lower levels of p53 protein compared to the normal counterpart. Sequencing of p53 cDNA for HEp-2 and 1483 lines showed no mutations, but northern analysis revealed that these cell lines expressed HPV-18 E6/E7 messages. Four cell lines (SCC-9, SCC-15, SCC-25, and Tu-139) expressed negligible amounts of p53 transcripts compared to the normal counterpart and undetectable levels of p53 protein. These cell lines contained mutations in the highly conserved open reading frames of the p53 gene as follows: the SCC-9 had a deletion of 32 base pairs between codons 274 and 285; the line SCC-15 had an insertion of five base pairs between codons 224 and 225; the line SCC-25 had a deletion of two base pairs in codon 209; and the Tu-139 line had a deletion of 46 base pairs between codons 171 and 186.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of UV-irradiation on cell cycle, viability and the expression of p53, gadd153 and gadd45 genes in normal and HPV-immortalized human oral keratinocytes.

We previously demonstrated neoplastic conversion of HPV-immortalized human oral keratinocytes by exposing cells to chemical carcinogens, but failed to transform normal human oral keratinocytes with same chemical carcinogens in vitro. Though the reason for different responses of normal and HPV-immortalized oral keratinocytes to chemical carcinogens remains speculative, the difference may be due to the capacity of normal cells and incapacity of HPV-immortalized cells for repairing damaged DNA induced by carcinogens. Since (1) the repair of damaged DNA takes place in G1/G2 phases of cell cycle, (2) wild type p53 plays major role in the induction of transient G1 and/or G2 arrests, and (3) the expression of gadd45 and gadd153 is also associated with the cell cycle arrest and DNA damage, we investigated transient cell cycle arrest and the expression of p53, gadd45 and gadd153 in normal human oral keratinocytes, HPV-immortalized oral keratinocytes, and an oral cancer cell line expressing mutant p53 after exposing cells to UV light. Normal cells demonstrated transient G1 arrest after exposure to UV light, but other tested cells did not. While UV-irradiation significantly increased the level of intranuclear wild type p53 protein in normal cells, it did not alter p53 protein levels in HPV-immortalized and oral cancer cells. The level of gadd45 transcripts was enhanced in all tested cells, but normal cells demonstrated higher increase in the level of gadd45 after UV-exposure compared to other tested cells. The level of gadd153 gene transcripts was only increased in normal oral keratinocytes after UV-irradiation. These data indicate that UV-induced transient G1 arrest in normal oral keratinocytes may be associated with both enhanced levels of intranuclear wild type p53 protein and gadd45 and gadd153 transcripts.

Recombinant RNA polymerase: inducible overexpression, purification and assembly of Escherichia coli rpo gene products.

The genes, rpoA, rpoB and rpoC of Escherichia coli, which encode the RNA polymerase alpha-, beta- and beta'-subunits, respectively, have been individually placed on expression plasmids under the control of the bacteriophage T7 promoter. Induction of the T7 RNA polymerase gene in host cells harboring each of the three plasmids resulted in the extensive overproduction of the three polypeptides. The overproduced subunits were purified and assembled into a functional enzyme, whose specific activity and dependence on the sigma-factor were indistinguishable from native RNA polymerase purified by conventional methods.