To determine whether integration of human papillomavirus (HPV) DNA sequences could lead to the deregulation of genes implied in oncogenesis, we analysed the HPV integration sites in a series of nine cell lines derived from invasive genital carcinomas. Using in situ hybridization, HPV16 or 18 sequences were found at chromosome band 8q24, the localization of MYC, in IC1, IC2, IC3, IC6 and CAC-1 cells and at other sites in IC4, IC5, IC7 and IC8 cells. We then localized viral sequences at the molecular level and searched for alterations of MYC structure and expression in these cells. MYC genomic status and viral integration sites were also analysed in primary tumors from which IC1, IC2, IC3 and IC6 cells were derived. In IC1, IC2 and CAC-1 cells, HPV DNA was located within 58 kb of MYC, downstream, upstream, or within MYC. In IC3 and IC6 cells, HPV DNA was located 400-500 kb upstream of MYC. Amplification studies showed that, in IC1, IC2 and IC3, viral and MYC sequences were coamplified in an amplicon between less than 50 and 800 kb in size. MYC amplification was also observed in primary tumors, indicating that this genetic alteration, together with viral insertion at the MYC locus, had already taken place in vivo. MYC was not amplified in the other cell lines. MYC mRNA and protein overexpression was observed in the five cell lines in which the HPV DNA was inserted close to the MYC locus, but in none of the lines where the insertion had occurred at other sites. MYC activation, triggered by the insertion of HPV DNA sequences, can be an important genetic event in cervical oncogenesis.

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We previously reported that mouse NIH 3T3 cells transformed by transfection of activated human c-Ha-ras become apparently normal upon treatment with the antibiotic azatyrosine. The revertant cells maintain their normal phenotype during prolonged culture in the absence of azatyrosine, although activated p21 is still expressed. The normal phenotype induced by azatyrosine could be due to activation of expression of some cellular gene(s) in the cells that results in suppression of ras function. To identify the genes with increased expression in the revertant cells, we adopted differential screening of recombinants from a phage cDNA library made from mRNA of the revertant cells, hybridized with 32P-labeled cDNAs made from mRNAs of the rs-transformed NIH 3T3 cells and the revertant cells. Two clones thus isolated were found to be almost identical to the ras recision gene (rrg), the clones were found to contain a sequence corresponding to that of the murine retrovirus-like intracisternal A particle. We speculate that azatyrosine activates several cellular genes in the ras-transformed cells and that some of these genes, including prg, act cooperatively to counteract ras function, resulting in reversion of the ras-transformed cells to the normal phenotype.Mouse NIH 3T3 cells that have been transformed by insertion of activated c-Ha-ras, c-Ki-ras, N-ras, c-raf, or c-erbB-2 (neu) can be converted to an apparently normal phenotype by incubation for 6 days in culture medium containing the antibiotic azatyrosine [L-P-(5-hydroxy-2-pyridyl)alanine] (ref. 1 and unpublished results). The apparently normal flat revertant cells show contact inhibition, cannot proliferate in soft agar, and scarcely form tumors in nude mice, although the activated oncogenes inserted are still expressed in the revertant cells. The reversion induced by azatyrosine is permanent, and the phenotype of the revertant cells does not change during prolonged culture in the absence of azatyrosine. Since the reversion efficiency is quite high (more than 85% of the cells showed reversion, and the other 15% were killed on treatment with azatyrosine), it is likely that an epigenetic change takes place in the revertant cells, and the gene(s) specifically expressed in the revertant cells counteracts ras, raf, or erbB-2 function, acting as a tumor-suppressor gene. To determine which genes are activated in the revertant cells, we prepared a AgtlO cDNA library from the revertant cells and screened it by differential hybridization with 32p-labeled cDNAs from the original ras-transformed NIH 3T3 cells and the revertant cells. Among 30 clones thus isolated, two were found to be nearly identical with the ras recision gene (rrg), § which was isolated as a tumor-suppressor gene by Contente et al. (2) and subsequently identified as the gene for lysyl oxidase (3). The other clones thus far identified were those of collagen type III and rhoB. They were not expressed in normal NIH 3T3 cells or in ras-transformed NIH 3T3 cells. Approximately half the 30 clones were found t...

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