Differential early viral gene expression in two stages of human papillomavirus type 16 DNA-induced malignant transformation.

Abstract

Human papiflomavirus (HPV) (6,11,34). Furthermore, specific diseases are associated with specific HPVs (17,22,26,28). For example, HPVs 16 and 18 are preferentially associated with human cervical and oral carcinomas (3,8,47,48). Analysis of cancer biopsies and some cell lines derived from human cervical carcinoma has demonstrated that integrated HPV 16 and 18 DNAs expressed transcriptional activity (10,20,35,47); specific transcripts have been mapped to the E6-E7 and El early gene regions (33,35,38,47). In a few cases, however, viral DNA was not detected in tumor cells (20,47) (Fig. 1); the pBR322 poison sequences (21) are absent in pdMMTneo.Transfection. The transfection procedure previously described (45) was modified; 2 to 5 ,ug of plasmid DNA for 1 x 105 to 5 x 105 NIH 3T3 cells was transfected without carrier DNA. Metals in the medium were sufficient to induce the mouse methallothionein promotor. For the focus-forming assay, transfected NIH 3T3 cells were maintained by refeeding in Dulbecco modified minimal essential medium supplemented with 10% bovine calf serum (GIBCO Laboratories). Cell lines derived from morphologically transformed foci were designated PM3T3Fo. A portion of the transfectants was selected with 400 ,g of G418 (GIBCO) per ml 10 to 14 days after transfection, and individual G418-resistant colonies were isolated. After selection and isolation, these lines (PM3T3G°) were expanded and grown without added G418.RNA extraction and enrichment of poly(A)-terminated mRNA. Subconfluent cultured cells were lysed with a 6 M guanidine thiocyanate solution containing 5 mM sodium citrate, 0.5% sodium sarcosyl, and 0.1 M ,B-mercaptoethanol. After high-molecular-weight cellular DNA was sheared by passing the lysate through a 20-gauge needle several times, the lysate was overlaid on a 6.1 M (1.52-g/ml) cesium trifluoroacetate cushion and centrifuged in an SW27 rotor at 25,000 rpm for 16 h at 15°C. Pelleted RNA was washed with 5 ml of 70% ethanol, dried, and dissolved in 1 ml of distilled water. After ethanol precipitation, RNA was redissolved in H20 and stored at -70°C. Poly(A)-terminated mRNA was 2165 on May 8, 2018 by guest

Characterization of Bovine Papillomavirus Type 1-transformed Clones which Show Distinct Transformed Phenotypes

“…Viral factors by themselves cannot specify the degree of the transformed phenotype in BPV-l-transformed Fisher rat embryo cells (Babiss & Fisher, 1986), a finding similar to that for HPV-16-transformed NIH3T3 cells (Yasumoto et al, 1987). Our study suggests a relationship between protein changes and transformed cell phenotypes using clones which show a distinct transformed phenotype.…”

Functional mapping of the human papillomavirus type 11 transcriptional enhancer and its interaction with the trans-acting E2 proteins.

“…Electron microscopic heteroduplex analyses (Croissant et al 1982;Beaudenon et al 1986;Broker and Chow 1986;Chow et al 1987c;Krubke et al 1987) and nucleotide sequence comparisons reveal that all papillomaviruses have similar genetic organization despite substantial nucleotide sequence divergence (Baker 1987). Consistent with this genetic homology, mRNAs of HPV-16, -1, -6, and-11 recovered from cervical carcinomas, plantar warts, and condylomata acuminata, respectively (Smotkin and Wettstein 1986;Chow et al 1987a, b;Nasseri et al 1987), and HPV-16 mRNAs from transformed mouse cells (Yasumoto et al 1987) are rather similar to those of bovine papillomavirus type 1 (BPV-1) present in fibropapillomas and in transformed mouse cells (Stenlund et al 1985;Yang et al 1985; and to those of cottontail rabbit papillomavirus in tumors (Nasseri and Wettstein 1984;Danos et al 1985;Phelps et al 1985). Production of viral messages relies upon alternative usage of promoters, mRNA splice sites, and polyadenylation sites for accessing the various open reading frames (ORFs) deduced from the DNA sequences.…”

Viral integration and fragile sites in human papillomavirus‐lmmortalized human keratinocyte cell lines

“…This activity has been mapped to the E6 and E7 viral genes (Bedell et al, 1989;Hawley-Nelson et al, 1989;Watanabe et al, 1989). Although these genes clearly have the capacity to immortalize, prolonged passage in culture or cooperation with activated rus has been necessary for full conversion to a malignant phenotype (Matleshewski et al, 1987;Yasumoto et al, 1987;Durst et al, 198713;Hurlin et al, 1991). Cells immortalized by transfection with HPV DNA generally contain a variable number of HPV sequences integrated at one or more chromosomal sites (Durst et al, 1987b;Kaur and McDougall, 1988;Schlegel et al, 1988;Watanabe et al, 1989).…”