Establishment of a human keratinocyte cell line carrying complete human papillomavirus type 1 genomes: lack of vegetative viral DNA synthesis upon keratinization.
A human papillomavirus type 1 (HPV-1)/pBR322 recombinant plasmid was constructed consisting of two complete HPV-1 genomes in a head-to-tail arrangement, inserted into the BamHI site of pBR322. To obtain established human keratinocytes keratinocytes carrying dimeric HPV-1, origin-minus simian virus 40 (SV40) DNA was used as a dominant transforming marker in co-transfection experiments performed with cultured human foetal keratinocytes. Using the Southern blotting technique, HPV-1 DNA was detected in one out of five SV40-transformed human keratinocyte cell lines which were obtained in this way. Further blotting experiments using SV40, pBR322 and HPV-1 DNA as probes revealed patterns of hybridization which were consistent with co-integration of SV40 DNA with between two and four copies of the HPV-1 genome. The HPV-1 sequences in this cell line were virtually non-methylated and were transcribed at a lower level than SV40 mRNAs in the same cultured cells. A low level of monomeric HPV form I DNA was detected by blotting of undigested total cellular DNA. No evidence for a stimulation of form I HPV DNA replication could be obtained by blotting analysis of total DNA extracted from keratinizing cysts, which were formed by subcutaneous inoculation of these cells into nude mice.
D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004261:DNA Replication; D004274:DNA, Recombinant / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics; D005260:Female; D005333:Fetus; D005814:Genes, Viral; D006801:Humans; D007633:Keratins / Q000096:biosynthesis*; D009693:Nucleic Acid Hybridization; D027383:Papillomaviridae / Q000235:genetics*; D011247:Pregnancy; D013539:Simian virus 40 / Q000235:genetics; D012867:Skin / Q000196:embryology
D004274:DNA, Recombinant; D004279:DNA, Viral; D007633:Keratins