The study of the human pathogen papillomaviruses (HPVs) has been hampered by the inability to propagate the virus in tissue culture. The addition of 12-O-tetradecanoyl phorbol-13-acetate to the media of organotypic (raft) cultures increased expression of physiological markers of keratinocyte differentiation and concomitantly induced production of virions. Capsid production was detected in differentiated suprabasal cells. Virions approximately 54 nanometers in size were observed by electron microscopy in raft tissue cross sections in the suprabasal layers. Virions purified through isopycnic gradients were found to contain type 31b DNA and exhibited an icosahedral shape similar to that of papillomaviruses found in clinical samples.

Primary human keratinocytes are useful for studying the pathogenesis of many different diseases of the cutaneous and mucosal epithelia. In addition, they can form organotypic tissue equivalents in culture that can be used as epidermal autografts for wound repair as well as for the delivery of gene therapy. However, primary keratinocytes have a finite lifespan in culture that limits their proliferative capacity and clinical use. Here, we report that treatment of primary keratinocytes (originating from 3 different anatomical sites) with Y-27632, a Rho kinase inhibitor, greatly increased their proliferative capacity and resulted in efficient immortalization without detectable cell crisis. More importantly, the immortalized cells displayed characteristics typical of primary keratinocytes; they had a normal karyotype and an intact DNA damage response and were able to differentiate into a stratified epithelium. This is the first example to our knowledge of a defined chemical compound mediating efficient cell immortalization, and this finding could have wide-ranging and profound investigational and medical applications. IntroductionSomatic cells have a limited lifespan, gradually slow in growth, and stop dividing, a process known as cellular senescence. This process is thought to limit the vulnerability of aging cells to disease. Human keratinocytes are invaluable for the study of skin biology and the pathogenesis of skin-related diseases, but their short lifespan in culture is a limitation. Different conditions have been developed to optimize the culture of keratinocytes; for example, the presence of fibroblast feeder cells increases the proliferative capacity of primary keratinocytes from approximately 20 to 40-60 population doublings (1, 2). Spontaneously immortalized keratinocyte lines, for example HaCaT (3) and NIKS (4), have been used for skin-related research. However, these cell lines have genetic abnormalities, such as mutations in p53 (5) or isochromosomes (4).Continuous replication of primary human cells is blocked by 2 separate events: mortality stage 1 (M1; replicative senescence) and mortality stage 2 (M2; crisis). At M1, signaling by shortened telomeres results in activation of the p53 and pRB pathways. M2 represents a critical period of genomic instability, with extremely eroded telomeres, resulting in chromosomal fusions and translocations. In retinal pigment epithelial cells and foreskin fibroblasts (which have decreased expression of components of the p16INK4A/pRB pathway), telomerase expression is sufficient to bypass both M1 and M2 and stabilize and elongate chromosome ends (6). However, telomerase expression is not sufficient for immortalization of keratinocytes, and p16INK4A function must also be disrupted (7,8).Keratinocytes expressing the E6 and E7 proteins encoded by high-risk human papillomavirus (HPV) types bypass both M1 and M2 blocks and become immortal (reviewed in ref. 9). E7 inactivates and degrades the pRB retinoblastoma tumor suppressor protein to induce G 1 /S phase progression of th...

Cellular and viral microRNAs (miRNAs) are the transcriptional products of RNA polymerase II and are regulated by transcriptional factors for their differential expression. The altered expression of miRNAs in many cancer types has been explored as a marker for possible diagnosis and therapy. We report in this study that oncogenic human papillomaviruses (HPVs) induce aberrant expression of many cellular miRNAs and that HPV18 infection produces no detectable viral miRNA. Thirteen abundant host miRNAs were specifically regulated by HPV16 and HPV18 in organotypic raft cultures of foreskin and vaginal keratinocytes as determined by miRNA array in combination with small RNA sequencing. The increase of miR-16, miR-25, miR-92a, and miR-378 and the decrease of miR-22, miR-27a, miR-29a, and miR-100 could be attributed to viral oncoprotein E6, E7, or both, all of which are known to target many cellular transcription factors. The examination of 158 cervical specimens, including 38 normal, 52 cervical intraepithelial neoplasia (CIN), and 68 cervical cancer (CC) tissues, for the expression of these eight miRNAs showed a remarkable increase of miR-25, miR-92a, and miR-378 with lesion progression but no obvious change of miR-22, miR-29a, and miR-100 among the HPV-infected tissues. Further analyses indicate that an expression ratio ≥1.5 of miR-25/92a group over miR-22/29a group could serve as a cutoff value to distinguish normal cervix from CIN and from CIN to CC. oncogenes E6 and E7 | noncoding RNAs | regulatory RNAs | virol oncogenesis | DNA tumor viruses