The human papillomaviruses (HPV) are major etiologic agents of several human cancers and have been found in essentially all cervical cancers and a significant number of other anogenital cancers as well as oral, laryngeal, and nasal cancers. Cervical cancers are associated with a high risk subset of HPVs of which HPV-16 is the most common. The E6 and E7 viral oncogenes interfere with the functions of p53 and pRb, respectively, and thus disrupt regulation of the cell cycle and promote genomic instability. The E6 protein has also been shown to induce expression of telomerase. Only a subset of individuals with HPV infection will develop persistent infection, and only some of those individuals will develop low-grade lesions and an even smaller number will develop high-grade lesions and invasive cervical cancer. It is likely that additional environmental and genetic factors predispose individuals to progression. In addition, progression of high risk HPV infection to high grade lesions and invasive cervical cancer requires significantly long lag periods, consistent with the accumulation of additional genetic and/or epigenetic changes. These changes are manifested by alterations in cellular gene expression as well as key changes in the biological behavior of the cells. The goals of this project are to use microarray gene expression profiling and other methods of assessing gene expression to: 1) Identify biomarkers that predict which lesions will progress to cancer and which will remain benign or regress. 2) Identify pathways disrupted during cervical cancer progression. 3) Identify changes in cellular gene expression induced by HPV infection. The first two goals are being pursued as part of a large molecular epidemiology study in collaboration with Drs. Sophia Wang, Mark Sherman, and Mark Schiffman of the Division of Cancer Epidemiology and Genetics, NCI. Biomarkers of risk for progressive cervical neoplasia: Ultimately we plan to use laser capture microdissection (LCM) of normal cervical epithelium, cervical pre-cancers, and invasive cancers coupled with microarray gene expression profiling to identify biomarkers for cervical cancer. During the past year we have adapted our amplification and target-labeling protocols to accommodate NCI's new spotted oligonucleotide arrays. We have also conducted work on two pilot projects. The first of these was designed to assess the best way to preserve cervical tissue specimens for subsequent laser capture microdissection and molecular analysis. Three sample preservation methods were tested: snap freezing in OCT, RNALater (Ambion), and fixation in ethanol followed by polyester wax embedding. The conclusion from this pilot is that snap freezing of tissue provides adequate histology for LCM while maintaining the best RNA quality and the greatest options for subsequent molecular analysis. The second pilot was designed to examine the effects of acetic acid and Lugol's solution on gene expression in cervical epithelium. Acetic acid and Lugol's solution (iodine) are widely used during culposcopy for the visualization of lesions. Microarray analysis of untreated and treated normal cervical tissue is currently being carried out. Finally, specimen collection for the main study is slated to begin in the fall of 2003. Effects of culture conditions on keratinocyte senescence, telomerase regulation, and immortalization of keratinocytes by high risk HPV oncoproteins: Several laboratories have previously shown that primary human foreskin keratinocytes (HFKs) grown in keratinocyte serum-free medium (K-SFM) on plastic substrates undergo a stress-induced p16INK4A-mediated senescence within a few passages. In contrast, p16INK4A levels are suppressed when keratinocytes are grown on feeder fibroblasts and the keratinocytes have an extended life span. We have confirmed these observations and investigated the role of telomerase in keratinocyte senescence. There is significant telomerase activity in freshly isolated HFKs, probably derived from the proliferating basal cell compartment. Although telomerase activity is quickly lost when HFKs are cultured in K-SFM, telomere shortening is unlikely to contribute to senescence of these cells. In contrast, we have found that culture of HFKs on feeder fibroblasts in serum-containing F-medium maintains telomerase activity at levels comparable to those in basal keratinocytes. Furthermore, these levels are sufficient to maintain telomere length for at least 43 population doublings (PDs). Beyond this point, however, both telomerase activity and telomere length decrease until the cells senesce, probably due to replicative senescence. We have also shown that telomerase activity is reversibly induced 14-fold when HFKs are transferred from K-SFM to F-medium and fibroblasts. The effects of culture conditions on immortalization of HFKs by HPV-16 E6 and E7 oncoproteins was also investigated. Induction of telomerase by the E6 protein is thought to be an important step in the immortalization of keratinocytes by high risk HPVs. We have shown that the feeder culture system strongly enhances the induction of telomerase by E6 and E7 proteins expressed from retroviral expression vectors, so that much higher telomerase levels can be achieved with less E6 and E7 protein. These superinduced telomerase levels were sufficient to maintain telomere length for at least 56 PDs, whereas telomeres were progressively eroded in HFK/16E6E7 cells grown in K-SFM on plastic. Furthermore, reversible superinduction of telomerase activity could be achieved by transfer of HFK/16E6E7 cells from K-SFM to F-medium. This superinduction requires both the E6 and E7 proteins, whereas only E6 is required for full induction of telomerase activity in HFKs cultured in K-SFM. We are currently studying the mechanisms involved in the regulation of telomerase by culture conditions and HPV oncogenes. Real time QRT-PCR assays for hTERT gave similar results to those obtained by telomerase activity assays and indicate that the regulation of telomerase activity mentioned above is at the RNA (and presumably transcriptional) level. Superinduction of telomerase in HFK/16E6E7 cells requires only F-medium, of which cholera toxin is a necessary but not sufficient component. In contrast, soluble factors and/or extracellular matrix secreted by the fibroblasts appear to be required for induction of telomerase in primary HFKs. Analysis of the differentiation status of HFKs in different culture systems suggests that the feeder system does not induce telomerase by blocking differentiation. Our work has significant implications for tissue culture studies of keratinocyte immortalization and help to explain many of the discrepancies in the papillomavirus literature. These studies are currently in press in Cancer Research. Identification and isolation of putative cervical stem cells based on cell surface phenotype: We are currently investigating the role of cervical stem cells in the progression of HPV infection to cervical cancer. The majority of cervical HPV infections are transient non-neoplastic productive viral infections which disappear within months. Persistent high risk HPV infections are much more likely to progress to premalignant and malignant cervical lesions. The crucial factors for the persistence of HPV infections in women with normal immune status are still unknown. One hypothesis is that viral persistence requires the infection of specific cervical cells such as stem cells. Cervical epithelial stem cells remain poorly characterized because specific molecular markers have not been identified that permit them to be distinguished from their differentiated progeny. We have identified a candidate cervical epithelial stem