Human papillomaviruses (HPVs) are a large family of viruses that infect mucosal or cutaneous epithelium,causing causing a spectrum of proliferative lesions. There is mounting evidence that the genus beta HPVsplay a role in the etiology of squamous cell skin cancers (SCSC), though the mechanism by which the E6and E7 proteins might contribute to carcinogenesis is not known. Unlike the high-risk alpha HPVs that areassociated with cervical cancers, the genus beta HPVs do not persist in every cell in the tumor, nor do E6/E7promote the degradation of p53 or Rb. Our working model is that E6/E7 expression causes cellularproliferation, thereby increasing the pool size of cells that are susceptible to UV mutagenesis. In response toUV damage E6 blunts repair and apoptotic processes, increasing the survival of cells with p53 or othermutations, which can progress to invasive cancer. To test these hypotheses we propose four specific aims:1. To investigate the ability of genus beta E6proteins to blunt the apoptotic response to UVdamage.We propose to: a) determine the mechanism of Bak degradation. We will determine whether E6AP, orsome other ubiquitin ligase, targets Bak for degradation; investigate the role of Bcl-XL and Mcl-1 in blockingBak degradation; and identify the sequences of E6 that mediate Bak binding and degradation (in aim 2); b)examine other aspects of the intrinsic and extrinsic apoptotic pathways to fully characterize deregulation bybeta E6 proteins; c) determine whether beta E6s inactivate p53 by blocking p300-induced acetylation; and d)examine markers of apoptosis studied in E6-HFKs in human skin cancers and pre-cancers. 2. To identifyboth conserved and novel functions of the beta E6 proteins. We propose to: a) identify regions ofE6involved in targeting Bak for degradation; b) identify the beta HPV E6s that are capable of inducingtelomerase and map the critical residues; and more broadly, c) we plan to identify new binding partners forthe beta E6 proteins using TAP-tagged E6, GST- E6 pull downs, and will also focus on the unique amino andcarboxy -terminal domains. 3. To investigate the ability of genus beta E7 proteins to promote S phaseentry and perturb epithelial cell differentiation. We will assess the ability of HPV 8 and 38 E7 to promoteentry into S phase in three growth assays; and we will examine whether p130 is degraded in monolayer andorganotypic culture. 4. To develop transgenic mouse models with regulated expression of E6/E7. Wepropose to develop regulatable 'Tet-On' bitransgenic mouse systems, K14-rtTA/TetRE-HPV8E6/E7, inwhich E6/E7 expression is off until the mice are exposed to doxycycline (Dox). We will determine whetherprecancerous lesions once initiated can be maintained or progress in the absence of HPV expression. Wewill also explore whether low doses of UV can stimulate tumorigenesis in HPV- ON mice comparednontransgenic littermates, and accelerate lesion formation compared to spontaneous development. We willdetermine the timing and duration of E6 expression that is required. Histology and markers of apoptosis willbe studied in the spontaneously occurring and UV-induced lesions.
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