Apoptosis is a mechanism of cellular demise which is believed to play a role in many physiological processes, and which when defective, can contribute to the pathogensis of cancer. At present, neither the regulation and/or alterations in the apoptotic pathway that occur during carcinogenesis are understood. Apoptosis is stimulated in cells by the p53 tumor suppressor protein, adenovirus E1A and Interleukin 1-beta converting enzyme (ICE), and is inhibited by overexpression of bcl-2 oncogene and adenovirus E1B 19 kD oncoprotein. A role for altered apoptosis in cancer comes from recent reports which have documented that inactivation of p53 and/or overexpression of bcl-2 occur in numerous cancers. Recently, work from our laboratory has shown that inactivation of p53 (either by physical complex formation between p53 and Human Papillomavirus (HPV) type 16- or 18- E6 protein or by mutation of p53), is associated with increased expression of bcl-2 in cervical carcinoma cells. The combination of inactive p53 and increased expression of bcl-2 may lead to inhibition of apoptosis and thereby contribute to the development or progression of cervical cancer. Thus, we wish to test the hypothesis that expression of high-risk HPV E6 and/or E7 results in epithelial cell transformation by altering the activity and/or expression of cellular proteins which regulate apoptosis. Therefore, our specific aims are: 1) Determine whether apoptosis is defective in cervical carcinoma cells and keratinocytes transfected with HPV-16 vs. HPV-6 E6 or E7; 2) Determine endogenous levels of p53, ICE, bcl-2 and bax in normal, HPV-6 and HPV-16 E6 and E7 transfected keratinocytes and cervical carcinoma cells; 3) Determine the effects of bcl-2 and bax expression on HPV-16 full length, E6 and E7 induced immortalization, transformation and apoptosis in keratinocytes; and 4) Determine whether Bcl-2 and Bax form hetero- and homodimers in intact normal, HPV-16 E6 and E7 transfected keratinocytes and cervical carcinoma cells. These studies will emphasize measurements of apoptosis, levels of p53, and bcl-2 and bax and their interactions using novel digitized video microscopic techniques, which will be combined with transfection and immortalization and transformation assays to determine the role of expression of specific apoptopic regulators in epithelial cell transformation. The proposed studies are designed to establish direct cause-and-effect relationships between expression of HPV early proteins and p53, ICE, bcl-2 and bax and altered apoptosis in epithelial cells. New information forthcoming from these studies will better define the role of apoptosis in cervical carcinogenesis and suggest potential sites of intervention to slow or prevent cervical cancer.
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