NON-CANONICAL FUNCTIONS OF HTERT IN CELL IMMORTALIZATION BY HPV

描述（由申请人提供）：细胞永生化是癌症发展的关键事件，致瘤性人乳头瘤病毒编码E6癌基因，该基因主要负责诱导端粒酶活性和随后的永生化。最近的研究表明，HPV E6蛋白通过其参与和激活hTERT启动子的能力介导端粒酶活性的增加，并直接与至少两种启动子结合蛋白Myc和NFX-1相互作用。E6还具有通过直接与hTERT蛋白结合来增加端粒酶活性的翻译后机制。在目前的研究中，我们发现原代角质形成细胞的hTERT永化独立于其端粒酶活性或延长端粒的能力。与最近的干细胞研究一致，我们新的初步发现表明，hTERT可以改变角质细胞基因的表达，并增加HPV基因的表达。我们假设hTERT的转录激活功能或其在改变凋亡反应中的潜在作用可能在细胞增殖中绕过M1/M2限制点从而促进细胞永生方面发挥重要作用。在本应用程序中，我们将使用一个可变hTERT表达向量库来评估这些可能性。我们将把这些突变蛋白有效绕过细胞衰老的能力与它们诱导逆转录酶活性、hTR结合、端粒结合、端粒延伸、启动子反激活和细胞凋亡调节的能力联系起来。这些研究将为癌症病因学中的一个基本事件提供新的见解。

DESCRIPTION (provided by applicant): Cell immortalization is a critical event in the development of cancer and the oncogenic human papillomaviruses encode an E6 oncogene that is primarily responsible for inducing telomerase activity and consequent immortalization. Recent studies indicate that the HPV E6 protein mediates the increase in telomerase activity via its ability to engage and activate the hTERT promoter and interact directly with at least two promoter-bound proteins, Myc and NFX-1. E6 also has a post-translational mechanism to increase telomerase activity by binding directly to hTERT proteins. In the current grant, we show that hTERT immortalization of primary keratinocytes is independent of its telomerase activity or ability to elongate telomeres. In agreement with recent work with stem cells, our new preliminary findings demonstrate that hTERT can alter the expression of keratinocyte genes as well as increase the expression of HPV genes. We hypothesize that the transcription activation function of hTERT or its potential role in altering apoptotic responses may play an essential role in bypassing the M1/M2 restriction points in cell proliferation and thereby facilitate cell immortalization. In this application, we will evaluate these possibilities using a library of mutan hTERT expression vectors. We will correlate the ability of these mutant proteins to efficiently bypass cellular senescence with their ability to induce reverse transcriptase activity, hTR binding, telomere binding, telomere elongation, promoter transactivation, and modulation of apoptosis. These studies will provide new insights into a basic event in the etiology of cancer.