NON-CANONICAL FUNCTIONS OF HTERT IN CELL IMMORTALIZATION BY HPV

描述(申请人提供)：细胞永生化是癌症发展过程中的关键事件，致癌的人类乳头瘤病毒编码E6癌基因，该基因主要负责诱导端粒酶活性并随后永生化。最近的研究表明，HPV E6蛋白通过参与和激活hTERT启动子并直接与至少两个启动子结合的蛋白Myc和NFX-1相互作用来介导端粒酶活性的增加。E6还具有翻译后机制，通过直接与hTERT蛋白结合来增加端粒酶活性。在目前的研究中，我们发现原代角质形成细胞的hTERT永生化与其端粒酶活性或延长端粒的能力无关。与最近对干细胞的研究结果一致，我们的新的初步发现表明，hTERT可以改变角质形成细胞基因的表达，也可以增加HPV基因的表达。我们推测，hTERT的转录激活功能或其在改变细胞凋亡反应中的潜在作用可能在绕过细胞增殖的M1/M2限制点从而促进细胞永生化方面发挥重要作用。在本申请中，我们将使用突变hTERT表达载体文库来评估这些可能性。我们将把这些突变蛋白有效地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、端粒延长、启动子反式激活和调节细胞凋亡的能力联系起来。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.