Analyzing mechanism of oncogenesis by human papillomavirus

E6的转化活性需要在高风险HPV E6蛋白中保守的羧基末端的PDZ结合基序。自从我们首次报道hDLG作为E6靶标以来，hScrib、MUPP 1和MUGI已被报道为类似的靶标。我们将PSD 95确定为新靶点。HPV阳性的宫颈癌细胞系中PSD 95的表达水平很低，这一事实使我们推测PSD 95可能具有肿瘤抑制作用。PSD 95在CaSki细胞中的过表达改变了在培养皿中的生长，但显著降低了在软琼脂培养基和裸鼠中的生长。研究还表明，PSD 95被E6/E6 AP复合物遍在化并被蛋白酶体降解。长期以来，人们一直不知道E6如何激活正常人角质形成细胞和乳腺上皮细胞中的端粒酶。我们发现E6/E6 AP复合物与转录抑制因子NFX-1结合。从NIX-1基因中，表达了123 kDa（NFX 1 -123）和91 kDa（NFX 1 -91）蛋白。我们发现E6/E6 AP复合物特异性地增强了hTERT启动子转录抑制因子NFX-1 -91的降解，这表明NFX-1的降解增强至少是E6激活端粒酶的一种机制。我们认为E6和E7上调hWAPL可能是E6和E7诱导正常细胞染色体不稳定的一种机制，hWAPL在宫颈癌中优先过表达。人WAPL（humanWAPL）是果蝇翅分离样基因的同源基因。该基因产物与异染色质结合，其过表达可诱导培养细胞中的染色体不稳定性。

Transforming activity of E6 requires the PDZ-binding motif at the carboxy terminus conserved among high risk HPV E6 proteins. Since we first reported the hDLG as the E6 target, hScrib, MUPP1, and MUGI have been reported as the similar targets. We identified PSD95 as a novel target. The fact that HPV positive cervical cancer cell lines showed very low expression levels of PSD95, allowed us speculate that PSD95 might function as tumor suppressor. Overexpression of PSD95 in CaSki cells unchanged the growth in a culture dish but significantly reduced the growth both in soft agar medium and nude mice. It was also suggested that PSD95 was ubiquitinated by E6/E6AP complex and degraded by proteosome.It has long been unknown how E6 activates telomerase in normal human keratinocyte and mammary epithelial cells. We found that E6/E6AP complex bound to a transcriptional repressor, NFX-1. From NIX-1 gene, 123 kDa (NFX1-123) and 91kDa (NFX1-91) proteins were expressed. We found E6/E6AP complex specifically enhanced degradation of NFX1-91 which functioned as transcriptional repressor of hTERT promoter, indicating that the enhanced degradation of NFX-1 is at least a mechanism to activate telomerase by E6.We suggest that the upregulation of hWAPL, which is preferentially overexpressed in cervical cancers, might be a mechanism which induces chromosomal instability in normal cells by E6 and E7. Human WAPL (hWAPL) is a human homologue of Drosophila wing apart-like gene. The gene product binds to heterochromatin and its overexpression can induce chromosomal instability in cultured cells.