Alteration of Rb in HTLV-1 transformed cells

描述(申请人提供)：人类T细胞白血病病毒I型(HTLV-I)是成人T细胞白血病(ATL)的病原体。ATL是一种复杂的多方面疾病，最初由病毒诱导的致癌事件演变而来，并导致与ATL侵袭性亚型相关的多种染色体异常。Tax是一种编码在病毒基因组Px区的病毒癌蛋白，通过靶向细胞周期蛋白D2、p21/waf1、p16/INK4a和p53，被认为是HTLV-I转化细胞细胞周期解除调控的主要因素。这种协调的放松调控，特别是在细胞周期的G1阶段，通过失去检查点控制和对抗有丝分裂原不敏感来确保细胞持续进展。 我们研究的长期目标是了解限制点调节器的税收依赖扰动是如何导致白血病发生的。真核细胞中G1限制点的一个重要调节因子是肿瘤抑制蛋白视网膜母细胞瘤(Rb)。Rb与p107和p130一起，是参与静息、细胞分裂、分化、衰老和细胞凋亡的蛋白质家族的一部分。我们最近发现，在HTLV-I转化细胞和ATL患者中，与未感染的对照组相比，Rb蛋白减少。具体地说，这种减少依赖于税收表达，并在翻译后水平上进行调节。我们发现Tax能够通过Tax的C末端的LXCXE或PENF同源基序与B结构域中的RB结合。我们进一步表明，通过体外降解试验，RB的降解依赖于Tax与26S蛋白酶体结合的能力。Rb的蛋白酶体降解似乎仅限于低磷酸化(或活性)的Rb物种，因为HTLV-I转化细胞中保留的主要Rb物种是Rb的非活性形式。因此，Rb活性物种的丧失使我们推测，这导致了一种类似于由于遗传/表观遗传改变而失去Rb活性的细胞的表型。 我们的假设是，税收对Rb的降解有助于HTLV-I感染细胞的永生化。我们进行这些研究的理由是基于数据，该数据表明HTLV-I病毒蛋白税通过将Rb蛋白靶向26S蛋白酶体进行降解来破坏Rb蛋白的稳定。我们认为，这种机制类似于HPV依赖E7的Rb失稳，导致Rb活性形式的丧失。P16/INK4a/Cyclin D通路中Rb的失控是HPV-16永生化的早期事件，它通过促进细胞周期进入和衰老原代细胞的增殖来实现。我们的初步数据显示，在HTLV-I转化的细胞中也观察到了这些表型，可能与ATL恶性有关。以下具体目标将解决我们的假设：(I)HTLV-I转化细胞中Rb的税收依赖性蛋白分解机制是什么？(2)RB的退化是导致依赖税收的不朽的早期事件吗？从这些研究中获得的数据将阐明税收和RB相互作用如何导致HTLV-I感染的ATL细胞永生化。

DESCRIPTION (provided by applicant): Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T-cell leukemia (ATL). ATL is a complex and multi-faceted disease that evolves initially from viral-induced carcinogenic events and leads to multiple chromosomal abnormalities that are associated with the aggressive subtypes of ATL. Tax, the viral oncoprotein encoded within the pX region of the viral genome, is considered a major contributor to cell cycle deregulation in HTLV-I transformed cells by targeting such cellular factors as cyclin D2, p21/waf1, p16/INK4a, and p53. This concerted deregulation, especially at the G1 phase of the cell cycle, ensures continuous cellular progression via the loss of checkpoint control and insensitivity to anti-mitogens. The long-term goal of our research is to understand how Tax-dependent perturbation of restriction point regulators leads to leukemogenesis. A significant regulator of the G1 restriction point in eukaryotic cells is the tumor suppressor protein retinoblastoma (Rb). Rb, with p107 and p130, are part of a family of proteins involved in quiescence, cellular division, differentiation, senescence, and apoptosis. We have recently found that within both HTLV-I transformed cells and ATL patients there are a decrease of Rb protein as compared to uninfected controls. Specifically, this decrease is dependent on Tax expression and regulated at the posttranslational level. We have found that Tax is able to bind to Rb within the B domain through either a LXCXE or PENF homology motif within the C terminus of Tax. We further show, through in vitro degradation assays, that Rb degradation is dependent on Tax's ability to associate with the 26S proteasome. The proteasome degradation of Rb appears to be restricted to the hypophosphorylated (or active) Rb species, since the predominant Rb species remaining in HTLV-I transformed cells is the inactive form of Rb. Therefore, loss of the active species of Rb has led us to speculate that this results in a phenotype that is similar to cells that have lost Rb activity due to genetic/epigenetic alterations. Our hypothesis is that Rb degradation by Tax contributes to the immortalization of HTLV-I infected cells. Our rationale for these studies is based on data that shows the HTLV-I viral protein Tax destabilizes the Rb protein by targeting this protein to the 26S proteasome for degradation. We believe this mechanism is analogous to HPV's E7-dependent destabilization of Rb that results in the loss of the active form of Rb. Rb deregulation within the p16/INK4a/cyclin D pathway is an early event in HPV-16 immortalization by promoting cell cycle entry and proliferation of senescent primary cells. These phenotypes are also observed in HTLV-I transformed cells as shown by our preliminary data and may contribute to ATL malignancy. The following specific aims will address our hypothesis: (I) what is the mechanism of Tax-dependent proteolysis of Rb in HTLV-I transformed cells? (II) Is degradation of Rb an early event that contributes to Tax-dependent immortalization? Data obtained from these studies will shed light on how Tax and Rb interaction leads to the immortalization of HTLV-I infected ATL cells.