HTLV-I Tax activates the anaphase promoting complex

描述（由申请人提供）：HTLV-I感染如何从临床潜伏期发展为成人t细胞白血病/淋巴瘤（ATL）尚不清楚，但涉及独特的病毒反激活物/癌蛋白Tax。与其他白血病细胞不同，ATL细胞通常是非整倍体，具有高度分叶或卷曲的细胞核，因此被称为“花”细胞。非典型淋巴细胞双核或含有裂核/脑状核也很容易在htlv - 1感染者的血液涂片中看到。这些病理结果表明，有丝分裂畸变伴随HTLV-I病毒复制，并可能在恶性肿瘤的发展中发挥重要作用。我们发现首次表达Tax的幼稚细胞（酿酒葡萄球菌、HeLa和人二倍体成纤维细胞）在S/G2/M进程中停滞不前。然后它们经历错误的有丝分裂，其特征是严重的染色体非整倍体，形成微核、双核和多核细胞。我们进一步表明，在酵母、啮齿动物和人类细胞中，Tax导致Pds1p/securin（后期抑制剂）和Clb2p/cyclin B水平显著降低。这种Pds1p/securin和Clb2p/cyclin B1的缺失发生在S/G2期间，并且与上述有丝分裂畸变密切相关。利用酿酒酵母的遗传学，我们发现Pds1p和Clb2p的非计划性降解可能与E3泛素连接酶Cdc20相关的后期促进复合物（APC[Cdc20]）的异常激活（通过Tax）有关，该复合物通过靶向细胞周期蛋白A、安全蛋白和细胞周期蛋白B1的及时多泛素化和破坏来调节中期到后期的转变。在酵母遗传学的指导下，我们进一步证明了Tax在S期直接结合并激活人类APC[Cdc20]，比正常时间提前。由Tax引起的细胞周期畸变并不仅仅以有丝分裂异常结束。我们最近发现，在经历上述有丝分裂危机后，Tax+ HeLa细胞立即在G1期永久停滞，并进入类似衰老的状态，p21[CIP1/WAF1]和p27[KIP1]水平急剧升高。重要的是，这种衰老表型可以通过APC[Cdc20]的非预定激活和Skp2（很可能是APC介导的）的缺失来解释，Skp2是SCF[Skp2]的底物识别亚基，是一种独特的E3泛素连接酶，靶向P21[CIP1/WAF1]和p27[KIP1]的降解。有趣的是，在先前的Tax-naive细胞中检测到的严重的有丝分裂危机和衰老在HTLV-I转化的t细胞中没有看到，HTLV-I转化的t细胞大量产生Tax。这表明HTLV-I转化的细胞可能含有特定的“抑制”突变，使它们能够逃脱由Tax诱导的衰老状态。在本应用中，我们试图进一步研究(1)Tax激活APC[Cdc20]的机制，(2)Tax诱导的衰老样状态的生物学特性和生化基础，以及(3)HTLV-I转化细胞中使其逃避Tax诱导的快速衰老的“抑制”机制。

DESCRIPTION (provided by applicant): How HTLV-I infection progresses from clinical latency to adult T-cell leukemia/lymphoma (ATL) is not well understood but involves the unique viral transactivator/oncoprotein, Tax. Unlike cells of other leukemia, ATL cells are often aneuploid with highly lobulated or convoluted nuclei, earning them the name of "flower" cells. Atypical lymphocytes that are binucleated or contain cleaved/cerebriform nuclei are also readily seen in the blood smears of HTLV-I-infected individuals. These pathological findings suggest that mitotic aberrations accompany HTLV-I viral replication and are likely to play an important role in the development of malignancy. We have discovered that naive cells (S. cerevisiae, HeLa, and human diploid fibroblast) that express Tax for the first time become stalled in S/G2/M progression. They then undergo faulty mitosis characterized by severe chromosome aneuploidy, and formation of micronucleated, binucleated and multinucleated cells. We have shown further that Tax causes a significant reduction in Pds1p/securin (the anaphase inhibitor) and Clb2p/cyclin B levels in yeast, rodent, and human cells. This loss of Pds1p/securin and Clb2p/cyclin B1 occurs during S/G2, and strongly correlates with the aforementioned mitotic aberrations. Taking advantage of the genetics of S. cerevisiae, we have found that the unscheduled degradation of Pds1p and Clb2p can be linked to the aberrant activation (by Tax) of an E3 ubiquitin ligase, the Cdc20-associated anaphase promoting complex (APC[Cdc20]), which regulates metaphase to anaphase transition by targeting the timely polyubiquitination and destruction of cyclin A, securin and cyclin B1. Guided by yeast genetics, we have further demonstrated that Tax directly binds and activates human APC[Cdc20] during S phase, ahead of the normal schedule. The cell cycle aberrations induced by Tax does not end with just mitotic abnormalities. We have discovered recently that immediately after experiencing the mitotic crisis described above, Tax+ HeLa cells become permanently arrested in G1, and enter into a senescence-like state with drastically elevated levels of p21[CIP1/WAF1 and p27[KIP1]. Importantly, this senescence phenotype can be explained by the unscheduled activation of APC[Cdc20] and the loss of Skp2 (most likely APC-mediated), the substrate-recognition subunit of SCF[Skp2], a distinct E3 ubiquitin ligase that targets P21[CIP1/WAF1] and p27[KIP1] degradation. Interestingly, the severe mitotic crisis and senescence detected in previously Tax-naive cells are not seen in HTLV-I transformed T-cells, which produce Tax abundantly. This suggests that HTLV-I transformed cells may harbor specific "suppressor" mutations that allow them to escape the senescence state induced by Tax. In this application, we seek to investigate further (1) the mechanism by which Tax activates APC[Cdc20], (2) the biological characteristics of and biochemical basis for the senescence-like state induced by Tax, and (3) the "suppressor" mechanism(s) in HTLV-I transformed cells that allow them to escape the rapid senescence induced by Tax.