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.
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