This project is to understand the processes used by the human T cell leukemia virus type 1 (HTLV-1)for the transformation of human cells. There are several concepts on how a cell can progress from normal physiology to achieve a transformed phenotype. To characterize transformation, we have studied cellular genetic integrity, cell cycle progression and the checkpoints within a cell that guard normal cellular division. We have studied several cellular proteins and cellular microRNAs whose functions are perturbed by infection with a transforming virus. In the 2011 - 2012 period, we have made the following scientifc findings as outlined below. Expression of the human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein rapidily induces a significant increase of micronuclei (MN) and unstabilized DNA breaks in cells. Unstabilized DNA breaks can have free 3'OH ends accessible to in situ addition of digoxygenin (DIG)-labeled dUTP using terminal deoxynucleotidyl transferase. We have examined the induction of MN and unstabilized DNA breaks in wild type cells and cells individually knocked out for Ku80, PKcs, XRCC4, and H2AX proteins. We found that cells mutated for PKcs, XRCC4 and H2AX showed increased frequency of MN and unstabilized DNA breaks in response to the expression of Tax, while cells genetically mutated for Ku80 were refractory to Tax's induction of these cytogenetic effects. Thus, HTLV-1 Tax is shown to interfere with a normal cellular protective mechanism for stabilizing DNA breaks. These DNA breaks, unprotected by Ku80, are unstable and are subject to erosion or end-to-end fusion, ultimately leading to additional chromosomal aberrations. Aging cells show some characteristic changes shared with cancer cells. We have developed a mouse model to study some of the causes of accelerated aging. The human LMNA gene mutations result in laminopathies that include Emery-Dreifuss muscular dystrophy (AD-EDMD) and Hutchinson-Gilford progeria, the premature aging syndrome (HGPS). The Lmna null (Lmna(-/-)) and progeroid Lmna9 mutant mice are models for AD-EDMD and HGPS, respectively. Both animals develop severe tissue pathologies with abbreviated life spans. We show in these mice that aging is linked to a failure of cells to properly degrade/remove over accumulated cellular proteins. This failure produces cellular stress and cytotoxicity. Autophagy, a general homeostatic process for degradation of cytosolic proteins or organelles, has been reported to modulate the replication of many viruses. The role of autophagy in Human T-cell Leukemia Virus type 1 (HTLV-1) replication has, however, been uncharacterized. We have found that HTLV-1 infection increases the accumulation of autophagosomes and that this accumulation increases HTLV-1 production. We also found that the HTLV-1 Tax protein increases cellular autophagosome accumulation by acting to block the fusion of autophagosomes to lysosomes, preventing the degradation of the former by the latter. Interestingly, the inhibition of cellular autophagosome-lysosome fusion using Bafilomycin A increased the stability of the Tax protein, suggesting that cellular degradation of Tax occurs in part through autophagy. Our current findings indicate that by interrupting the cells autophagic process, Tax exerts a positive-feedback on its own stability. In FY 2013, using genetically altered mice, we reported that Tax expression does not achieve a functional equivalence of p53 inactivation as that seen with genetic mutation of p53 (i.e., a p53-/- genotype). Thus, we found statistically significant differences in tumorigenesis between Tax+p53+/+ versus Tax+p53-/- mice. We also found a role contributed by the cellular Wip1 phosphatase protein in tumor formation in Tax transgenic mice. Notably, Tax+Wip1-/-mice show statistically significant reduced prevalence of tumorigenesis compared to Tax+Wip1+/+ counterparts. These findings provide new insights into contributions by p53 and Wip1 in the in vivo oncogenesis of Tax-induced tumors in mice.
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