RB has a critical role in mediating the in vivo checkpoint response, mitigating secondary DNA damage and suppressing liver tumorigenesis initiated by aflatoxin B1.

Hepatocellular carcinoma (HCC) is a significant worldwide health concern that is associated with discrete etiological events, encompassing viral infection, metabolic stress and genotoxic compounds. In particular, exposure to the genotoxic hepatocarcinogen aflatoxin B1 (AFB1) is a significant factor in the genesis of human liver cancer. Presumably, genetic events associated with HCC could influence the effect of environmental insults, yielding a predilection for tumor development. The retinoblastoma (RB) tumor suppressor pathway is functionally inactivated in HCC through discrete mechanisms; however, the role of RB in suppressing tumorigenesis in this disease is poorly understood. Therefore, we analysed how RB status affects the response to AFB1 in reference to acute exposures and tumor development reflective of chronic exposure. Liver-specific Rb deletion resulted in an aberrant proliferative response to AFB1. This cell-cycle induction was associated with increased levels of secondary genetic damage and failure in appropriate cell-cycle coupling. This effect of RB loss was unique to AFB1 and involved the induction of a non-canonical proliferative pathway, and was not merely reflective of the overall cell-cycle deregulation or aberrant regenerative responses. The acute responses to AFB1 exposure presaged aberrations in hepatocyte nuclear morphology and ploidy with RB loss. Correspondingly, RB-deficient livers showed significantly enhanced susceptibility to liver tumorigenesis initiated by AFB1. Combined, these studies show that RB has a critical role in mediating checkpoint responses in liver tissue to maintain genome integrity and in suppressing tumorigenesis.

Loss of the retinoblastoma tumor suppressor: differential action on transcriptional programs related to cell cycle control and immune function.

Functional inactivation of the retinoblastoma tumor suppressor gene product (RB) is a common event in human cancers. Classically, RB functions to constrain cellular proliferation, and loss of RB is proposed to facilitate the hyperplastic proliferation associated with tumorigenesis. To understand the repertoire of regulatory processes governed by RB, two models of RB loss were utilized to perform microarray analysis. In murine embryonic fibroblasts harboring germline loss of RB, there was a striking deregulation of gene expression, wherein distinct biological pathways were altered. Specifically, genes involved in cell cycle control and classically associated with E2F-dependent gene regulation were upregulated via RB loss. In contrast, a program of gene expression associated with immune function and response to pathogens was significantly downregulated with the loss of RB. To determine the specific influence of RB loss during a defined period and without the possibility of developmental compensation as occurs in embryonic fibroblasts, a second system was employed wherein Rb was acutely knocked out in adult fibroblasts. This model confirmed the distinct regulation of cell cycle and immune modulatory genes through RB loss. Analyses of cis-elements supported the hypothesis that the majority of those genes upregulated with RB loss are regulated via the E2F family of transcription factors. In contrast, those genes whose expression was reduced with the loss of RB harbored different promoter elements. Consistent with these analyses, we found that disruption of E2F-binding function of RB was associated with the upregulation of gene expression. In contrast, cells harboring an RB mutant protein (RB-750F) that retains E2F-binding activity, but is specifically deficient in the association with LXCXE-containing proteins, failed to upregulate these same target genes. However, downregulation of genes involved in immune function was readily observed with disruption of the LXCXE-binding function of RB. Thus, these studies demonstrate that RB plays a significant role in both the positive and negative regulations of transcriptional programs and indicate that loss of RB has distinct biological effects related to both cell cycle control and immune function.

Short-term treatment with novel ribonucleotide reductase inhibitors Trimidox and Didox reverses late-stage murine retrovirus-induced lymphoproliferative disease with less bone marrow toxicity than hydroxyurea.

We evaluated the ability of a short course of treatment with the ribonucleotide reductase (RR) inhibitor hydroxyurea (HU) and two novel RR inhibitors Trimidox (TX) and Didox (DX) to influence late-stage murine retrovirus-induced lymphoproliferative disease. LPBM5 murine leukaemia virus retrovirus-infected mice were treated daily with HU, TX or DX for 4 weeks, beginning 9 weeks post-infection, after development of immunodeficiency and lymphoproliferative disease. Drug effects on disease progression were determined by evaluating spleen weight and histology. Effects on haematopoiesis were determined by measuring peripheral blood indices (white blood cells and haematocrit) and assay of femur cellularity and femoral and splenic content of colony-forming units granulocyte-macrophage (CFU-GM) and burst-forming units-erythroid (BFU-E). HU, TX and DX partially reversed late-stage retrovirus-induced disease, resulting in spleen weights significantly below pre-treatment values. Spleen histology was also improved by RR inhibitor treatment (DX>TX>HU). However, as expected, HU was significantly myelosuppressive, inducing a reduction in peripheral indices associated with depletion of femoral CFU-GM and BFU-E. In contrast, although TX and DX were moderately myelosuppressive, both drugs were significantly better tolerated than HU. In summary, short-term treatment in late-stage murine retroviral disease with HU, TX or DX induced dramatic reversal of disease pathophysiology. However, the novel RR inhibitors TX and DX had more effective activity and significantly less bone marrow toxicity than HU.

Regulation of heat shock transcription factor 1 by stress-induced SUMO-1 modification.

Heat shock transcription factor 1 (HSF1) mediates the induction of heat shock protein gene expression in cells exposed to elevated temperature and other stress conditions. In response to stress HSF1 acquires DNA binding ability and localizes to nuclear stress granules, but the molecular mechanisms that mediate these events are not understood. We report that HSF1 undergoes stress-induced modification at lysine 298 by the small ubiquitin-related protein called SUMO-1. Antibodies against SUMO-1 supershift the HSF1 DNA-binding complex, and modification of HSF1 in a reconstituted SUMO-1 reaction system causes conversion of HSF1 to the DNA-binding form. HSF1 colocalizes with SUMO-1 in nuclear stress granules, which is prevented by mutation of lysine 298. Mutation of lysine 298 also results in a significant decrease in stress-induced transcriptional activity of HSF1 in vivo. This work implicates SUMO-1 modification as an important modulator of HSF1 function in response to stress.

In vivo and in vitro comparison of the short-term hematopoietic toxicity between hydroxyurea and trimidox or didox, novel ribonucleotide reductase inhibitors with potential anti-HIV-1 activity.

Inhibitors of the cellular enzyme ribonucleotide reductase (hydroxyurea, [HU]) have been proposed as a new therapeutic strategy for the treatment of HIV type-1 (HIV-1) infection. However, HU use may be limited by the frequent development of hematopoietic toxicity. We report here short-term hematopoietic toxicity in mice receiving HU when compared to either of two more potent enzyme inhibitors, didox (DX) and trimidox (TX). High dose HU, DX, and TX monotherapy (500, 460, and 220 mg/kg/day respectively) was administered by daily i.p. injection (Monday-Friday) to C57BL/6 mice for 10 weeks. Effects on hematopoiesis were established by quantitating peripheral blood indices (hematocrit, hemoglobin, mean corpuscular volume, mean cell hemoglobin, mean corpuscular hemoglobin concentration, RBC, and WBC) and numbers of colony-forming units-granulocyte-macrophage (CFU-GM) and BFU-E from bone marrow and spleen. HU produced rapid induction of a macrocytic hypochromic anemia and altered white blood cell kinetics associated with myelosuppression defined as reduced marrow organ cellularity and induction of splenic extramedullary hematopoiesis. Compared to HU, TX and DX induced fewer changes in peripheral blood indices and CFU-GM and BFU-E per hematopoietic organ. In vitro human and murine marrow CFU-GM and BFU-E colony formations were assayed in the presence of dose escalation HU, DX, or TX (0, 1, 10, 50, 100, and 200 microM). HU inhibited colony formation more than either DX or TX. These in vivo and in vitro studies suggest that novel ribonucleotide reductase inhibitors TX and DX may provide an effective alternative to HU in HIV-1 therapy because they demonstrate reduced hematopoietic toxicity.