In the past year significant progress has been made in understanding the role of DNA damage in cellular aging and senescence and in the understanding of the Deleted in Liver Cancer 1, (DLC1), genes tumor suppressive function and its potential use for therapeutic interventions. Accumulation of DNA damage may play an essential role not only in the process of aging but also in induction of cellular senescence. The ability of cells to sense and repair DNA damage declines with age. However, the underlying molecular mechanism for this age-dependent decline is still elusive. To understand quantitative and qualitative changes in the DNA damage response during human aging, the nature and dynamics of phosphorylated histone H2AX (gamma-H2AX) foci, which occur specifically at sites of DNA double-strand breaks (DSB), were examined in cells from normal individuals of different age and from patients with Werner syndrome (WS), a disorder associated with premature aging, genomic instability and increased incidence of cancer. This study showed that the incidence of gamma-H2AX foci generally increases with donors age, although the cells derived from patients with Werner syndrome (WS) exhibited considerably higher incidence of gamma-H2AX foci than those taken from normal donors of comparable age. Further increases in gamma-H2AX focal incidence occurred in culture as both normal and WS fibroblasts progressed toward senescence. The rates of recruitment of DSB repair proteins to gamma-H2AX foci correlated inversely with age for both normal and WS donors, perhaps due in part to the slower growth of gamma-H2AX foci in older donors. Since genomic stability may depend on the efficient processing of DSBs where the rapid formation of gamma-H2AX foci and the rapid accumulation of DSB repair proteins may be instrumental, our findings suggest that increasing inefficiency in these processes may contribute to genome instability associated with normal and pathological aging. The DLC1 gene, isolated and characterized by our group, has recently been independently confirmed as a potent bona fide tumor suppressor gene. Silencing or underexpression of this gene plays an important role in several common human cancers such as liver, breast, lung and prostate cancer and, most significantly, signaling pathways modulated by DLC1 provide wide range of targets for therapeutic interventions. Induction of apoptosis is a major attribute of tumor suppressor gene. In two independent studies with lung and prostate tumor cells, we identified cellular and molecular alterations indicative of the induction of programmed cell death by DLC1. DLC1 has a dramatic inhibitory effect on in vitro cell proliferation and prevented in vivo the development of tumors after inoculation of human non-small cell lung carcinoma cells in nude mice. Transduction of DLC1 in lung cancer cells invariably induced changes in cell morphology corroborative of the tumor suppressor function of the gene. DLC1-mediated actin cytoskeleton-based morphological alterations, and DLC1 protein nuclear translocation occur prior to inhibition of cell migration and before to the induction of apoptosis. These observations led to the conclusion that DLC1 functions in the cytoplasm as an inhibitor of tumor cell proliferation and migration, but in the nucleus as an inducer of apoptosis. Based on our evidence, DLC1 is now considered a metastasis suppressor gene. The process of tumor cell dissemination from the locally growing primary tumors to metastasis in anatomically distant sites is the leading cause of cancer mortality, representing over 90% of cancer casualties. Metastatic disease is a complex process involving various cellular and genetic alterations. We showed that DLC1 inhibits the spread of liver cancer cells to distant organs. The inhibition of liver cancer cell dissemination to distant organs was associated with reduction of RhoA activity, cytoskeleton alterations, down regulation of osteopontin (OPN) and matrix metalloproteinase-9 (MMP9) expression, which are highly up regulated in most primary liver tumors with associated metastases. Over-expression of OPN and MMP-9 and silencing of DLC1 may provide a prognostic marker for unfavorable prognosis of liver cancer. DLC1 gene therapy using adenoviral vector, as the most useful vehicle for gene transfer, is a realistic prospect for prevention of HCC dissemination. Also, searching for dietary constituents and pharmacological agents that up-regulate DLC-1 gene expression could lead to the development of drugs that might be useful for prevention of metastasis, the most harmful event during liver cancer progression. In prostate cancer, we found that recurrent downregulation and inactivation of DLC1 by epigenetic mechanisms may serve as an marker for early detection of prostate cancer and subsequently we showed that adenovirus-mediated DLC1 transfer in androgen-independent aggressive and metastatic prostate tumor cells inhibited cell proliferation in vitro, their tumorigenicity in nude mice, and induced apoptosis. It also induced cell cycle arrest, inhibited the activation of RhoA and the formation of actin stress fibers. In prostate tumor cells that are resistant to histone deacetylase inhibitors-induced cell death, restoration of DLC1 expression induced apoptosis only after chemical inhibition of the antiapoptotic Bcl-2 protein expression. These results suggest that adenovirus-mediated DLC1 transfer, alone or together with other agents such as inhibitors of Bcl-2, or histone deacetylases that were already tested in clinical trials might prove effective in the treatment of aggressive androgene independent and metastatic prostate cancer.
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