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Research
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- Molecular Regulators of Cell Cycle Progression and Radiation- and Anticancer Drug-Induced Mitotic Catastrophe in Cancer Promotion and Progression
Our studies are aimed at characterizing mitotic catastrophe as both a new and different mode of cell death than apoptosis, and as a potential player in the induction of genomic instability and cell transformation. An important observation that we have made is that radiation-induced mitotic catastrophe preferentially occurs in cells lacking p53 function. Mitotic catastrophe is the outcome of premature entry of cells into mitosis driven by over accumulation of the pro-mitotic cell cycle kinase complex cyclin B1/cdk1 which allows the cell to escape the G2/M checkpoint control, and it is characterized by the presence of spontaneous premature chromosome condensation (SPCC) and nuclear fragmentation. Studies conducted using the anthracycline antibiotics also show that cells treated with these anti-cancer drugs undergo mitotic catastrophe in a fashion that mirrors the induction of the same after radiation and mild-heat treatments. We have also described the presence of a de novo delayed DNA damage in cells that undergo mitotic catastrophe. As de novo DNA strand breaks are generated many hours following cell irradiation, these results suggest a novel mechanism for the generation of DNA damage which occurs a day or more following irradiation. We have also observed that a small percentage of cells undergoing mitotic catastrophe is able to survive for many generations post-irradiation. Measurement of cell viability, assayed through trypan-blue staining, has shown that 91 % of irradiated HeLa cells are still able to exclude the dye at 48 h post 5 Gy X-irradiation, a time where some 80 % of the cells contain fragmented nuclei. Further, digital time-lapse analysis performed with the Large Scale Digital Cell Analysis System (LSDCAS) demonstrates that a small percentage of cells undergoing mitotic catastrophe is able to divide for many generations post-irradiation and form what a conventional clonogenic assay would define as a viable colony. Thus, mitotic catastrophe may lead to a delayed DNA damage phenotype. In cells that might survive mitotic catastrophe, this DNA damage, if not repaired with fidelity, might contribute to mutations that can cause delayed cell death or can represent a first step towards the acquisition of genomic instability and may contribute to the acquired resistance of tumor cells which is often reported in clinical and experimental outcome. Studies in my laboratory are underway, using RT-PCR among other techniques, to determine the spectrum of mutations occurring in the genome of cells undergoing mitotic catastrophe and to correlate this mutated genome with the cells’ fate. Further, large scale experiments using LSDCAS are also underway to determine if human tumor-derived cell lines undergoing mitotic catastrophe following a variety of treatments are able to retain clonogenicity. Our goals are to characterize post-translational mutations occurring in the p53 gene and to mechanistically link these to deregulation of cyclin B1 gene expression and to deregulation of other target genes involved in cell proliferation, cell senescence and cell death. These studies are of great interest as p53, among all its functions, has also been shown to regulate G2 arrest via inhibition of mitotic entry when DNA synthesis is inhibited, and to regulate intracellular levels of cyclin B1 through its effects on cyclin B1 promoter activity. Thus, abrogation of this p53 regulated pathway may lead to mitotic catastrophe and other subsequent effects on cell cycle regulation. We consider it possible that p53 functions to repress radiation-induced mitotic catastrophe through its activity as a modulator of the G2 checkpoint mechanisms. On the other hand, it has also been proposed that lack of p53 promotes mitotic catastrophe as a mechanism for removing damaged cells from populations following genotoxic stress. The transcriptional repression mechanisms by which p53 mediates decrease of intracellular levels of cyclin B1 and of cyclin B1 promoter activity are lost in the absence of p53 function. Thus, under condition of genotoxic stress cyclin B1 protein can accumulate, cyclin B1/cdk1 complexes can be activated, the G2 checkpoint block is consequently abrogated and the cells enter mitosis prematurely undergoing mitotic catastrophe. In my lab we are focused on defining the mechanisms underlying the role of p53 in mitotic catastrophe as we believe that this approach will lead to strategies to improve clinical response for those human tumors with defects in p53 or p53-related pathways.
In line with what discussed above, under the auspices of NASA (that has funded a million dollar project – PI F. Ianzini) we are commencing experiments aimed at identifying the role of high-LET radiation-induced mitotic catastrophe in mutagenesis and its implication in carcinogenesis. The rationale of these studies is based upon the notion that during space flight astronauts are exposed to various types of radiation and concerns have been raised regarding the genotoxic effects of such exposure. In particular, radiation hazards in the space environment include solar flares (or solar particle events, SPE), geomagnetically trapped radiation, galactic cosmic radiation (GCR), and secondary radiation. Solar storms periodically emit bursts of energetic charged particles. These solar storms normally consist of protons (85 %), α-particles (5-10 %), and heavy charged ions (HZE) (5-10 %). A large SPE could result in exposure to an HZE fluence of 3x10< with energies above 20 MeV. GCR consists of protons (85 %), α-particles (14 %), and HZE particles (1-2 %) ranging in energy from 100 MeV to 10 GeV. At geosynchronous orbit, the GCR is essentially isotropic. HZE particles (ions having a charge greater than 2 and an energy exceeding 50 MeV/nucleon) deposit energy as a function of the square of the charge (Z) and the inverse of velocity. Consequently, even though they exist in low abundance, GCR particles with Z greater than 3 are responsible for an increased percentage of dose. As GCR enters the atmosphere, it collides with atmospheric nuclei and breaks into pions and protons. The pions subsequently decay into muons before striking the Earth. GCR traversing the shielding of the spacecraft will also produce secondary radiation consisting of HZE, pions, neutrons and protons. Secondary radiation will also be produced when SPE protons and geomagnetically trapped electrons and protons will interact with the spacecraft. Thus, the resulting dose buildup from the bremsstrahlung radiation may be significant. In fact, in tissue the buildup factor for protons of 500 MeV is greater than 3 at a depth of 20 cm. Moreover, due to the unpredictability of SPE, it is possible that acute radiation exposures in excess of 2 Gy may occur to the skin of occupants of orbiting space satellites or during distant flights and explorations. Our studies will be driven by our major hypothesis that iron and proton ions of varying energies (providing high-LET values) induce mutations in exposed human cells via processes occurring in cells that undergo mitotic catastrophe. In particular, we will test the hypotheses that a) high-LET radiation leads to an enhanced incidence of mitotic catastrophe; b) a small proportion of irradiated cells undergoing mitotic catastrophe escapes death and form viable colonies and c) mutations and altered nucleosomal organization of chromatin (DNA damage) are persistent in survivors of mitotic catastrophe cells thus making these cells prone to cell transformation.
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