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Molecular Mechanisms Of Cycle Arrest And Apoptosis In Lymphoid Cells Elicited By Cytolethal Distending Toxin, A Genotoxin Produced By Campylobacter Jejuni And Helicobacter Hepaticus
It is well-established that the genotoxin, cytolethal distending toxin (CDT) which is produced by nearly two dozens of clinically-important bacterial pathogens causes DNA damage in a wide range of eukaryotic cells in vitro. More recently, it has become clear that irrespective of the origin of CDT, genotoxic damage results in a robust DNA damage response (DDR) in which the serine/threonine protein kinase ataxia telangiectasia mutated (ATM) plays a central role. The CDT-induced DDR closely resembles the response to DNA double strand breaks (DSBs) elicited by ionizing radiation (IR) which can lead to irreversible cell cycle arrest and apoptosis of mammalian cells. It is currently believed that CDT-induces ATM-dependent arrest at the G1/S and G2/M transitions of the cell cycle by activation of p53/Chk2 and p21(for G1/S) and Chk2 and CDC25C (for G2/M) respectively. By contrast, the mechanism of CDT-induced apoptosis remains incompletely understood; however, there is some evidence that it results from activation of the intrinsic apoptotic pathway through upregulation of Bax together with down-regulation of Bcl-2 and the release of mitochondrial cytochrome c causing down-stream activation of caspase-9. Moreover, on the basis of a higher sensitivity of hematopoietic cells to the cytotoxic effects of CDT, it has been suggested that CDT modulates the host immune response in vivo. To provide a well-characterized in vitro model system that complements in vivo studies in a mouse model of defective DNA damage, we first generated several cells lines from spontaneous thymic lymphomas of Atm-/- and p53-/- mice. Then by comparing the responses of primary thymic lymphocytes with that of thymic lymphoma cell lines originated from Atm-/- and p53-/- mice, we clearly showed that treatment with H. hepaticus CDT causes a transient reduction in DNA synthesis which is characteristic of activation of the ATM-dependent intra-S checkpoint. By demonstrating an ATM-dependent reduction in phosphorylated histone H3, we further confirmed CDT-mediated G2/M cell cycle arrest. These are the first conclusive evidences that CDT is capable of inducing cell cycle arrest in all phases of lymphoid cell cycle. Finally, we used our Atm-/- and p53-/- mouse thymic lymphoma cells lines to show that CDT-induced apoptosis is also p53-dependent. Next, we examined the in vitro DDR of several human leukemia and lymphoma cell lines to genotoxic damage induced by CDT obtained from the human intestinal pathogen Campylobacter jejuni. While all human leukemia and lymphoma cell lines irrespective of their p53 status display comparable levels of activation of the ATM effector [gamma]-H2AX in response to CDT treatment, two leukemic cell lines, MOLT-3 and MOLT-4 with wild-type p53 showed time- and dose-dependent increased susceptibility to C. jejuni CDT-induced apoptosis when compared to Jurkat, CEM and CA46 (also Bax deficient) cell lines with mutated p53. Demonstration of CDTinduced Chk1 activation in human leukemic cell lines suggests for the first time the involvement of ATR in the DDR to CDT-induced genotoxic damage. Taken together, we again conclude that p53 mutational status is a major determinant of the variable susceptibility of human lymphoma and leukemia cell lines to CDT. Activation of intra-S checkpoint by CDT may contribute to bacterial virulence by modulating rapidly dividing host lymphocytes, and thus, host adaptive immune response following bacterial infection. To address this question directly, we assessed the adaptive humoral immune response following H. hepaticus infection of laboratory mice with a defective DDR due to a mutation in Atm. While infected wild type and heterozygous mice had high levels of IgG1 antibodies to H. hepaticus whole cell lysate (WCL), Atm null mice had approximately 60% reduction in serum antibody responses to both WCL and CdtB. The data suggest that DDR mechanisms are essential for bacterial and CDT-specific host adaptive immune responses.
Duhamel, Gerald E.
Appleton, Judith Ann; Weiss, Robert S.; Altier, Craig
Ph.D. of Veterinary Medicine
Doctor of Philosophy
dissertation or thesis