Mechanisms of apoptotic induction by iron chelators

The orally bioavailable hydroxypyridinone iron chelator, CP20 (L1 or deferiprone) is known to induce bone marrow hypoplasia and thymic aplasia in laboratory animals and apoptosis in thymocytes and leukaemic cell lines, although the mechanisms are unclear. Experiments contained within this thesis have sought to elucidate how iron chelators CP20 and Desferrioxamine (DFO) induce apoptosis in murine thymocytes, human leukaemic cells and haemopoietic progenitor cells. Inhibition of the iron (III) containing enzyme, ribonucleotide reductase (RR) with consequent inhibition of DNA synthesis has been examined as a possible mechanism of apoptotic induction. The apoptotic effects of the RR inhibitor, hydroxyurea, have been compared with those of the iron chelators in thymocytes, human leukaemic HL60 cells and human haemopoietic progenitors. Apoptosis has been compared in different cell types by quantitative flow cytometry. DNA synthesis inhibition has been assessed by the incorporation of both BrdU and 3H-thymidine. Whereas iron chelators induce thymocyte apoptosis as early as 4 hours, hydroxyurea showed no effect, suggesting that RR inhibition is not the primary apoptotic mechanism in this cell type. By contrast, both the chelators and hydroxyurea induced apoptosis in proliferating HL60 cells where BrdU analysis showed that for both HU and chelators the apoptotic population was derived from cells which had recently entered S phase. In haemopoietic progenitors derived from CD34+ peripheral blood cells in liquid culture, apoptosis was induced by HU and chelators only when the cells were in cycle (> days 2 or 9 days of culture). These findings are consistent with inhibition of RR being causative in apoptotic induction in proliferating cells but not in thymocytes. In thymocytes, induction of apoptosis by chelators requires RNA and protein synthesis because actinomycin D and cycloheximide respectively abrogate this process. Chelator induced apoptosis was equal in p53 knockout and wild-type thymocytes, suggesting that primary DNA damage is not the apoptotic trigger. A possible link between chelation of zinc and the induction of apoptosis was also investigated. In thymocytes, zinc was shown to abrogate the apoptotic effects of chelators in vitro and in vivo. Furthermore prolonged exposure of thymocytes to chelators deprives the cells of intracellular zinc, indicating that zinc chelation may contribute to the apoptosis. The bidentate hydroxypyridinones interact with intracellular zinc pools at low concentrations (1uM CP20) in a fundamentally different manner from the hexadentate iron chelator DFO. Unlike the latter chelator, CP20 can shuttle zinc from inaccessible sites within cells onto larger zinc chelating molecules thereby enhancing apoptosis. In conclusion, the findings in this thesis show that proliferating cells in S-phase are particularly susceptible to apoptotic induction by iron chelators. Furthermore because of the similarity in terms of cell specificity and kinetics of apoptosis between HU and iron chelators, inhibition of RR is a likely mechanism of apoptotic induction in proliferating cells. However in thymocytes which are predominantly non-proliferating, a different mechanism of apoptotic induction must be invoked, which may in part involve the chelation of zinc

Mnt Loss Triggers Myc Transcription Targets, Proliferation, Apoptosis, and Transformation

Myc oncoproteins are overexpressed in most cancers and are sufficient to accelerate cell proliferation and provoke transformation. However, in normal cells Myc also triggers apoptosis. All of the effects of Myc require its function as a transcription factor that dimerizes with Max. This complex induces genes containing CACGTG E-boxes, such as Ornithine decarboxylase (Odc), which harbors two of these elements. Here we report that in quiescent cells the Odc E-boxes are occupied by Max and Mnt, a putative Myc antagonist, and that this complex is displaced by Myc-Max complexes in proliferating cells. Knockdown of Mnt expression by stable retroviral RNA interference triggers many targets typical of the “Myc” response and provokes accelerated proliferation and apoptosis. Strikingly, these effects of Mnt knockdown are even manifest in cells lacking c-myc. Moreover, Mnt knockdown is sufficient to transform primary fibroblasts in conjunction with Ras. Therefore, Mnt behaves as a tumor suppressor. These findings support a model where Mnt represses Myc target genes and Myc functions as an oncogene by relieving Mnt-mediated repression

Identification of sensitive serum microRNA biomarkers for radiation biodosimetry.

Exposure to ionizing radiation through environmental, occupational or a nuclear reactor accident such as the recent Fukushima Daiichi incident often results in major consequences to human health. The injury caused by radiation can manifest as acute radiation syndromes within weeks in organs with proliferating cells such as hematopoietic and gastrointestinal systems. Cancers, fibrosis and degenerative diseases are also reported in organs with differentiated cells, months or years later. Studies conducted on atom bomb survivors, nuclear reactor workers and animal models have shown a direct correlation of these effects with the absorbed dose. Physical dosimeters and the available radio-responsive biologics in body fluids, whose responses are rather indirect, have limitations to accurately evaluate the extent of post exposure damage. We have used an amplification-free, hybridization based quantitative assay utilizing the nCounter multiplex platform developed by nanoString Technologies to compare the levels of over 600 miRNAs in serum from mice irradiated at a range of 1 to 12 Gy at 24 and 48 hr time points. Development of a novel normalization strategy using multiple spike-in oligonucleotides allowed accurate measurement of radiation dose and time dependent changes in serum miRNAs. The response of several evolutionarily conserved miRNAs abundant in serum, were found to be robust and sensitive in the dose range relevant for medical triage and in patients who receive total body radiation as preparative regimen for bone marrow transplantation. Notably, miRNA-150, abundant in lymphocytes, exhibited a dose and time dependent decrease in serum, which we propose as a sensitive marker indicative of lymphocyte depletion and bone marrow damage. Our study has identified several markers useful for evaluation of an individual's response by minimally invasive methods, relevant to triage in case of a radiation accident and evaluation of toxicity and response during and after therapeutic radiation

c-Myc is essential for vasculogenesis and angiogenesis during development and tumor progression

c-Myc promotes cell growth and transformation by ill-defined mechanisms. c-myc(−/−) mice die by embryonic day 10.5 (E10.5) with defects in growth and in cardiac and neural development. Here we report that the lethality of c-myc(−/−) embryos is also associated with profound defects in vasculogenesis and primitive erythropoiesis. Furthermore, c-myc(−/−) embryonic stem (ES) and yolk sac cells are compromised in their differentiative and growth potential. These defects are intrinsic to c-Myc, and are in part associated with a requirement for c-Myc for the expression of vascular endothelial growth factor (VEGF), as VEGF can partially rescue these defects. However, c-Myc is also required for the proper expression of other angiogenic factors in ES and yolk sac cells, including angiopoietin-2, and the angiogenic inhibitors thrombospondin-1 and angiopoietin-1. Finally, c-myc(−/−) ES cells are dramatically impaired in their ability to form tumors in immune-compromised mice, and the small tumors that sometimes develop are poorly vascularized. Therefore, c-Myc function is also necessary for the angiogenic switch that is indispensable for the progression and metastasis of tumors. These findings support the model wherein c-Myc promotes cell growth and transformation, as well as vascular and hematopoietic development, by functioning as a master regulator of angiogenic factors