Maclean, Kirsteen Helen; (1999) Mechanisms of apoptotic induction by iron chelators. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

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.

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