Ronald D. Snyder

Photogenotoxicity of fluoroquinolones in Chinese hamster V79 cells: dependency on active topoisomerase II.

Abstract— The Chinese hamster V79 lung cell in vitro micronucleus assay was adapted to detect and quantify phototoxicity and photogenotoxicity of fluoroquinolones. Using this assay, the quinolones were ranked in terms of decreasing phototoxicity: clinafloxacin > lomefloxacin, sparfloxacin > trovafloxacin, nalidixic acid, ofloxacin, ciprofloxacin > enoxacin, norfloxacin. This rank order agrees well with published studies utilizing various other phototoxicity models and establishes this approach as a fast and sensitive way to characterize the phototoxic potential of quinolones. Nearly complete inhibition of phototoxicity was observed if the cells were pretreated for as little as I min with 10–20 mM sodium azide prior to the addition of quinolone. An identical azide effect was seen in unirradiated quinolone‐and etoposide‐treated cells. These findings are consistent with a model in which sodium azide renders DNA topoisomerase II catalytically inactive. In this state, topoisomerase II cannot initiate DNA strand cleavage and the DNA/topoisomerase complex becomes insensitive to quinolones and other topoisomerase II inhibitors. The fact that azide reduces both UV‐dependent and UV‐independent toxicity and clastogenicity strongly suggests a common mechanism of toxicity dependent on the formation of topoisomerase‐induced DNA double‐strand breaks.

Synthesis and DNA-binding properties of polyamine analogues

The synthesis of a series of novel polyamine analogues is reported. The DNA binding of these compounds and a variety of other polyamines were compared with their IC50 values against HeLa cell. There seemed to be no apparent correlation between the DNA binding and toxicity against HeLa cells.

Effects of sodium selenite on DNA and carcinogen-induced DNA repair in human diploid fibroblasts

Sodium selenite induces substantial DNA damage in human fibroblasts. This damage appears to consist of true DNA breaks rather than alkali-labile sites and to not arise via free oxygen radical production. Cloning efficiency and DNA strand breakage show dramatically enhanced sensitivity to selenite if the treatments are carried out in the presence of reduced glutathione or, to a lesser degree, serum, supporting the notion that a glutathione-selenite conjugant is required for activation to a genotoxic form. In addition, the notion that selenium anticarcinogenicity involves enhancement of cellular DNA repair, has been examined. No evidence for enhancement (or inhibition) of repair of methyl methanesulfonate (MMS)-, UV- or bleomycin-induced DNA damage was observed in human fibroblasts treated with selenite.

Effects of polyamine analogs on the extent and fidelity of in vitro polypeptide synthesis

A series of polyamine analogs has been examined for their ability to support protein synthesis in an in vitro rabbit reticulocyte translation system. Diamines were found to stimulate protein synthesis to the greatest extent (8-12 fold). Triamines, tetraamines and pentaamines only stimulated 2-4 fold under these conditions although much lower concentrations were required. At elevated temperatures (45 degrees C), diamines were somewhat more active than at lower temperature but activity of longer chain polyamines was elevated very significantly. Polyamines with terminal benzyl or smaller alkyl groups had diminished activity. It is concluded that both charge and charge distribution determine the ability of polyamines to stimulate translation. Fidelity studies identified two classes of polyamines: those which are able to lower the optimal Mg2+ concentration required for amino acid misincorporation while not affecting extent of misincorporation relative to Mg2+ alone, and those which are sparing for Mg2+ and also stimulate extent of misincorporation.

EFFECT OF POLYAMINE DEPLETION ON DNA DAMAGE and REPAIR FOLLOWING UV IRRADIATION OF HeLa CELLS

Abstract— Treatment of HeLa cells with the polyamine biosynthesis inhibitors, methylglyoxal bis(guanylhydrazone) (MGBG), difluoromethylornithine (DFMO) or a combination of the two, resulted in reduction in cellular polyamine levels. Analysis of UV light‐induced DNA damage and repair in these polyamine depleted cells revealed distinct differences in the repair process relative to that seen in cells possessing a normal polyamine complement. Initial yield of thymine dimers and rate of removal of these lesions from cellular DNA appeared normal in pofyamine‐depleted cells. However, depleted cells exhibited retarded sealing of DNA strand breaks resulting from cellular repair processes, reduced repair synthesis and an increased sensitivity to UV killing. Incision at damaged sites was not affected since ara‐C repair‐dependent breaks accumulated in a normal fashion. Molecular analysis of inhibited repair sites by exonuclease III and T4 DNA ligase probes suggest that the strand interruptions consist of gaps rather than ligatable nicks, consistent with an interpretation of the repair defect being at the gap‐filling stage rather than the ligation step. Observed patterns of differential polyamine depletion by DFMO and MGBG, and partial reversal of repair inhibition by polyamine supplementation, suggests that polyamine depletion per se, rather than some secondary effect of inhibitor treatment, is responsible for the inhibition of repair.

Alterations in repair of alkylating agent-induced DNA damage in polyamine-depleted human cells

Treatment of HeLa cells with the polyamine biosynthesis inhibitors difluoromethylornithine (DFMO) and/or methylglyoxal bis(guanylhydrazone) (MGBG) results in marked depression in levels of the cellular polyamines putrescine, spermidine and spermine. Cells in this polyamine-depleted state exhibited increased sensitivity to monofunctional alkylating agents, manifested as decreased cloning ability and retardation of the DNA excision repair process. DFMO treatment did not alter the initial level of interaction of radiolabeled alkylating agent with cellular DNA, but combined treatment with DFMO and MGBG reduced covalent binding, probably through effects on cell cycling. Polyamine supplementation had no effects on initial yield of DNA single-strand breaks in drug-treated cells. The repair defect appeared similar to that observed previously in polyamine-depleted cells following X-irradiation and UV irradiation, namely retarded sealing of DNA strand breaks. It was not possible to reverse the effects of these inhibitors by short periods of polyamine loading, despite the fact that all three polyamines could be restored to near-normal levels. These findings provide the first demonstration of altered response of polyamine-depleted cells to monofunctional alkylating agents and contribute to our understanding of altered responses of polyamine-depleted cancer cells to a variety of DNA-reactive chemotherapeutic drugs.

Effects of Polyamines on the Binding of Hoechst 33258 to Calf Thymus DNA

Abstract The ability of polyamines to displace the minor groove-binding dye Hoechst 33258 from calf thymus DNA was investigated. Polyamines displace non-specific DNA phosphate bound Hoechst in a charge-dependent fashion, but show very little ability to displace the high affinity binding of Hoechst in the minor groove of DNA. This high affinity binding is, however, sensitive to ethidium bromide and the minor groove binding drug berenil. These studies suggest that polyamines probably bind DNA in the minor groove very weakly, if at all, relative to known minor groove binding agents.