Sequence-Specific Double-Strand Cleavage of DNA by Fe-Bleomycin. 2. Mechanism and Dynamics

Abstract

The mechanism of iron-bleomycin-mediated ds-cleavage of DNA has been investigated at specific sites within specific sequences using hairpin oligonucleotides (Absalon et al., 1995) and our recently developed technique for determining sequence-specific isotope effects upon oxidative degradation of DNA (Kozarich et al., 1989; Worth et al., 1993). Isotope effects upon ds-cleavage have been observed when the C-4' hydrogen of either nucleotide involved in the ds-break was substituted with deuterium. The values of the isotope effects determined for ss and ds events occurring at the same site were indistinguishable at four sites examined in detail. The results are consistent with a mechanism of ds-cleavage in which the pathways leading to ss- and ds-cleavage partition from a common intermediate subsequent to abstraction of the C-4' hydrogen from the first nucleotide involved in the cleavage. Deuterium substitution at the primary cleavage site of a ds-break failed to result in an equivalent effect on the amount of cleavage at the secondary cleavage site, suggesting that ds-cleavage may be initiated from either of the nucleotides involved in the ds-cleavage event. A kinetic preference for cleavage initiated at the 1 degree site, however, is probable. The requirement in the ds-cleavage process for O2, in addition to that needed to form "activated BLM", has been clearly demonstrated by the absence of ds-cleavage products in reactions performed under anaerobic conditions in which ss-cleavage still occurs. These results support, in part, the basic model for ds-cleavage proposed by Steighner and Povirk [(1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8350-8354], in which a single molecule of BLM effects ds-cleavage and requires reactivation to effect cleavage at the second strand. The essential factor establishing the ratio of ss- to ds-cleavage at a specific site may be related to the efficiency by which Fe-BLM can be reactivated and/or repositioned at a second site for cleavage.

DNA Cleavage/Repair and Signal Transduction Pathways in Irradiated Breast Tumor Cells

“…We failed to observe any direct evidence of cell killing in MCF-7 cells with either l,25-(OH) 2 -D 3 orEB 1089 with exposure times as long as 96 h, although it is possible that the reduction in the number of cells over time reflects both growth inhibition and (a limited degree of) cell death. While other laboratories have reported apoptosis in breast tumor cells (including MCF-7 cells) treated with vitamin D 3 compounds (26)(27)(28)(29), the susceptibility to apoptosis appears to be limited to a relatively small fraction of the target cell population in these studies (i.e. between 5 and 15%) (26,27).…”

“…It is noteworthy that p21 antisenseexpressing cells exposed to ara-C displayed a relatively modest enhancement in ROS generation, consistent with the notion that induction of ROS lags behind and represents a consequence of earlier rnitochondrial damage (41). It is also important to note that the increased susceptibility of p21 WAF1/CIP1 antisense-expressing cells to ara-C-mediated rnitochondrial dysfunction was accompanied by a loss in leukemic cell self-renewal capacity, particularly in view of reports suggesting a discordance between apoptosis and loss of clonogenic potential (27,30, 44).…”

“…While other laboratories have reported apoptosis in breast tumor cells (including MCF-7 cells) treated with vitamin D 3 compounds (26)(27)(28)(29), the susceptibility to apoptosis appears to be limited to a relatively small fraction of the target cell population in these studies (i.e. between 5 and 15%) (26,27). As a consequence of the prolonged survival of the bulk of the tumor cell population, most studies (both in vitro and in vivo) indicate that l,25-(OH) 2 -D 3 , (28)(29)(30)(31)(32)(33)(34).…”

“…It is noteworthy that p21 antisenseexpressing cells exposed to ara-C displayed a relatively modest enhancement in ROS generation, consistent with the notion that induction of ROS lags behind and represents a consequence of earlier rnitochondrial damage (41). It is also important to note that the increased susceptibility of p21 WAF1/CIP1 antisense-expressing cells to ara-C-mediated rnitochondrial dysfunction was accompanied by a loss in leukemic cell self-renewal capacity, particularly in view of reports suggesting a discordance between apoptosis and loss of clonogenic potential (27,30, 44).Although disruption of p21 WAF1/CIP1 function has been shown to increase the sensitivity of tumor cells to various agents (8-10), this has not been a universal finding. For example, antisense-mediated disruption of p21 WAF1/CIP1 reduced antioxidant-related apoptosis and potentiation of 5-FU-mediated lethality in human colon cancer cells (HCT 15 and HCT 116) in a p53-independent manner (45).…”

Synthesis, Structure Characterization and Antitumor Activity Study of a New Iron(III) Complex of 5-Nitro-8-hydroxylquinoline (HNOQ)

“…10) For instance, the iron complex of the glycopeptide bleomycin, which is clinically used in cancer treatment, can cause double-strand breaks in DNA. 11) This successful application of an iron complex has motivated investigations of new iron complexes for cancer treatment. During the last decade, a number of iron complexes, involving hydroxyferrocifen iron complexes, 12) salophen iron complexes 13) and dipyridophenazine iron complexes, 14) were synthesized and studied for their antitumor activities.…”