Lawrence F. Povirk

DNA damage and mutagenesis by radiomimetic DNA-cleaving agents : bleomycin, neocarzinostatin and other enediynes

Bleomycin and the enediyne antibiotics effect concerted, simultaneous site-specific free radical attack on sugar moieties in both strands of DNA, resulting in double-strand breaks of defined geometry and chemical structure, as well as abasic sites with closely opposed strand breaks. The hypersensitivity of several mammalian double-strand break repair-deficient mutants to these agents confirms the role of these double-strand breaks in mediating cytotoxicity. In bacteria, mutagenesis by both bleomycin and neocarzinostatin appears to result from replicative bypass of abasic sites, the repair of which is blocked by the presence of closely opposed strand breaks. However, in mammalian cells, such abasic sites decompose to form double-strand breaks, and mutagenesis consists primarily of small deletions, large deletions, and gene rearrangements, all of which probably result from errors in double-strand break repair by a nonhomologous end-joining mechanism. Studies with the radiomimetic antibiotics emphasize the importance of this end-joining repair pathway, and these agents provide useful probes of its mechanistic details, particularly the effects of chemically modified DNA termini on repair.

Effects of bryostatin 1 and other pharmacological activators of protein kinase C on 1-[β-d-arabinofuranosyl]cytosine-induced apoptosis in HL-60 human promyelocytic leukemia cells

We have demonstrated previously that bryostatin 1, a macrocylic lactone with putative protein kinase C (PKC)-activating properties, synergistically augments the antileukemic actions of the deoxycytidine analog 1-[beta-D-arabinofuranosyl]cytosine (ara-C) in HL-60 human promyelocytic leukemia cells (Grant et al., Biochem Pharmacol 42: 853-867, 1991), and that this effect appears to be related to sensitization to ara-C-induced apoptosis (Grant et al., Cancer Res 52: 6270-6278, 1992). In the present studies, we have assessed the extent of this damage by quantitative spectrofluorophotometry of small molecular weight, double-stranded DNA fragments in order to provide: (a) a more complete characterization of the interaction between ara-C and bryostatin 1, and (b) a direct comparison of the relative effects of bryostatin 1 treatment with other pharmacological manipulations known to modulate protein kinase C activity. Exposure of cells to ara-C (10(-9) to 10(-4) M; 1-24 hr) induced time- and concentration-related increases in the extent of DNA fragmentation. Treatment with bryostatin 1 (10(-11) to 10(-7) M; 1-24 hr) alone failed to induce DNA damage, but promoted substantial time- and concentration-related increases in the extent of fragmentation induced by a subsequent 6-hr exposure to ara-C. Maximal potentiation of fragmentation (e.g. 2- to 3-fold greater than that obtained with ara-C alone) was observed following a 24-hr pretreatment with 10(-8) M or 10(-7) M bryostatin 1, and correlated closely with enhanced inhibition of HL-60 cell clonogenicity. The stage-1 tumor-promoter phorbol dibutyrate potentiated the effects of ara-C in a biphasic manner, maximally augmenting the response at 2.5 x 10(-8) M, but exerting no effect at 10(-7) M, whereas the stage-2 tumor-promoter mezerein failed to augment ara-C-related DNA fragmentation at low concentrations, and antagonized ara-C action at high concentrations. In contrast, ara-C-related DNA fragmentation was attenuated or abolished either by continual preexposure to synthetic diglyceride or by pretreatment with exogenous phospholipase C at all concentrations tested. Increased DNA fragmentation was not specifically related to recruitment of cells into S-phase or enhancement of ara-C-related cellular differentiation. Finally, concentrations of bryostatin 1 that maximally potentiated ara-C-related DNA fragmentation were associated with virtually complete down-regulation of total cellular PKC activity, whereas diglyceride and phospholipase C, which suppressed the response to ara-C, moderately increased total PKC activity.(ABSTRACT TRUNCATED AT 400 WORDS)

End-joining of free radical-mediated DNA double-strand breaks in vitro is blocked by the kinase inhibitor wortmannin at a step preceding removal of damaged 3' termini.

Both mammalian cells and Xenopus eggs possess activities for the joining of nonhomologous DNA ends, and such activities may play a major role in double-strand break repair. In order to dissect the biochemical processing of breaks with oxidatively modified ends, vectors containing various site-specific double-strand breaks with 3′-phosphoglycolate termini were constructed and treated with Xenopus egg extracts. These vectors were rejoined by the extracts at rates 30-100 times slower than comparable 3′-hydroxyl vectors. Vectors with blunt or cohesive 3′-phosphoglycolate ends yielded single repair products corresponding to simple phosphoglycolate removal followed by ligation, while a vector with mismatched ends was also rejoined but yielded a mixture of products. Addition of the kinase inhibitors wortmannin and dimethylaminopurine not only blocked rejoining, but also suppressed phosphoglycolate removal, implying an early, essential, kinase-dependent restriction point in the pathway. The results suggest that double-strand breaks with oxidatively modified ends are repaired in Xenopus eggs by a highly conservative and stringently regulated end-joining pathway, in which all biochemical processing of the breaks is contingent on both end alignment and a specific phosphorylation event. Several lines of indirect evidence suggest DNA-dependent protein kinase as a likely candidate for effecting this phosphorylation.

Ionizing Radiation-induced DNA Strand Breakage and Rejoining in Specific Genomic Regions as Determined by an Alkaline Unwinding/Southern Blotting Method

A recently developed, combined alkaline unwinding/Southern blotting assay was utilized to examine DNA damage and repair induced by ionizing radiation within specific large-scale genomic regions. Following treatment of MCF-7 breast tumour cells with 2-10-Gy gamma-rays, strand breakage and rejoining were measured in bulk DNA, in the centromeric alpha-satellite region of chromosome 17, and in the chromatin regions containing the unexpressed beta-globin gene and the expressed c-myc oncogene, which is known to be important for growth in the MCF-7 cell line. Damage in both the c-myc and beta-globin regions was markedly greater than in either alpha-satellite or bulk DNA. However, the kinetics of strand break repair were approximately the same in c-myc as in alpha-satellite or bulk DNA. Surprisingly, the radiomimetic antibiotic bleomycin, which also induces free-radical-mediated strand breakage, showed considerably less heterogeneity of DNA damage among the genomic regions examined than did radiation. The results suggest that actively transcribed genes, as well as at least some inactive genes, are surrounded by large-scale domains of radiosensitive chromatin. With no apparent enhancement of rejoining, the increased incidence of strand breaks in these regions persists until rejoining is essentially complete. Changes in the integrity of specific chromatin regions may be an important aspect of DNA damage-induced cell death.

Effect of in vitro cleavage of apurinic/apyrimidinic sites on bleomycin-induced mutagenesis of repackaged lambda phage

Previous studies have revealed bleomycin to be a potent base-substitution mutagen in repackaged phage lambda. In order to assess the role of apurinic/apyrimidinic (AP) sites in bleomycin-induced mutagenesis, bleomycin-damaged lambda DNA was treated with putrescine or endonuclease IV to effect cleavage of bleomycin-induced AP sites. The DNA was then packaged, the phage grown in SOS-induced E. coli, and the frequency of clear-plaque mutants in the progeny was determined. Bleomycin-induced mutagenesis was decreased approx. 2-fold by treating the DNA with putrescine, but was unaffected by endonuclease IV. The results are consistent with the production of bleomycin-induced mutation at certain AP sites having a closely opposed single-strand break, since such sites are cleaved by putrescine but not by endonuclease IV.

Influence of amsacrine (m-AMSA) on bulk and gene-specific DNA damage and c-myc expression in MCF-7 breast tumor cells

In the MCF-7 human breast tumor cell line, the aminoacridine, m-AMSA, induces protein-associated DNA strand breaks consistent with inhibition of topoisomerase II. However, neither single-strand nor double-strand breaks in DNA, determined using conventional assays, show a consistent relationship with m-AMSA-induced inhibition of growth. In contrast, when DNA strand breaks are determined by alkaline unwinding under the high salt conditions of the alkaline unwinding/Southern blotting (AU/SB) assay, developed by our laboratories, damage to DNA corresponds closely with growth inhibition. The AU/SB assay, which is capable of assessing breaks within large-scale domains (upwards of 1 megabase) surrounding genes of interest, was further utilized to explore the capacity of m-AMSA to induce damage within specific genomic regions that may regulate cell growth. Regions encompassing the transcriptionally active oncogenes, c-myc and c-fos, were found to be more susceptible to m-AMSA-induced strand breaks than the region encompassing the non-transcribed alpha-satellite DNA or the genome as a whole (bulk DNA). These findings demonstrate that m-AMSA may produce more pronounced damage within specific genomic regions than in bulk DNA, m-AMSA also preferentially altered expression of the c-myc oncogene; at an m-AMSA concentration where growth was inhibited by between 70 and 80%, steady-state c-myc mRNA levels declined to approximately 10-15% of control levels within 2-3 hr; furthermore, concentration-dependent reductions in c-myc expression appeared to coincide with growth inhibition. In addition, inhibition of [3H]thymidine incorporation after 2 hr directly paralleled inhibition of growth, suggesting an early effect at the level of DNA biosynthesis, possibly related to the down-regulation of c-myc expression. It is proposed that specific lesions, e.g., in regions surrounding the c-myc gene, as well as generalized lesions in DNA may lead to growth inhibition mediated by down-regulation of the expression of select growth regulatory genes, such as c-myc.

Bleomycin-induced mutagenesis in repackaged lambda phage: base substitution hotspots at the sequence C-G-C-C

DNA isolated from lambda phage was treated with bleomycin A2 plus Fe2+. The bleomycin-damaged DNA was added to lambda packaging extracts and the resulting phage were grown in SOS-induced E. coli. Under these conditions, treatment of the DNA with 0.8 microM bleomycin reduced the viability of the repackaged phage to 3% and increased the frequency of clear-plaque mutants in the progeny by a factor of 16. Bleomycin-induced mutations which mapped to the DNA-binding domain of the cI gene were subjected to DNA-sequence analysis. The most frequent events were single-base substitutions at G:C base pairs, nearly all of which occurred at cytosines in the sequence Py-G-C. Cytosines in the third position of the sequence C-G-C-C were particularly susceptible to mutation. At A:T base pairs, mutations were less frequent and were a mixture of single-base substitutions and -1 frameshifts, occurring primarily at G-T and A-T sequences. Thus, the overall specificity of bleomycin-induced mutations matches that of bleomycin-induced DNA lesions (strand breaks and apyrimidinic sites), which are formed at G-C (particularly Py-G-C), G-T and, to a lesser extent, A-T sequences. Furthermore, the frequency of various types of substitutions was consistent with selective incorporation of A and T residues opposite apyrimidinic sites at these sequences. The highly selective nature of bleomycin-induced mutations may explain the lack of mutagenesis by this compound in a number of reversion assays.

Targeted base substitutions and small deletions induced by neocarzinostatin at the APRT locus in plateau-phase CHO cells

Treatment of confluence-arrested CHO-D422 cells for 48 h with low concentrations (0.5-3 nM) of the radiomimetic antibiotic neocarzinostatin resulted in an increase in up to 11-fold in the frequency of mutations at the hemizygous APRT locus. Analysis by PCR and DNA sequencing revealed that the mutations were a mixture of base substitutions, small deletions, and large-scale rearrangements. base substitutions occurred preferentially at sequence positions where the drug is known to produce abasic sites with closely opposed strand breaks, e.g., AGT, TGT and AGC, where the abasic site occurs at the underlined base and the strand break occurs opposite the first base in each triplet. These results suggest that the substitutions were produced by replicative bypass of the abasic sites, perhaps during attempted repair of the accompanying strand break. Single-base deletions, which comprised nearly half of all deletions, were targeted to these same sequence positions, suggesting that they may have been generated either by replicative bypass of the abasic sites, or by end-joining repair of double-strand breaks, which are induced the same sites. Quantitative analysis of neocarzinostatin-induced damage to APRT DNA in vitro confirmed the association between lesions involving concommitant damage to both DNA strands, and mutations. The results are consistent the hypothesis that agents which induce such bistranded DNA damage can produce biologically significant levels of mutagenesis even in nondividing cells.

A highly conservative, cyclically permuted, non-homologous exchange among three unrelated DNA sequences in bleomycin-treated CHO cells

Molecular analysis of a bleomycin-induced rearrangement of the aprt gene in CHO cells revealed that it consisted of a nearly perfect three-way exchange among non-homologous sequences, consistent with a mechanism involving cyclically permuted misjoining of the six ends of three double-strand breaks.

Monofunctional adenine N-3 adducts of melphalan : Occurrence at a mutational hotspot sequence and resistance to removal by alkA protein

Previous work showed that a CTAAA sequence in the supF gene of the shuttle plasmid pZ189 was a hotspot for mutagenesis by the aromatic nitrogen mustards melphalan and chlorambucil, and indirect evidence suggested adenine N‐3 adducts as premutagenic lesions. In order to characterize the adducts formed at this sequence more directly, a substrate was prepared in which the three adjacent adenines in the CTAAA sequence were 3H‐labeled. Following treatment of this substrate with [14C]melphalan, thermolabile adducts were depurinated and analyzed by HPLC. Only a single peak bearing both 3H and 14C label was detected and it coeluted with the single major adduct formed by the reaction of melphalan with free adenine base. Various spectrometric analyses of this species were all consistent with its identification as a monofunctional adenine N‐3 adduct of melphalan. There was no evidence for any bifunctional adducts involving the labeled adenines. There was little if any release of the adenine N‐3 adduct of melphalan by Escherichia coli AlkA protein, under conditions where 3‐methyladenine was quantitatively released. The results support the proposal that monofunctional adenine N‐3 adducts are intermediates in the generation of T → A and T → G transversions by aromatic nitrogen mustards. Environ. Mol. Mutagen. 31:333–339, 1998