Alteration and activation of sequence-specific cleavage of DNA by bleomycin in the presence of the antitumor drug cis-diamminedichloroplatinum(II).

Abstract

The antitumor drug cis-diamminedichloroplatinum(II) dramatically alters the sequence-specific cleavage of the bleomycin Ar-iron(11)-02 system. Preferred bleomycin cleavage sites adjacent to oligo(dG) regions on two restriction fragments of plasmid pBR322 DNA were masked by pretreatment with cis-diamminedichloroplatinum(II). trans-Diamminedichloroplatinum-(II), which is inactive as an antitumor drug, showed similar but not identical behavior. The DNA-cleaving activity of bleomycin was substantially modified by cis-diamminedichloroplatinum(11), and a number of specific new cutting sites in guanine-rich parts of the sequence were activated by both isomers of the platinum complex. The -sults further emphasize the possibility that the synergism found when cis-diamminedichloroplatinum(II) and bleomycin are used in combination chemotherapy may be due to interactions at the level of DNA-drug binding.Bleomycin and cis-diamminedichloroplatinum(II) (cis-DDP) ( Fig. 1) are used in combination chemotherapy to treat malignant melanoma (2), head-neck carcinoma (3), and testicular carcinoma (4). The two drugs exhibit synergism in the clinical management of head and neck cancer (5). The biological target for both and bleomycin (7) is believed to be DNA.Bleomycin cleaves double-stranded DNA specifically at 5'-*3' G-C and G-T sequences in the presence of ferrous ion and molecular oxygen (1,(8)(9)(10)(11)(12), and the anticancer activity of this glycopeptide antibiotic is related to DNA strand scission. cis-DDP, on the other hand, binds covalently to DNA. Attachment of cis-DDP to poly(dG) regions through intrastrand crosslinking of guanosine N7 atoms is thought to be responsible for its various biological activities (13)(14)(15)(16)(17) DNA-Drug Reactions and Analysis. Reaction samples containing the 165-bp 32P-labeled DNA piece and sonicated calf thymus carrier DNA (1 mg/ml) were incubated with cis-or trans-DDP at a drug-to-nucleotide (D/N) mole ratio of 0.33, 0.165, 0.083, or 0.041 in 1 mM sodium phosphate/5 mM sodium chloride buffer, pH 7.4, at 37°C for 3 hr. In studies with the 327-bp fragment, the carrier DN and cis-DDP concentrations were 20 ,ug/ml and 40-,uM, respectively, corresponding to D/N = 0.67. Sodium chloride was added to 0.2 M final concentration to stop the reaction and the platinated DNA was precipitated by addition of sodium acetate (0.3 M) and ethanol. The pellets were rinsed with cold 75% (vol/vol) ethanol and dried (15). The ratio of bound platinum per nucleotide for both cis-and trans-DDP was determined by flameless atomic absorption spectroscopic analysis of carrier DNA platinated under identical conditions. Next, bleomycin A2 samples containing bound Fe(II) (2 and 4 u.M in two different experiments) and 2-mercaptoethanol (20 mM) in deaerated water were added to the platinated DNA dissolved in 20 mM Tris HCI buffer, pH 8.3, and the mixtures were incubated at 37°C for 10 min (327 bp) or 4 min (165 bp). A 10 ,uM solution of Na2EDTA was added to stop the reaction and the DNA was precipitated by addit...

DNA damage response (DDR) pathway engagement in cisplatin radiosensitization of non-small cell lung cancer

“…While the number of γ-H2Ax foci persisting at 24 hours is likely more than could be explained by the random distribution of complex CDDP-DSB lesions, the presence of a CDDP lesion could differentially influence the position of DSB distribution so that complex CDDP-DSB lesions are more frequent than would be expected by random distribution, leading to persistent γ-H2Ax foci at the site of unrepaired DSBs. This is supported by studies suggesting CDDP causes differential distribution of DSBs following treatment with IR and bleomycin 49,50 and an increased number of IR-induced DSBs 51 , although this remains controversial. 52 Additionally, H460 cells treated with CDDP-IR were able to progress through the G2/M checkpoint prior to the G2 arrest induced by CDDP treatment alone (Figure 3).…”

Site-specific cleavage of RNA by Fe(II).bleomycin.

“…Clearly, the dramatic difference in susceptibilities of these two tRNA precursors to degradation by Fe(II)-BLM argues for selective recognition of the tRNAHiS precursor at the level of tertiary structure; in fact, an alternate conformation for tRNATYr precursor has been suggested (20). This would parallel the alteration of DNA sequence selectivity as DNA conformation was altered (13,14,21).…”

“…Treatment with Fe(II)-BLM at a BLM to RNA nucleotide ratio similar to that used for tRNA His precursor effected cleavage at four sites, CCU-AACATA. subset of all 5'-GC-3' and 5'-GT-3' sites, although it has become increasingly clear that BLM is also responsive to DNA conformational alterations (13,14,19,21,23). Recognition of B-form DNA by Fe(II)-BLM primarily involves recognition of two adjacent bases and is optimal for 2 of the 10 possible distinguishable duplex sequences for a 2-base binding site (24).…”

Complex Cisplatin-Double Strand Break (DSB) Lesions Directly Impair Cellular Non-Homologous End-Joining (NHEJ) Independent of Downstream Damage Response (DDR) Pathways

“…However, there is both experimental and mathematical data indicating that the combination of IR and cisplatin does not result in the random distribution of damage. In vitro, the presence of cisplatin-DNA lesions alters the position of both a bleomycin induced DSB and IR induced DNA damage (33,34). Extrapolation of these in vitro data would suggest that the formation of a DSB in close proximity to a cisplatin-DNA lesion occurs more frequently than by random occurrence and is a physiologically important phenomenon occurring with combined cisplatin-IR treatment.…”