Alan Eastman

A system in mouse liver for the repair of O6-methylguanine lesions in methylated DNA

An activity from mouse liver with catalyzes the disappearance of O6-methylguanine from DNA methylated with methylnitrosourea has been partially purified by ammonium sulfate fractionation and DNA-cellulose chromatography. The activity does not require divalent metal ions and is not affected by EDTA. It is specific for the repair of O6-methylguanine lesions and does not affect the removal of 7-methylguanine, 7-methyladenine or 3-methyladenine. The disappearance of O6-methylguanine is linear with respect to the concentration of protein and is dependent on incubation temperature. The kinetics and substrate dependence experiments suggest that the protein factor is product-inactivated. Amino acid analysis of hydrolysates of protein obtained after incubation of methylated DNA with the protein factor indicates the presence of radiolabeled S-methyl-L-cysteine, suggesting that during the repair of O6-methylguanine from methylated DNA, the methyl group is transferred to a sulfhydryl of a cysteine residue of a protein. This represents the first such demonstration in a mammalian system.

The significance of DNA cross-linking to cis-diamminedichloroplatinum(II)-induced cytotoxicity in sensitive and resistant lines of murine leukemia L1210 cells

The cross-linking of DNA by cis-diamminedichloroplatinum(II) (cis-DDP) has been studied in a murine leukemia L1210 line (L1210/0) and a 30-fold resistant subline (L1210/DDP2) utilizing the technique of alkaline elution. The kinetics of cross-link formation were similar in both cell lines. After 1-h treatment with cis-DDP, cross-linking continued to increase to a maximum at 12 h posttreatment. Although cross-linking decreased by 24 h some lesions were seen to persist. After 72 h interstrand cross-links were completely removed by both cell lines, however 50% of the DNA-protein cross-links still remained in the sensitive cells even though they were undetectable in the resistant subline. To correlate cross-linking with cytotoxicity, a range of drug concentrations were used for concomitant growth inhibition studies and alkaline elution analyses. In addition to the 30-fold resistant subline, two other sublines developed in our laboratory which show 20- and 50-fold resistance to cis-DDP were included in these determinations. All the resistant sublines exhibited the same degree of cross-linking at the same applied drug doses although these doses elicited markedly different cytotoxicities; cross-linking varied only with applied drug concentrations. The degree of cross-linking in L1210/0 was also directly related to the dose of cis-DDP. However, the sensitive cell line displayed equivalent total cross-linking at a 5-fold lower applied dose. This may implicate an uptake phenomenon as a contributor to resistance. However, such a phenomenon can account for only part of the resistance observed as there existed 6-fold more total cross-links and 9-fold more interstrand cross-links in L1210/DDP2 at doses that were equitoxic to L1210. This suggests the existence of a more critical cytotoxic lesion that was not detectable by alkaline elution, probably interstrand cross-links. Resistance could be due to a differential removal of these lesions.

Comparison of in vivo and in vitro binding of polycyclic hydrocarbons to DNA

The in vivo binding of [3H]benzo(a)pyrene (BP) and 3-[3H]methylcholanthrene (3MC) to liver and lung DNA was studied in A/J mice. Only in liver was there any reduction in total DNA-bound radioactivity between 4 h and 24 h after administration of the hydrocarbon. DNA was fractionated on Sephadex LH-20 after enzymatic digestion. A single deoxyribonucleoside-BP adduct was detected whereas two major 3MC-adducts were observed. Wtih both BP and 3MC, three additional peaks of radioactivity eluted rapidly in the lung DNA experiments while a fourth was noted with liver DNA. The nucleoside-bound adducts from lung represented a much larger proportion of the total radioactivity than with liver. In vitro analysis of 3MC binding to DNA showed the nucleoside-bound adducts to be predominantly deoxyguanosine-dependent but that the early peaks were independent of base suggesting binding to another part of the DNA molecule, perhaps phosphate, i.e, phosphotriesters.

Comparison of the interaction of cis- and trans-diamminedichloroplatinum(II) with DNA by a simple filter binding assay.

A nitrocellulose filter binding assay has been used to assess the interaction of cis- and trans-diamminedichloroplatinum(II) with calf thymus DNA. Local regions of denaturation resulted from cis-DDP interaction and these were attributed to the concerted effect of close platinations in DNA. Single-strand specific S1 nuclease and nitrocellulose filters had the same sensitivity for detecting these lesions. About 1150cis-DDP lesions were DNA interstrand crosslinks and these were resistant to S1 nuclease. About 1150 platinations resulting from trans-DDP caused interstrand crosslinks. Very little local denaturation was detected except at high trans-DDP levels, because of the geometric constrains that prevent trans-DDP from forming DNA-intrastrand crosslinks.

A technique for the measurement of breakage and repair of DNA alkylated in vivo.

The alkaline elution technique has been adapted for use in the assessment of DNA damage induced in the livers and lungs of mice after administration of an alkylating agent, methylemthanesulfonat (MMS). At 4 h after administration of MMS, damage ot DNA was readily demonstrable; the damage was repaired in liver by 24 h. The lung, particularly of the A/J mouse, exhibited an increased alkaline elution rate when compared to C57BL/6J, and repair was not entirely complete (as judged from the rate of alkaline elution of DNA) by 24 h. The rate of elution was dependent upon temperature. It is believed that this adaptation should have great utility in examining DNA repair in vivo.

DNA-protein crosslinks induced in a hamster tracheal epithelial cell line by benzo(a)pyrene

Abstract The formation of DNA crosslinks in a hamster tracheal epithelial cell line after benzo(a)pyrene treatment was studied using the technique of alkaline elution. DNA strand breaks were introduced with 450 rads of x-rays at 0°C in order to facilitate its elution. The effect of prior treatment with benzo(a)pyrene was to reduce the rate of elution compared to that observed with control irradiated cells. The crosslinking effect induced by benzo(a)pyrene was reversed by incubation of the cell lysates with proteinase K prior to alkaline elution, implying that the crosslinks were formed between DNA and protein. The crosslinking effect was shown to increase with time of exposure to benzo(a)pyrene. After 8 hours cells were postincubated in the absence of the carcinogen for periods of up to 48 hours. No diminution in the level of crosslinks was observed.

DNA crosslinking induced by 1,2-diaminocyclohexanedichloroplatinum(II) in murine leukemia L1210 cells and comparison with other platinum analogues

Abstract 1,2-Diaminocyclohexanedichloroplatinum(II) (DCDP) is an analogue of the clinically efficacious cancer chemotherapeutic drug cis-diamminedichloroplatinum(II) (cis-DDP). DCDP is presently undergoing clinical trials at least in part because a cis-DDP-resistant murine leukemia L1210 cell line is sensitive to its action. The alkaline elution technique was used to measure DNA-protein and DNA-interstrand crosslinks induced by DCDP in sensitive and resistant L1210 cells. This was compared to the action of cis-DDP and its clinically ineffective isomer trans-DDP. The action of DCDP was similar to that for cis-DDP with maximum crosslinking occurring between 6 and 12 h after a 1 h treatment. Both cis-DDP and DCDP exhibited proportionately higher levels of interstrand crosslinking than trans-DDP. Near complete removal of both classes of DCDP-induced crosslinks was seen by 72 h. While the extent of crosslinking was different for each compound, little difference between the two cell lines was noted with respect to crosslinking by either DCDP or trans-DDP. These cell lines exhibit a 2-fold resistance to both DCDP and trans-DDP and at equitoxic doses of both drugs the resistant cells demonstrated twice the interstrand crosslinks that were seen in the sensitive cells. The extent of crosslinking related directly to the concentration of drug. When treated with equitoxic doses of DCDP, cis-DDP or trans-DDP, the resistant cells consistently exhibited more interstrand crosslinks than sensitive cells, suggesting the existence of a more critical cytotoxic lesion which was not detectable by the alkaline elution technique. These lesions could be either intrastrand crosslinks or monofunctional platination. Resistance must be due to a differential sensitivity to the lesions that form, which may be due to an altered capacity to repair the lesions.