SummaryThe chemotherapeutic agents 2'-deoxy-5-azacytidine (DAC) and cisplatin (cDDP) have been shown in vitro to be synergystic in their cytotoxicity toward human tumour cells. We have investigated possible molecular mechanisms underlying this synergy using the plasmid pSVE3 in vitro and after transfection into CMT3 cells. Increased binding of cDDP to DAC-substituted DNA generated in vivo was confirmed by flameless atomic absorption spectrophotometry (FAAS). The Many factors are considered when selecting the drugs to be included in a regimen of combination chemotherapy. Frequently, agents are chosen based on toxicity profiles and demonstrated efficacy in clinical trials. Occasionally, in vitro cytotoxicity assays will suggest a synergistic interaction (i.e. the effect of the two drugs together is greater than the predicted additive effect), and this information can be used in designing treatment protocols. Previous studies from our laboratory, using human tumour cell lines, showed that synergistic cytotoxicity can be demonstrated between 2'-deoxy-5-azacytidine (DAC) and cis-dichlorodiaminoplatinum (cDDP) (Frost et al., 1990). The mechanism for synergy between cDDP and DAC remains to be elucidated. Theoretically, synergy between two drugs can occur at several cellular levels. One drug may increase cellular uptake of a second drug, or inhibit its removal from the cell. Metabolism of the second drug could be altered in a way that the drug persists in an active form for a longer time. For compounds that act at the level of DNA, incorporation or binding may be enhanced. The synergistic interaction between cDDP and DAC may likely take place at the DNA level, as this is where both drugs are known to exert their effects. cDDP binds directly to DNA, producing both intrastrand and interstrand crosslinks (Bird, 1978;Caradona et al., 1982;Pinto & Lippard, 1985). The intrastrand crosslinking is most frequent at N7 of adjacent guanosines. DAC is an analog of deoxycytidine, substituting a nitrogen for carbon 5 of the pyrimidine ring. DAC is incorporated into DNA (Vesely & Cihak, 1977) and functions as a DNA methyltransferase inhibitor (Creusot et al., 1982). The resulting DNA hypomethylation has been shown to be associated with changes in gene expression and cell differentiation (Jones & Taylor, 1980;Razin & Riggs, 1980 Materials and methods Plasmid preparationThe plasmid pSVE3 (Hartman et al., 1982), was isolated from E. coli DH5 cells (Hanahan, 1983) using the alkaline extraction method followed by CsCl density centrifugation and extensive dialysis as previously described (Sambrook et al., 1989).Cells and culture conditions CMT3 cells (Gerard & Gluzman, 1985) were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Grand Island, NY) supplemented with L-glutamine (292 yg ml-'), penicillin (500 U ml-1), streptomycin sulphate (100 fig ml-'), 10% fetal bovine serum (Hazleton, Lenexa, KS) at 37°C in a 10% CO2 humidified incubator.Determination of DAC incorporation into DNA CMT3 cells were plated in two 35 mm diameter...