2′,2′-Difluorodeoxycytidine (gemcitabine, dFdC) and cis -diammine-dichloroplatinum (cisplatin, CDDP) are active agents against ovarian cancer and non-small-cell lung cancer (NSCLC). CDDP acts by formation of platinum (Pt)–DNA adducts; dFdC by dFdCTP incorporation into DNA, subsequently leading to inhibition of exonuclease and DNA repair. Previously, synergism between both compounds was found in several human and murine cancer cell lines when cells were treated with these drugs in a constant ratio. In the present study we used different combinations of both drugs (one drug at its IC 25 and the other in a concentration range) in the human ovarian cancer cell line A2780, its CDDP-resistant variant ADDP, its dFdC-resistant variant AG6000 and two NSCLC cell lines, H322 (human) and Lewis lung (LL) (murine). Cells were exposed for 4, 24 and 72 h with a total culture time of 96 h, and possible synergism was evaluated by median drug effect analysis by calculating a combination index (CI; CI < 1 indicates synergism). With CDDP at its IC 25 , the average CIs calculated at the IC 50 , IC 75 IC 90 and IC 95 after 4, 24 and 72 h of exposure were < 1 for all cell lines, indicating synergism, except for the CI after 4 h exposure in the LL cell line which showed an additive effect. With dFdC at its IC 25 , the CIs for the combination with CDDP after 24 h were < 1 in all cell lines, except for the Cls after 4 h exposure in the LL and H322 cell lines which showed an additive effect. At 72 h exposure all Cls were < 1. CDDP did not significantly affect dFdCTP accumulation in all cell lines. CDDP increased dFdC incorporation into both DNA and RNA of the A2780 cell lines 33- and 79-fold ( P < 0.01) respectively, and tended to increase the dFdC incorporation into RNA in all cell lines. In the AG6000 and LL cell lines, CDDP and dFdC induced > 25% more DNA strand breaks (DSB) than each drug alone; however, in the other cell lines no effect, or even a decrease in DSB, was observed. dFdC increased the cellular Pt accumulation after 24 h incubation only in the ADDP cell line. However, dFdC did enhance the Pt–DNA adduct formation in the A2780, AG6000, ADDP and LL cell lines (1.6-, 1.4-, 2.9- and 1.6-fold respectively). This increase in Pt–DNA adduct formation seems to be related to the incorporation of dFdC into DNA ( r = 0.91). No increase in DNA platination was found in the H322 cell line. dFdC only increased Pt–DNA adduct retention in the A2780 and LL cell lines, but decreased the Pt–DNA adduct retention in the AG6000 cell line. In conclusion, the synergism between dFdC and CDDP appears to be mainly due to an increase in Pt–DNA adduct formation possibly related to changes in DNA due to dFdC incorporation into DNA. © 1999 Cancer Research Campaign

In tumour cells the pharmacological basis for multidrug resistance (MDR) often appears to be a reduced cellular cytostatic drug accumulation caused by the drug efflux protein, P-glycoprotein (Pgp MDR), or by other drug transporters (non-Pgp MDR). Here we report the reversal of the decreased daunorubicin (DNR) accumulation in five non-Pgp MDR cell lines (GLC4/ADR, SW-1573/2R120, HT1080/DR4, MCF7/Mitox and HL60/ADR) by genistein. Genistein inhibited the enhanced DNR efflux in the GLC4/ADR cells. In these cells the decreased VP-16 accumulation was also reversed by genistein. Three other (iso)flavonoids biochanin A, apigenin and quercetin also increased the DNR accumulation in the GLC4/ADR cells. In contrast to the effects on non-Pgp MDR cells, 200 microM genistein did not increase the reduced DNR accumulation in three Pgp MDR cell lines (SW-1573/2R160, MCF7/DOX40 and KB8-5) or in the parental cell lines. In conclusion the use of genistein provides a means to probe non-Pgp related drug accumulation defects. Images Figure 1 Figure 6

Summary Multidrug resistance (MDR) in tumour cells is often caused bv the overexpression of the plasma membrane drug transporter P-glycuprutein (P-gp) or the recently discosered multidrug resistance-associated protein (MRP). In this studs we insvestigated the specificity and sensitivity of the fluorescent probes rhodamine 123 (R123). daunorubicin (DNR) and calcein acetoxymethvl ester (calcein-AM) in order to detect the function of the drug transporters P-gp and MRP. using flow cytometrv. The effects of modulators on the accumulation and retention of these probes were compared in seseral pairs of sensitise and P-gp-as w-ell as MRPoserexpressing cell lines. R123. in combination with the modulator PSC833. provided the most sensitise test for detecting P-gp-mediated resistance. Moreoser. in a 60 min drug accumulation assay RI 23 can be regarded as a P-p-specific probe. since R123 is not ver-efficiently effluxed bv MRP. In contrast to R123. a 60 mm DNR or calcein-AM accumulation test could be used to detect MRP-mediated resistance The MRP-specific modulator genistein could be used in combination with DNR. but not w-ith calcein-AM. Vincristine (VCR) can be used to increase the cellular uptake of calcein-AM in MDR cells. but is not specific for MRP Thus.although the combination of DNR with genistein appeared to be as sensitive as the combination of calcein-AM with VCR. the former mav be used to probe specific MRP activity wshereas the latter provides a combined (P-gp + MRP) functional MDR parameter. With these functional assavs the role and relatise importance of P-p and MRP can be studied in. for example. haematological malignancies

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Pgp expression in tumor cells, and especially when accompanied by Pgp expression in fibroblasts in desmoplastic stroma, has prognostic value in primary breast cancer patients and is likely to be a marker of a more malignant phenotype. Pgp expression of tumor cells might play a role in tamoxifen resistance. These findings may have important implications for teh treatment of breast cancer patients, and warrant further prospective investigation in a larger patient population.