BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 234, 573–577 (1997) ARTICLE NO. RC976674 Deficient Apoptotic Process in Cisplatin-Resistant L1210 Cells Cannot Account for the Cellular Response to Various Drug Treatments Yvan Canitrot, Philippe Frit, and Bernard Salles 1 Institut de Pharmacologie et de Biologie Structurale, UPR CNRS 9062, 205 route de Narbonne, 31077 Toulouse Cedex, France Received April 7, 1997 cells to induction of apoptosis during chemotherapy is Apoptosis is a major determinant of the effectiveness a major determinant in the outcome of the therapy, of antitumor chemotherapy since most of the drugs and the inability to activate the apoptotic program used in cancer treatment provoke cell death by this could result in a cell insensitivity to cytotoxic treat- process. We selected L1210/0.7R (7-fold) and L1210/3R ment. In the case of CDDP-resistance, different mecha- (16-fold) murine leukemia cells resistant to cisplatin nisms such as decreased intracellular accumulation [7], (CDDP) by adaptation of parental L1210/S cells to in- enhanced drug inactivation by gluthatione [8] or metal- creasing drug concentration. L1210/0.7R exhibited a lothioneins [9], and increased DNA repair [10,11] have decreased apoptosis response to CDDP compared to been shown to play a role. Moreover, apoptosis cannot parental L1210/S, while it was totally defective in be triggered in CDDP-resistant cell lines, suggesting L1210/3R as analyzed by cell morphology, DNA frag- an involvement of apoptosis control processes in the mentation, and poly(ADP-ribose) polymerase cleav- mechanism of resistance [12]. age. This default in apoptosis did not result from dif- In order to check the role of apoptosis in the cell ferential expression of the antiapoptotic protein bcl-2 response to anticancer drugs, we selected leukemia or from altered expression of p53. L1210/3R was resis- L1210 cells with CDDP and examined the relationship tant to other cross-linking agents and sensitive to to- poisomerase II inhibitors and microtubule poisons. between apoptosis and drug sensitivity phenotype. Whatever the drug sensitivity phenotype to these agents, L1210/3R was totally defective in apoptosis in MATERIALS AND METHODS response to drug treatment, showing that apoptosis control cannot be directly involved in the resistance Cell lines and culture conditions. L1210 murine leukemia cell process of these cell lines.  1997 Academic Press lines were grown in suspension in humidified incubator at 37°C with 5% CO 2 in RPMI 1640 medium supplemented with 10% fetal calf serum and penicillin (2110 5 U/l)/streptomycin (25 mg/l). Two CDDP- resistant variants were generated by continuous exposure of the pa- rental cell line L1210/S to increasing CDDP concentrations up to Cisplatin (CDDP), a drug widely used in the treat- 0.7 mg/ml or 3 mg/ml and designated as L1210/0.7R and L1210/3R, respectively. Resistant cells were cultured in presence of CDDP, 0.7 ment of various human solid tumors, binds to DNA and 3 mg/ml for L1210/0.7R and L1210/3R, respectively. CDDP was leading to the formation of platinum-DNA adducts, ei- omitted on the 2 last passages before experiment. ther intra- or interstrand cross-links [1]. Since DNA Cytotoxicity studies. All the drugs were purchased from Sigma. repair deficient mutants, such as xeroderma pigmento- Cytotoxicity was measured using the MTT assay. Briefly, cells were sum cell lines, are sensitive to CDDP [2], it is commonly seeded into 96-well plates at 7110 3 cells/well (in 100 ml). Forty-eight accepted that DNA adducts are responsible for cell tox- hours after the addition of drugs (50 ml), MTT (50 ml of 1 mg/ml in icity. As reported for numerous anticancer drugs, PBS) (Sigma) was added, and the plates were incubated for 3 h at 37°C. Plates were centrifuged, the supernatant discarded, and the CDDP has been shown to trigger cell death by apoptosis resulting formazan crystals were solubilized by addition of DMSO [3]. Apoptosis is a genetically controlled process in (100 ml) and gentle shaking. The plates were scanned at a wavelength which numerous genes are involved either as inducers of 570 nm with a Titertek Multiskan Plus plate reader. Drug cytotox- or inhibitors [4-6]. Thus, the susceptibility of cancer icity was expressed as the ratio of the absorbance of treated versus untreated cells. Results were expressed as curves (absorbance ratio versus drug concentration) from which the drug IC 50 (i.e. the concen- tration that reduces the absorbance value to 50% of the control) was 1 Corresponding author. Fax: 33-04.61. 17.59.94. E-mail: salles@ ipbs.fr. determined. The resistance factor is the ratio of IC 50 from the resis- 0006-291X/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved. 573