Biochemical Pharmacology, Vol. 32, No. 6, pp. 1037-1044, 1983. 0006-2952/83/961037-08 $03.00/0 Printed in Great Britain. ~) 1983 Pergamon Press Ltd. UPTAKE AND CYTOFLUORESCENCE LOCALIZATION OF ELLIPTICINE DERIVATIVES IN SENSITIVE AND RESISTANT CHINESE HAMSTER LUNG CELLS JEAN-YvEs CHARCOSSET, BERNARDSALLES* and ALAIN JACQUEMIN-SABLON'~ Unit6 de Biochimie et Enzymologie, Institut Gustave Roussy, 94800 Villejuif, France (Received 5 July 1982; accepted 25 October 1982) Abstract--Uptake of two eUipticine derivatives, 2-N-methyl-ellipticinium (NME) and 2-N-methyl-9- hydroxy-ellipticinium, by sensitive and resistant Chinese hamster lung cells was studied. The results show that uptake and retention of these molecules by both types of cells were identical, thus indicating that the resistance to ellipticines, in this system, is not related to an impaired permeability of the cells to the drugs. However, influx and efflux kinetics, as well as experiments at increasing external concns, showed that both drugs accumulate within the cells in different ways. A cellular overconcentration of the drugs, which does not require an energy-dependent process, is observed. Fluorescence microscopy showed that, in both sensitive and resistant cells, NME is mainly, if not entirely, located in the cytoplasm. Chinese hamster lung cells resistant to the DNA- intercalating antitumoral drugs from the ellipticine series have been selected by growing the cells in the presence of step-wise increasing concns of 9-OH-E$ [1]. In these conditions, two sublines, with respec- tively about 10- and 12-fold resistance to this drug, were isolated. Our results showed that, in parallel to the development of resistance to 9-OH-E, the resistant cells undergo several changes: among them were modifications of the morphology and growth parameters, a decreased oncogenic potential, and a cross-resistance to a variety of anti-tumoral agents. In the same cellular system, resistance to actinomycin D and anthracyclin antibiotics was associated with similar modifications of the cell properties [2, 3], and in these cases the drug resistance was attributed to a change in the cell membrane that resulted in a decreased permeability of the cells to the drug. In order to study the mechanism of cellular resist- ance to ellipticine derivatives, we investigated the uptake of two ellipticine derivatives by the sensitive and resistant cells: one, NME, is a fluorescent mol- ecule, and the other, NMHE, was radioactively labeled. Uptake and retention of these molecules by the sensitive and resistant ceils were identical, thus indicating that the resistance mechanism is not related to an impaired permeability of the cells to the drugs. The uptake kinetics of these derivatives, which only differ from one another by one hydroxyl group at position 9, are markedly different. Yet, * Present address: Laboratoire de Toxicologie et Phar- macologie Fondamentale, Toulouse, France. t To whom correspondence should be addressed at: Unit6 de Biochimie et Enzymologie, Institut Gustave Roussy, 94800 Villejuif, France. ~t Abbreviations: 9-OH-E, 9-hydroxy-ellipticine; NME, 2-N-methyl-ellipticinium acetate; NMHE, 2-N-methyl-9- hydroxy-ellipticinium acetate; TPB, tetraphenylboron; MEM, Eagle's minimum essential medium modified; PBS, phosphate buffer saline. both drugs appear to be overconcentrated in the cells by an energy-independent process. Finally, the cel- lular localization of NME was studied by fluor- escence microscopy which showed that, in both sen- sitive and resistant cells, most, if not all the drug was located in cytoplasm. MATERIALS AND METHODS Cells and culture medium. The Chinese hamster lung cells, DC-3F, and the 9-OH-E resistant sublines have been previously described [1]. Monolayer cul- tures were maintained in MEM, supplemented with 7% fetal calf serum, streptomycin (50 #g/ml) and penicillin (100 I.U./ml). Resistant sublines DC-3F/ 9-OH-E 0.3 and 9-OH-E 0.6 were permanently grown respectively in the presence of 0.3 and 0.6/~g/ml 9-OH-E. The drug was removed from the medium for 10-15 days before each experiment. Chemicals. The structures of both ellipticine derivatives used in this work are shown in Fig. 1. NME was a generous gift from Drs Nguyen Dat- Xuong and E. Lescot (Institut de Chimie des Sub- stances Naturelles, Gif-sur-Yvette, France). NMHE, carrying a 14C-labeled methyl group at position 2 (2mC~/mmole, 600/~g/ml), was synthetized and kindly provided by Dr Van Bac [4] (Institut de Chimie des Substances Naturelles). All chemicals were of reagent grade and obtained from commercial sources. Uptake of drugs. For kinetics experiments, the cells were plated, about 16 hr before the beginning of the assay, on 35-mm dia. tissue culture dishes (Coming, No. 25000), containing 2 ml of growth medium. The number of plated cells was adjusted to have about 1 x 106 cells/dish at the time of drug exposure. At zero time, the medium was replaced by l ml of drug-containing medium [NME or [14C]NMHE (1 #g/ml)]. The cells were then incu- bated either at 4 ° or 37 ° in a humidified incubator 1037