Biochimica et Biophysica Acta, 1029 (1990) 191-195 191 Elsevier BBAMEM 70663 BBA Report Ultrastructural alterations in plasma membranes from drug-resistant P388 murine leukemia cells Luis M. Garcia-Segura 1, Jose A. Ferragut 2, Antonio V. Ferrer-Montiel 2, Pablo V. Escriba 2 and Jose M. Gonzalez-Ros 2 I Cajal Institute, C.S.LC., Madrid and 2 Department of Neurochemistry (School of Medicine), University ofAlicante, Alicante (Spain) (Received 4 May 1990) Key words: Freeze-fracture; Cellular multidrug resistance; Intramembrane particle; Membrane protein; Chemotherapy drug; Anthracycline; Daunomycin Freeze-fracture studies of daunomycin-sensitive and daunomycin-resistant P388 cell lines, reveal a significant increase in the numerical density of intramembrane particles at both, the protoplasmic and the exoplasmic leaflets of the plasma membrane from the drug-resistant cells. Such change in plasma membrane architecture is not accompanied by overexpression of P-glycoproteins. Furthermore, drug-sensitive cells exhibited an increased number of exo-endocytotic images when compared to drug-resistant cells. Our observations suggest that there are global changes in the structural organization of the plasma membrane, which are related to the acquisition of the cellular drug-resistant phenotype. The plasma membrane of tumour cells is receiving increasing attention in regard to cellular multidrug re- sistance (MDR) [1,2]. MDR cell lines exhibit a reduced net accumulation of drug relative to the parental drug- sensitive cell lines and most authors have explained these observations in terms of differences in membrane transport of the drug in and out of the cells [3,4]. Moreover, the degree of drug resistance in several cell lines and tumors, has been correlated with the overex- pression of P-glycoproteins, a well known family of plasma membrane, high molecular weight proteins (re- viewed in Ref. 5). These proteins predictably have the characteristics of pore-forming Proteins and act as ATP-dependent pumps to actively eliminate drug and other hydrophobic substances from cells [2,5,6]. The above paragraph emphasizes the importance of alterations in plasma membrane phenomena and/or plasma membrane components in MDR. In spite of this, information on how the pleitropic changes found in MDR cells are reflected on the ultrastructural organi- zation of the plasma membrane, is scarce. Arsenault and co-workers [7], reported freeze-fracture studies in colchicine-resistant Chinese Hamster Ovary (CHO) cell lines and in a vinblastine-resistant human leukemia cell line, and concluded that an increase in the numerical density of intramembrane particles (IMPs) in the proto- plasmic leaflet of the plasma membrane, accompanies resistance. The drug-resistant CHO and human cell Correspondence: J.M. Gonzalez-Ros, Department of Neurochemistry m (School of Medicine), University of Alicante, 03080 Alicante, Spain. lines used in those studies overexpressed P-glycopro- teins and in fact, the authors explain their results based on a correlation between the number of IMPs and P-glycoprotein overexpression. We report here freeze-fracture studies of plasma membranes from daunomycin-sensitive P388 murine leukemia cells (P388/S) and from a stable, daunomy- cin-resistant subline (P388/R) (approx. 50-fold resis- tance in in vitro cytotoxicity assays) (see legend to Fig. 1 for details and experimental conditions). Examples of the appearance of fracture faces of plasma membranes from P388/S and P388/R cells, are given in Fig. 1. Plasma membranes from P388/S and P388/R cells differ in the frequency of appearance of exo-endocytotic images (Fig. 1), a finding which was not apparent in the CHO cells study [7]. These images, pits in the proto- plasmic face and domes in the exoplasmic face, were quantitated in both fracture faces. Plasma membranes from P388/S cells exhibit more than a 2-fold increase in the number of exo-endocytotic images, as compared to those from P388/R cells (Table I). Skovsgaard and co-workers have proposed that drug resistance results from an increased exocytosis of drug previously trapped in endosomal/lysosomal complexes and in fact, they reported an increased plasma membrane traffic in drug-resistant P388 [8] and Ehrlich ascites cells [9]. Although these are measurements of membrane recy- cling dynamic processes, which can not be formally related to our observations, one would expect that dif- ferences in plasma membrane traffic should be reflected somehow in the morphology of the plasma membrane. Therefore, it is tempting to speculate that our observa- 0005-2736/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)