[CANCER RESEARCH 63, 3084 –3091, June 15, 2003] P-Glycoprotein, Expressed in Multidrug Resistant Cells, Is Not Responsible for Alterations in Membrane Fluidity or Membrane Potential 1 Claudina Alema ´n, Jean-Philippe Annereau, Xing-Jie Liang, Carol O. Cardarelli, Barbara Taylor, Jun Jie Yin, Adorjan Aszalos, and Michael M. Gottesman 2 Laboratory of Cell Biology, National Cancer Institute, NIH, Bethesda, Maryland 20892-4254 [C. A., J-P. A., X-J. L., C. O. C., A. A., M. M. G.]; FACS Core Facility, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255 [B. T.]; and Instrumentation and Biophysics Branch, Center for Food Safety and Applied Nutrition, College Park, Maryland 20740-3835 [J. J. Y.] ABSTRACT Expression of P-glycoprotein (P-gp), the multidrug resistance (MDR) 1 gene product, can lead to MDR in tumors. However, the physiological role of P-gp in normal tissues is not well understood. Previous studies on multidrug-resistant cells have suggested changes in membrane fluidity and membrane potential associated with P-gp expression, but interpreta- tion of these studies is difficult, because most experimental cells have been selected for long periods in the presence of cytotoxic drugs and may have other host alterations. Therefore, we created two cell lines in which a transfected human MDR1 cDNA is repressed by tetracycline and induced in the absence of tetracycline. One cell line was derived from a mouse embryonic fibroblast cultured from a double (mdr1a/1b) knockout mouse, and the other was from a human HeLa cell line. Analysis of the kinetics of expression of P-gp showed that the mRNA had a half-life of 4 h, and the protein had a half-life of 16 h. P-gp cell surface expression (measured with monoclonal antibody MRK-16) and P-gp function (measured with a fluorescent substrate, rhodamine 123) was characterized by using fluores- cence-activated cell sorting. No differences in membrane potential using the fluorescent probe oxonol or in membrane “fluidity” using fluorescent anisotropy probe or electron spin resonance probe were observed in the tet-repressible P-gp-expressing cells. In contrast, several drug-selected cells that express P-gp showed an increase in membrane fluidity and membrane potential. These results suggest that expression of P-gp per se has little effect on membrane fluidity or membrane potential, and it does not have H  pump activity. The changes in these parameters observed in drug-selected cells must reflect other host adaptations to drug selection. INTRODUCTION P-gp 3 belongs to the class of ABC transporters (ABCB1) and is the protein product of the MDR1 gene. It is a drug transporter with broad specificity for many amphipathic, organic substrates (1–3). In hu- mans, P-gp is constitutively expressed in the intestine, liver, kidney, placenta, and the blood brain barrier (4). It has been shown to have a protective effect against xenobiotics that enter the body through the intestines or the brain through the blood brain barrier (5). The com- plete physiological role and the endogenous substrates of P-gp are not known. Mice lacking mdr1a and mdr1b have normal life spans in the laboratory under protective care (5). In many cancers, P-gp is over- expressed contributing to resistance to clinically important chemo- therapeutic drugs that are P-gp substrates, including doxorubicin, vinblastine, and Taxol. In the laboratory, many different cell lines have been used to demonstrate effects of P-gp on the plasma membrane, on drug resist- ance, on apoptosis, and on resistance to viruses in an effort to elucidate the physiological role of P-gp (6 –12). It is not surprising that results obtained from different cell lines and expression systems are not consistent with each other. The question arises whether the results obtained are because of the selection pressure on the cells, or other unknown phenomena that occur in cells that have been in culture, in some cases, for a number of years. We have developed one mouse and one human cell line in which human MDR1 is under tetracycline control. In these cells, addition of tetracycline rapidly turns off transcription of MDR1 mRNA, and over a period of a few days P-gp disappears from the cells, allowing a comparison of the same cells with and without P-gp in the plasma membrane. In this study, we characterize the P-gp mRNA half-life, P-gp protein half-life, and measure membrane properties, such as potential and fluidity. To assess plasma membrane biophysical characteristics, such as fluidity, of normal and tumor cells, ESR was used. ESR was used previously to measure membrane fluidity of transplanted melanoma cells (13), human lung tumor and normal tissues (14), and cisplatin- sensitive and -resistant cells (15). Also, high and low metastatic Lewis lung cancer cells were shown to have different plasma membrane fluidity by using ESR (16). Fluidity of plasma membranes was also studied using lipid soluble fluorescence probes, such as DPH (15). Wanten and Naber (17) used DPH and TMA-DPH in their studies, which we used in our investigation here. Measurement of plasma membrane potential is indicative of the general biological status of cells. For example, alteration of membrane connected proteins changes membrane potential (18, 19), and antiproliferative signals induce plasma membrane depolarization in lymphocytes (20). Using these techniques, we show that drug-selected cells frequently show changes in membrane properties, whereas P-gp expression alone does not affect these properties. MATERIALS AND METHODS Insertion of MDR1 cDNA into Tet-repressible Vectors. The plasmids, pRevTRE and pRevTet-Off were purchased from Clontech (CLONTECH Laboratories, Inc., Palo Alto, CA). The human MDR1 gene was cloned into BamH1 and Cla1 sites in the multiple cloning site of the pRevTRE plasmid. The sequence was verified using an ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA) in a Perkin-Elmer GeneAmp System 9600. The conditions were: 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min. A series of six primers were used to sequence the entire insert. The forward direction primers used were MDR516 (5': CAC-CCG-ACT-TAC-AGA-TGA), MDR1159 (5': AAT-ATT-AAG-GGA- AAT-TTG-GAA), and MDR3447 (5': AAA-GGA-GGC-CAA-CAT-ACA). The reverse direction primers used were MDR964R (5': AGA-ATA-TTC- CCC-TGA-GAG), MDR1340R (5': CAC-TGA-CCA-TCC-CCT-CTG), and MDR3747R (5': GTG-CCA-TGC-TCC-TTG-ACT-CTG). The reactions were column purified with Auto Seq G-50 to eliminate the excess primers (Amer- sham Pharmacia Biotech, Inc., Piscataway, NJ). The reactions were electro- phoresed in the National Cancer Institute Sequencing Core Facility. After Received 10/22/02; accepted 4/15/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the NIH. 2 To whom requests for reprints should be addressed, at Laboratory of Cell Biology, National Cancer Institute, NIH, Building 37, Room 1A09, 37 Convent Drive, MSC 4254, Bethesda, MD 20892-4254. 3 The abbreviations used are: P-gp, P-glycoprotein; MDR, multidrug resistance; ESR, electron spin resonance; DPH, 1,6-diphenyl-1,3,5-hexatriene; TMA, trimethyl-ammoni- um; FBS, fetal bovine serum; FACS, fluorescence-activated cell sorter; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; UTR, untranslated region; RT, reverse tran- scription-PCR; Cp, crossing point; AOSA, 6-(9-anthroyloxy)stearic acid; T-SASL, 2,2,6,6-tetramethyl piperidin-1-oxyl-4-yl-octadecenoate; 5-doxyl-SA, 5-doxyl stearic acid; mAb, monoclonal antibody. 3084 Research. on November 9, 2015. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from