FEBS 15734 FEBS Letters 368 (1995) 373-376 Cell-specific effects of RAS oncogene and protein kinase C agonist TPA on P-glycoprotein function T.R Stromskaya a, I.A. Grigorian a, V.S. Ossovskaya b, E.Y. Rybalkina a, RM. Chumakov b, B.P. Kopnin a'* ~Institute of Cancerogenesis, Cancer Research Center, Kashirskoye shosse 24, Moscow, 115478, Russian Federation bEngelhardt Institute of Molecular Biology, Moscow, Russian Federation Received 17 April 1995; revised version received 19 May 1995 Abstract We compared the influence of exogenous N-ras onco- gene and treatment with PKC agonist 12-O-tetradecanoylphor- bol-13-acetate (TPA) on P-glycoprotein (Pgp) function in various human, rat and dog cell lines. Two approaches were used: (a) flow cytometry analysis of Rhodamine 123 (Rh123) exclusion; and (b) sensitivity to cytotoxic action of colchicine. We have found that in Ratl fibroblasts, rat IAR2 epithelial cells and rat McA RH 7777 (hepatoma), ras activates Pgp function, while in MDCK (dog kidney), K562 (human chronic myelogenous leukaemia) and LIM1215 (human colon carcinoma) cells it either has no effect or even acts in opposite direction. TPA-induced Pgp function shows dissimilar pattern of cell specificity. It is assumed that PKC and ras oncogene regulate mdrl gene expression through at least partially distinct signalling pathways. Key words." RAS oncogene; Protein kinase C; Multidrug resistance, P-glycoprotein I. Introduction Acquisition of MDR in vitro and in vivo usually results from increased expression of the mdrl (pgyl) gene encoding so-called Pgp, the 170-180 kDa transmembrane glycoprotein that func- tions as an energy-dependent efflux pump for various lipophilic compounds 1-3. A lack of response of human tumors to chemotherapy is often due to increased mdrl gene transcription 2. The regulation of mdrl gene activity is studied poorly. Recently it was shown that PKC-activating agents induce mdrl expression and as a result enhance Pgp function 4. Also, it was found that ras oncogenes are able to activate a reporter gene expression driven by human mdrl gene promoter in murine immortalized fibroblasts 5. However, the functional signifi- cance of this finding was unclear. Meanwhile an understanding the role of ras-mediated signalling pathway in regulation of mdrl gene activity might be important for prediction of re- sponse to therapy with definite drugs since activation of ras genes is characteristic of many types of human tumors 6. To study whether activated ras oncogene is really able to induce Pgp-mediated MDR we created the retroviral construct ex- pressing human N-ras a'pl2 eDNA, introduced it into various human and rodent cell lines and analyzed the changes in Pgp * Corresponding author. Abbreviations: MDR, multidrug resistance; FCM, flow cytometry; Pgp, P-glycoprotein; PKC, protein kinase C; TPA, 12-O-tetradecanoylphor- bol-13-acetate; PBS, phosphate-buffered saline; Rh123, Rhodamine 123; FCS, fetal calf serum; PCR, polymerase chain reaction. function in the resultant cells. In addition, in order to elucidate whether the same signalling pathway might be responsible for ras- and PKC-induced MDR we compared the effects of ex- ogenous ras and TPA treatment in cells of different origin. 2. Materials and methods 2.1. Cell lines LIM1215 (human colon carcinoma) 7, K562 (human chronic my- elogenous leukaemia) 8, MDCK (dog kidney cells) 9, McA RH 7777 (rat hepatoma) 10, IAR2 (minimally transformed rat epithelial cells) 1l and Ratl (immortalized fibroblasts) were used. The cells were cultivated in DMEM or RPMI 1640 (for LIM 1215, K562 and McA RH 7777) media supplemented with 10% of FCS. 2.2. Expression vectors, isolation of cell sublines expressing exogenous RAS In order to transfer RAS oncogene into different cell lines, including those already expressing plasmids with neo gene, we prepared on the basis of pPS retroviral vector 12 the construct containing human activated N-ras a'p12 and selectable hygro (resistance to hygromycin) genes. The N-ras ~splz cDNA 13 was subcloned as BamHI-HindII! fragment from the pBS-ras plasmid (provided by Dr. A. Tatosyan, Cancer Research Center, Moscow) into appropriate cloning sites of pPS-3/hygro vector. The resulting construct was introduced by CaZ+P- transfection into murine amphitropic packaging ~CRIP 14 cells. After selection with hygromycin (200/,tg/ml for three weeks) the obtained clones were combined and used as producers of recombinant pPS/ hygro-ras virions. The culture medium in which these cells (about 105 per ml) were cultivated for one day was filtered through 0.45 /zm membranes (Millipore), diluted (1 : 3-1 : 5) by medium containing 1% of FCS and 8/zg/ml of polybrene (Serva), and added to LIM1215, K562, McA RH 7777, IAR2, Ratl or MDCK cells (1 ml of mixture per 105 cells). The next day it was changed for fresh growth medium. After additional cultivation for 24-48 h the cells were plated 1 : 5 or 1 : 10 onto 60 mm dishes containing selective media (hygromycin, 150 250/lg/ml). After 2 3 weeks with weekly medium change the resulting resistant cultures were trypsinised and replated. To minimise the influence of clonal variability we developed from all parental cell lines 1-2 inde- pendent sublines each derived from dozens of individual clones. The cultures were tested for expression of introduced N-ras by cDNA-PCR analysis. For this purpose total cell RNA was isolated by guanidine thiocyanate extraction. Synthesis of cDNA specific for re- combinant construct was performed on RNA template by using 24 bp primers that anneal just downstream of pPS/hygro cloning sites. The reaction mixture containing 15 pmol of primers, 1 /zg of RNA and 5 units of TET-Z Tth DNA-polymerase (BioMaster, Moscow) in 20,ul of reverse transcription buffer 16.6 mM (NH4)2SO4, 67 mM tris.HCl pH 8.8, 1 mM MnCIz, 2.5% glycerol, 200 ,uM each of dATP, dGTP, dCTP and dTTP was incubated for 15 rain at 70°C. Aliquots (2-5/A) from reverse transcription reaction were then used as DNA templates for PCR reaction using a pair of 24 bp primers specific to the regions of pPS/hygro flanking cloning site. Changes in morphology and growth parameters of Rat 1 cell sublines transformed by pPS/hygro-ras were described by us earlier 15. 2.3. FCM analysis of Pgp function Evaluation of Rh123 efflux from various cells was performed ac- 0014-5793195l$9.50 © 1995 Federation of European Biochemical Societies. All rights reserved. SSD1 0014-5793(95)00662-1
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`374 TP Stromskaya et aL IFEBS Letters 368 (1995) 373-376 cording to the described procedures 16,17• The cells were detached from plastic, incubated with 5 mg/ml of Rh123 (Sigma) for 10 min at 37°C, washed twice with PBS and incubated for 30 min in dye-free medium• The cell fluorescence was evaluated by the FACScan flow cytometer (Becton-Dickinson). 2.4. Sensitivity to cytotoxic drugs This was determined by colony formation assay or cell counting after cultivation in media containing different concentrations of colchicine (Merck). 3. Results and discussion Rat 1 cell derivatives transformed by pPS/hygro-ras construct showed significant increase in resistance to colchicine (Fig. 1). The LDs0 (drug dosage diminishing the number of clonogenic cells by half) for all independently selected sublines that express exogenous N-ras was increased 3 to 4.5-fold as compared with parental Ratl or Rat/neo cells (Table 1). FCM analysis ofefflux of Rh123, a Pgp-transported fluorescent mitochondrial dye, showed that in Ratl sublines expressing N-ras the proportion of Rh123-dull cells is higher than that in the parental cells. Pgp inhibitor verapamil abolished ras-induced stimulation of Rh123 efflux (Fig. 2). So, in agreement with the data showing activation of mdrl gene promoter by ras oncogene 5 we ob- served in ras-transformed Rat 1 cell sublines stimulation of Pgp function. However several other cell lines (MDCK dog kidney, K562 human myelogenous leukaemia, and LIM1215 human colon carcinoma cells) expressing the same pPS/hygro-ras retroviral construct showed neither decreased colchicine sensi- Rat/ras loql ~ ...... ~ ~Ratl o \° \='~ ~, oklM1215-ras2 80 C~\(~\ \ "~, ~. ~ ', -B- K562-ras 40 ~ I '" o i "', 2Ts s ~Is~3'os'0 Colchicine, no/rnl Fig. I. Influence of expression of exogenous N-ras "sN2 on sensitivity of various cell lines to colchicine. Cell survival was estimated as a ratio of a number of colonies (for Ratl sublines) or cells (for K562 and LIM1215 sublines) grown at given colchicine dosage to those grown in drug-free medium. For clonogenic assay 3 x 102 cells were seeded in duplicate onto 60 mm dishes for 14 days. In cell yield assay 5 × 104 cells were plated into 25 cm 2 flasks for 7 days• For each pair of cell sublines the data of one of typical experiments are presented. tivity, nor enhanced Rh123 exclusion (Figs. 1,2; Table 1). Meanwhile, according to the data of cDNA-PCR analysis (Fig. 3) the levels of exogenous ras mRNA in these cells were compa- rable with those found in rat cell lines showing either pro- nounced (Rat/neo fibroblasts; IAR2 rat epithelial cells) or RaUneo Rat./neo-ras2 Rat/neo-ras2 + Vet "; ~';---"~'"l iI,.,~ ........ lO0 | • t M DC K-ras/A4 I MDCK-ras/A4+Ver 101 102 103 104 100 101 102 10J Rhodamine123 IAR2 IAR2-ras/C5 IAR2-ras/C5+Ver 104 100 l01 |02 103 104 Fig. 2. Effects of N-ras oncogene on Pgp activity in Rat 1, M DCK, and IAR2 cells• Logarithmic fluorescence intensity of ras-expressing and parental was studied after staining with Rh123 as described in section 2. N-ras stimulated Rh123 efflux in Rat/neo-ras2 and IAR2-ras/C5 cells, while in MDCK-ras/A4 its expression was accompanied by some inhibition of Pgp activity• Incubation in the presence of 30 mM of verapamil (the lower row) caused complete reversion of ras-induced activation of Pgp function in Rat I and IAR2 cell derivatives whereas in MDCK-ras/A4 cells it did not influence Rh123 efflux.
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`7~P. Stromskaya et al./FEBS Letters 368 (1995) 373-376 375 21.7 5.15 0.94 0.83 12345 678910 Fig. 3. cDNA-PCR analysis of cell sublines developed as a result of retrovirus mediated transfer of pPS/hygro-ras construct, cDNAs spe- cific for mRNAs produced by exogenous constructs were synthesized and amplified as described in section 2, separated on l% agarose gel and visualized by ethidium bromide staining. In all the sublines selected with hygromycin after introduction of pPS/hygro-ras (tracks 1-3, 7 10 K562-ras, IAR2-ras/F10, LIM1215-rasl, MDCK-ras/B2, IAR2-ras/ C5, MDCK-ras/C4, and Rat/neo-rasl, respectively) the fragment of expected molecular weight (about 900 bp) is seen (arrow). In Rat/hygro cells (track 6) infected with insert-free pPS/hygro construct a low- molecular weight fragment (about 300 bp) corresponding to the vector adapter region was amplified. Parental K562 (track 4) and LIM1215 (track 5) do not show the pPS-specific amplification products. Left the position of the marker fragments is indicated. slight (McA RH 7777 rat hepatoma) induction of Pgp function (Table 1). Such differences in activation of Pgp function may reflect either species-specific (rodent vs. dog and human), or tissue-specific distinctions in response to ras oncogene. In pre- vious studies cell-specific stimulation of mdrl gene expression was observed after treatment with cytotoxic drugs 18 and PKC agonists 4. Interestingly that while the former study Table 1 Cell-specific induction of P-glycoprotein-mediated MDR by activated N-ras oncogene Cells pPS/hygro-ras Relative Rh123 expression drug-resistance* exclusion Ratl - 1.0 + Rat/ras + 3.1 +++ Rat/neo - 1.0 + Rat/neo-rasl + 4.5 ++++ Rat/neo-ras2 + 4.0 ++++ IAR2 - 1.0 + IAR2-ras/C5 + n.d. +++ McA RH 7777 - 1.0 + McA RH 7777-ras2 + 2.1 ++ MDCK - 1.0 + MDCK-ras/B2 + n.d. - MDCK-ras/A4 + n.d. - K562 - 1.0 + K562-ras + 0.7 - LIM1215 - 1.0 + LIMl215-rasl + 0.8 - LIM 1215-ras2 + 0.8 - *Relation of LDs0 of colchicine for given cell subline to LDs0 for parental cells. ¢3 (o ¢j RaUneo L Rat/neo + TPA MDCK ,~ ~ q iT~., , 111~4,, , ,J.l., I 10 10 2 10 3 I( ' MDCK + TPA 10 0 10 4 10 ° 10! 10~ 10 3 10 ~ Rhodamine123 Fig. 4. Differential effects of TPA and activated RAS on Pgp activity in Ratl and MDCK cells. Unlike N-ras asps2 TPA stimulates Rh123 efflux in MDCK but not in Rat/neo cells where it causes slight inhibi- tion of Pgp function. revealed some distinctions between human and rodent cells, the latter observed differences in mdrl gene response in various cell lines of human origin. The results obtained to date are insuffi- cient to understand the basis of differential susceptibility of various cell lines to ras-mediated stimulation of Pgp activity. However they indicate that ras gene mutations may have differ- ent prognostic significance in prediction of response to chemo- therapy of various types of human tumors. In cells transformed by ras oncogenes activation of PKC is often observed 19. To elucidate whether the same signalling pathway might be responsible for ras- and PKC-induced activa- tion of mdrl gene we compared the effects of exogenous N- ras "Sp~2 and TPA (an efficient activator of PKC) on Pgp func- tion in a variety of cell types. We found that majority of cell lines tested showed differential sensitivity to Pgp-related effects of ras and TPA. For example, expression of N-ras aspl2 in MDCK cells causes no increase in Pgp function (Fig. 2) while treatment of MDCK cells with TPA increases the proportion of Rh123-dull cells (Fig. 4). Differential response of Pgp-medi- ated Rh123 efflux to ras and TPA was observed also in Ratl and LIMI215 cell lines (Figs. 2,4; Table 2). These findings suggest that ras oncogenes and PKC probably regulate mdrl gene expression through different (at least in part) signalling pathways. The detailed mechanisms of such regulation are a subject for future studies. Table 2 Comparison of the effects of exogenous N-ras oncogene and TPA in various cell lines Cell line Stimulation of Rh123 efflux N-ras TPA Rat/neo ++++ - McA RH 7777 + ++ MDCK - + LIM1215 - +
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`376 T.P Stromskaya et al./FEBS Letters 368 (1995) 373376 Acknowledgements: We thank Dr. T. Zabotina (Cancer Research Cen- ter, Moscow) and Dr. A. Poletaev (Engelhardt Institute of Molecular Biology, Moscow) for help in FCM analysis of Rh 123 efflux. This work was supported in part by grants from the Russian Fund for Basic Research and the International Science Foundation. References 1 Endicott, J.A. and Ling, V. (1989) Annu. Rev. Biochem. 58, 137 171. 2 Roninson, I.B. (1992) Biochem. Pharmacol. 43, 95-102. 3 Chin, K.-V., Pastan, I. and Gottesman, M.M. (1993) Adv. Cancer Res. 60, 157 180. 4 Chaudhary, P.M. and Roninson, I.B. (1992) Oncol. Res. 4, 281 290. 5 Chin, K.-V., Ueda, K., Pastan, I. and Gottesman, M.M. (1992) Science 255, 459462. 6 Bos, J.L. (1989) Cancer Res. 49, 4682-4689. 7 Whitehead, R.H., Macrae, F.A., St. John, D.J.B. and Ma, J. (1985) J. Natl. Cancer Res. 74, 75%765. 8 Lozzio, C.B. and Lozzio, R.B. (1975) Blood 45, 321-334. 9 Lever, J. (1979) Proc. Natl. Acad. Sci. USA 76, 1323-1327. 10 Becker, J., De Nechaud, B. and Potter, V.E (1976) in: Onco- Developmental Gene Expression (Fisfman, W.H., Sell, S., Eds.) NY, Academic Press, pp. 259-270. 11 Montesano, R., Saint Vincent, L., Drevon, C. and Tomatis, L. (1975) Int. J. Cancer 16, 550-558. 12 Prassolov, V.S. and Chumakov, P.M. (1988) Mol. Biol. 22, 1371- 1380 (in Russian). 13 Souyri, M., Koehne, C.F., O'Donnel, EV., Aldrich, T.H., Furth, M.E. and Fleissner, E. (1987) Virology 158, 6%78. 14 Danos, O. and Mulligan, R.C. (1988) Proc. Natl. Acad. Sci. USA 85, 6460 6464. 15 Khramtsova, S., Stromskaya, T., Potapova, G., Chumakov, P. and Kopnin, B. (1993) Biochem. Biophys. Res. Commun. 194, 383 390. 16 Neyfakh, A.A. (1988) Exp. Cell. Res. 174, 168-176. 17 Chaudhary, P.M. and Roninson I.B. (1991) Cell 66, 85-94. 18 Chin, K.-V., Chauhan, S.S., Pastan, I. and Gottesman, M.M. (1990) Cell Growth Diff. 1,361-365. 19 Weinstein, I.B., Borner, C.M., Krauss, R.S., O'Driscoll, K., Choi, P.M., Moritomi, M., Hoshina, S., Hsieh, L.-L., Tshou-Wong, K.-M., Guadagno, S.N., Ueffing, M. and Guillem, J. (1991) in: Origins of Human Cancer: A Comprehensive Review (Brugge, J., Curran, T., Harlow, E. and McCormick, F., Eds.) Cold Spring Harbor, Cold Spring Harbor Laboratory Press, pp. 113-124.
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