P-glycoprotein (MDR1) Expression in Leukemic Cells Is Regulated at Two Distinct Steps, mRNA Stabilization and Translational Initiation
2003; Elsevier BV; Volume: 278; Issue: 12 Linguagem: Inglês
10.1074/jbc.m211093200
ISSN1083-351X
AutoresErnesto Yagüe, Angel L. Armesilla, Georgina B. Harrison, James I. Elliott, Alessandro Sardini, Christopher F. Higgins, Selina Raguz,
Tópico(s)DNA Repair Mechanisms
ResumoMultidrug resistance in acute myeloid leukemia is often conferred by overexpression of P-glycoprotein, encoded by the MDR1 gene. We have characterized the key regulatory steps in the development of multidrug resistance in K562 myelogenous leukemic cells. Unexpectedly, up-regulation ofMDR1 levels was not due to transcriptional activation but was achieved at two distinct post-transcriptional steps, mRNA turnover and translational regulation. The short-lived (half-life 1 h) MDR1 mRNA of naı̈ve cells (not exposed to drugs) was stabilized (half-life greater than 10 h) following short-term drug exposure. However, this stabilized mRNA was not associated with translating polyribosomes and did not direct P-glycoprotein synthesis. Selection for drug resistance, by long-term exposure to drug, led to resistant lines in which the translational block was overcome such that the stabilized mRNA was translated and P-glycoprotein expressed. The absence of a correlation between steady-state MDR1 mRNA and P-glycoprotein levels was not restricted to K562 cells but was found in other lymphoid cell lines. These findings have implications for the avoidance or reversal of multidrug resistance in the clinic. Multidrug resistance in acute myeloid leukemia is often conferred by overexpression of P-glycoprotein, encoded by the MDR1 gene. We have characterized the key regulatory steps in the development of multidrug resistance in K562 myelogenous leukemic cells. Unexpectedly, up-regulation ofMDR1 levels was not due to transcriptional activation but was achieved at two distinct post-transcriptional steps, mRNA turnover and translational regulation. The short-lived (half-life 1 h) MDR1 mRNA of naı̈ve cells (not exposed to drugs) was stabilized (half-life greater than 10 h) following short-term drug exposure. However, this stabilized mRNA was not associated with translating polyribosomes and did not direct P-glycoprotein synthesis. Selection for drug resistance, by long-term exposure to drug, led to resistant lines in which the translational block was overcome such that the stabilized mRNA was translated and P-glycoprotein expressed. The absence of a correlation between steady-state MDR1 mRNA and P-glycoprotein levels was not restricted to K562 cells but was found in other lymphoid cell lines. These findings have implications for the avoidance or reversal of multidrug resistance in the clinic. multidrug resistance reverse transcription 12-O-tetradecanoylphorbol-13-acetate threshold cycle luciferase Epstein-Barr virus MDR1 is the most common impediment to successful chemotherapy for a variety of cancers (1Gottesman M.M. Fojo T. Bates S.E. Nat. Rev. Cancer. 2002; 2: 48-58Crossref PubMed Scopus (4443) Google Scholar). The most frequent form of drug resistance in relapsed acute leukemia is overexpression of P-glycoprotein (2McKenna S.L. Padua R.A. Br. J. Haematol. 1997; 96: 659-674Crossref PubMed Scopus (77) Google Scholar, 3Ivy S.P. Olshefski R.S. Taylor B.J. Patel K.M. Reaman G.H. Blood. 1996; 88: 309-318Crossref PubMed Google Scholar). P-glycoprotein is a member of the ATP-binding cassette superfamily of active transporters and functions as an energy-dependent efflux pump that reduces the intracellular concentration of cytotoxic compounds and, hence, their toxicity. P-glycoprotein has a broad substrate specificity and can confer resistance to a wide range of different cytotoxic compounds (4Gottesman M.M. Pastan I. Annu. Rev. Biochem. 1993; 62: 385-427Crossref PubMed Scopus (3547) Google Scholar). Most pre-clinical and clinical efforts to overcome MDR aim to modulate P-glycoprotein activity. However, clinical trials of compounds that inhibit P-glycoprotein activity have had limited success and led to adverse pharmacokinetic side effects (1Gottesman M.M. Fojo T. Bates S.E. Nat. Rev. Cancer. 2002; 2: 48-58Crossref PubMed Scopus (4443) Google Scholar). It may, therefore, be more appropriate to target MDR1 expression. Indeed,MDR1 transcription has been targeted with Ecteinascidin 743 in pre-clinical studies (5Jin S. Gorfajn B. Faircloth G. Scotto K.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6775-6779Crossref PubMed Scopus (201) Google Scholar) and more recently by modulation of the nuclear receptor SXR (6Synold T.W. Dussault I. Forman B.M. Nat. Med. 2001; 7: 584-590Crossref PubMed Scopus (748) Google Scholar). Strategies involving antisense and transcriptional decoy (7Marthinet E. Divita G. Bernaud J. Rigal D. Baggetto L.G. Gene Ther. 2000; 7: 1224-1233Crossref PubMed Scopus (36) Google Scholar) and the use of anti-MDR1 mRNA hammerhead ribozymes have also been suggested (8Wang F.S. Kobayashi H. Liang K.W. Holland J.F. Ohnuma T. Hum. Gene Ther. 1999; 10: 1185-1195Crossref PubMed Scopus (33) Google Scholar). Stresses such as short-term exposure to cytotoxic drugs results in the up-regulation of MDR1 mRNA levels in many cell lines (9Hu Z. Jin S. Scotto K.W. J. Biol. Chem. 2000; 275: 2979-2985Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 10Madden M.J. Morrow C.S. Nakagawa M. Goldsmith M.E. Fairchild C.R. Cowan K.H. J. Biol. Chem. 1993; 268: 8290-8297Abstract Full Text PDF PubMed Google Scholar, 11Chaudhary P.M. Roninson I.B. J. Natl. Cancer Inst. 1993; 85: 632-639Crossref PubMed Scopus (360) Google Scholar, 12Thorgeirsson S.S. Huber B.E. Sorrell S. Fojo A. Pastan I. Gottesman M.M. Science. 1987; 236: 1120-1122Crossref PubMed Scopus (247) Google Scholar, 13Marino P.A. Gottesman M.M. Pastan I. Cell Growth Differ. 1990; 1: 57-62PubMed Google Scholar) and in human metastatic sarcomas in vivo (14Abolhoda A. Wilson A.E. Ross H. Danenberg P.V. Burt M. Scotto K.W. Clin Cancer Res. 1999; 5: 3352-3356PubMed Google Scholar). This is frequently due to transcriptional activation of the MDR1gene and has been reported in many cell lines after different physical and chemical stimulations and in cells selected for resistance to a variety of cytotoxic drugs (5Jin S. Gorfajn B. Faircloth G. Scotto K.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6775-6779Crossref PubMed Scopus (201) Google Scholar, 9Hu Z. Jin S. Scotto K.W. J. Biol. Chem. 2000; 275: 2979-2985Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 10Madden M.J. Morrow C.S. Nakagawa M. Goldsmith M.E. Fairchild C.R. Cowan K.H. J. Biol. Chem. 1993; 268: 8290-8297Abstract Full Text PDF PubMed Google Scholar, 15Vilaboa N.E. Galan A. Troyano A. de Blas E. Aller P. J. Biol. Chem. 2000; 275: 24970-24976Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 16Chin K.V. Tanaka S. Darlington G. Pastan I. Gottesman M.M. J. Biol. Chem. 1990; 265: 221-226Abstract Full Text PDF PubMed Google Scholar). In cell lines selected for drug resistance, increased MDR1 gene expression is also the result of amplification of the MDR1 locus and the appearance of self-replicating episomes (4Gottesman M.M. Pastan I. Annu. Rev. Biochem. 1993; 62: 385-427Crossref PubMed Scopus (3547) Google Scholar). Gene rearrangements that constitutively activate MDR1 transcription have also been associated with refractory acute lymphocytic leukemia (17Knutsen T. Mickley L.A. Ried T. Green E.D. du Manoir S. Schrock E. Macville M. Ning Y. Robey R. Polymeropoulos M. Torres R. Fojo T. Genes Chromosomes Cancer. 1998; 23: 44-54Crossref PubMed Scopus (70) Google Scholar, 18Mickley L.A. Spengler B.A. Knutsen T.A. Biedler J.L. Fojo T. J. Clin. Invest. 1997; 99: 1947-1957Crossref PubMed Scopus (93) Google Scholar). Although regulation due to changes in the MDR1 mRNA stability (19Lee C.H. Bradley G. Ling V. J. Cell. Physiol. 1998; 177: 1-12Crossref PubMed Scopus (41) Google Scholar), P-glycoprotein turnover (20Zhang W. Ling V. J. Cell. Physiol. 2000; 184: 17-26Crossref PubMed Scopus (36) Google Scholar), or trafficking (21Meschini S. Calcabrini A. Monti E. Del Bufalo D. Stringaro A. Dolfini E. Arancia G. Int. J. Cancer. 2000; 87: 615-628Crossref PubMed Scopus (70) Google Scholar) have been suggested, transcriptional regulation is widely considered to be the key step accounting for the complex spatio-temporal pattern of expression in vivo (22van Kalken C. Giaccone G. van der Valk P. Kuiper C.M. Hadisaputro M.M. Bosma S.A. Scheper R.J. Meijer C.J. Pinedo H.M. Am. J. Pathol. 1992; 141: 1063-1072PubMed Google Scholar, 23Nakayama M. Wada M. Harada T. Nagayama J. Kusaba H. Ohshima K. Kozuru M. Komatsu H. Ueda R. Kuwano M. Blood. 1998; 92: 4296-4307Crossref PubMed Google Scholar). It has also generally been assumed that up-regulation of MDR1 mRNA leads to an increase in P-glycoprotein. For example, human renal carcinoma (16Chin K.V. Tanaka S. Darlington G. Pastan I. Gottesman M.M. J. Biol. Chem. 1990; 265: 221-226Abstract Full Text PDF PubMed Google Scholar) and rat liver cells (24Schrenk D. Gant T.W. Michalke A. Orzechowski A. Silverman J.A. Battula N. Thorgeirsson S.S. Carcinogenesis. 1994; 15: 2541-2546Crossref PubMed Scopus (48) Google Scholar) up-regulate both MDR1 mRNA and P-glycoprotein following different stresses and are consequently transiently resistant to vinblastine. In this study, we show that in K562 leukemic cells the levels ofMDR1 mRNA increase in a dose- and time-dependent manner upon transient exposure to a variety of cytotoxic drugs. However, in contrast to the general prevailing models, we show that this is due to stabilization of mRNA and not because of transcriptional activation. Furthermore, the newly stabilized mRNA is not translated and so does not result in expression of P-glycoprotein and drug efflux. Only on subsequent long-term selection for drug resistance does the stabilized mRNA associate with polyribosomes, permitting translation of P-glycoprotein and drug efflux. The finding that drug resistance is a two-step post-transcriptional process, mediated by changes in bothMDR1 mRNA stability and translation, suggests new possibilities for treatment regimes to circumvent MDR in leukemia. K562, CCRF-CEM, and MANN cells were cultured in RPMI 1640 medium (Invitrogen) with 10% fetal calf serum and 2 mml-glutamine; KB-V1 cells (25Ueda K. Clark D.P. Chen C.J. Roninson I.B. Gottesman M.M. Pastan I. J. Biol. Chem. 1987; 262: 505-508Abstract Full Text PDF PubMed Google Scholar) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) with 20% fetal calf serum and 110 μmvinblastine (Sigma). All drugs were obtained from Sigma. For transient drug treatments (inductions), exponentially growing cells were seeded at 1 × 106 cells/ml and incubated with drug for the indicated times. The drug concentrations used were determined previously to cause macroscopic changes in cell morphology, indicative of cytotoxic stress, such as swelling, and changes in shape and granularity, in greater than 50% of cells. Unless otherwise stated, drug inductions were for 3 days using 3.4 nm doxorubicin, 22 μm vinblastine, 2.5 nm colchicine, 1.34 μm colcemid, or 100 μm cytarabine. Drug-resistant K562 sublines were obtained in a one-step selection by exposure to concentrations of the following cytotoxic drugs, which had been shown to result in 99.9% cell death after 14 days in culture in preliminary experiments: 40 pm colchicine, 30 pm doxorubicin, or 25 μm1-β-d-arabinofuranosylcytosine (cytarabine). Lines resistant to these concentrations of drug were designated KC40, KD30, and KA25 lines, respectively. RNA was prepared from cells by RNAzol extraction (Biogenesys, Poole, United Kingdom), reverse-transcribed (Roche Molecular Biochemicals), and amplified by PCR. Semi-quantitative RT-PCR estimation of MDR1 mRNA levels was performed as described (26Noonan K.E. Beck C. Holzmayer T.A. Chin J.E. Wunder J.S. Andrulis I.L. Gazdar A.F. Willman C.L. Griffith B. Von and Hoff D.D. et al.Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7160-7164Crossref PubMed Scopus (783) Google Scholar). Real-time quantitative PCR (Taqman; PerkinElmer Life Sciences) used the following primers within the MDR1coding sequence: sense, 5′-TTGTTCAGGTGGCTCTGGAT-3′; antisense, 5′-CTGTAGACAAACGATGAGCTATCACA-3′; and probe, 5′-AGGCCAGAAAAGGTCGGACCACCA-3′. Taqman Universal PCR Master Mix and control amplimers for GAPDH and 18 S ribosomal RNAs were used as recommended by the supplier (PerkinElmer Life Sciences). Results were collected and analyzed with an ABI Prism 7700 sequence detection system (PerkinElmer Life Sciences) as follows: the PCR cycle number that generated the first fluorescence signal above a threshold (threshold cycle, CT; 10 standard deviations above the mean fluorescence generated during the baseline cycles) was determined, and a comparative CT method was then used to measure relative gene expression. The following formula was used to calculate the relative amount of the transcript in the sample and normalized to an endogenous reference (GAPDH or 18 S rRNA): 2−ΔΔCT, where ΔCT is the difference in CT between the gene of interest and GAPDH or 18 S rRNA, and ΔΔCT for the sample = ΔCT of the actual sample − ΔCT of the lowest expressing sample. K562, KC40, and KD30 cells were incubated with 12.5 μg/ml actinomycin D to inhibit transcription. This concentration of actinomycin D was determined empirically in a series of pilot experiments as that which inhibited greater than 95% of [3H]uridine incorporation in both the naı̈ve and drug-resistant K562 cell lines, and thus nascent transcription, within 1 h (27Ogretmen B. McCauley M.D. Safa A.R. Biochemistry. 1998; 37: 11679-11691Crossref PubMed Scopus (30) Google Scholar). Cells were harvested at different times after actinomycin D addition, and total RNA was isolated as above. Total RNA, or the poly(A)+ fraction isolated by using an Oligotex kit (Qiagen), was used in real-time Taqman RT-PCR assays. In addition, Northern blots (28Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) of 10 μg of total RNA from the above samples were hybridized with probes derived from gene-specific sequences from the MDR1, GAPDH,Id2, or RAR-α genes (29Ishiguro A. Spirin K.S. Shiohara M. Tobler A. Gombart A.F. Israel M.A. Norton J.D. Koeffler H.P. Blood. 1996; 87: 5225-5231Crossref PubMed Google Scholar, 30Kizaki M. Koeffler H.P. Lin C.W. Miller C.W. Leuk. Res. 1990; 14: 645-655Crossref PubMed Scopus (14) Google Scholar). Hybridization signals were quantified with a PhosphorImager (AmershamBiosciences). Genomic DNA was isolated from cells by proteinase K digestion and phenol/chloroform extraction, digested with EcoRI, Southern blotted, and hybridized to nick-translated radioactive probes by standard procedures (28Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Probes were derived from the MDR1 5′-end region (975-bpPstI fragment comprising the transcription start point, 5′-untranslated region, and first intron) (31Ueda K. Pastan I. Gottesman M.M. J. Biol. Chem. 1987; 262: 17432-17436Abstract Full Text PDF PubMed Google Scholar) and the β-globin locus (3.0-kb EcoRI fragment containing DNase I hypersensitive site V) (32Blom van Assendelft G. Hanscombe O. Grosveld F. Greaves D.R. Cell. 1989; 56: 969-977Abstract Full Text PDF PubMed Scopus (196) Google Scholar). β-Globin to MDR1 ratios were obtained by quantitation using a PhosphorImager. Nuclei were prepared from naı̈ve, drug-induced, or drug-resistant K562 sublines as described (27Ogretmen B. McCauley M.D. Safa A.R. Biochemistry. 1998; 37: 11679-11691Crossref PubMed Scopus (30) Google Scholar). Nuclear run-on transcription and hybridization methods have been described elsewhere (33Yague E. Wood D.A. Thurston C.F. Mol. Microbiol. 1994; 12: 41-47Crossref PubMed Scopus (25) Google Scholar). For theMDR1 promoter-luciferase transcriptional assays, K562, KC40, and KD30 cells were transiently transfected with pMDR1(−1202), carrying the luciferase reporter gene from pGL2B (Promega, Madison, WI) under the control of MDR1promoter (34Jin S. Scotto K.W. Mol. Cell. Biol. 1998; 18: 4377-4384Crossref PubMed Google Scholar), together with pEFlacZ carrying the bacterial β-galactosidase gene under the control of the EF1αpromoter. After 2 days, cells extracts were prepared, and luciferase activity was determined with a luciferase assay system (Promega) and normalized against β-galactosidase expression (28Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The AP-1-dependent luciferase reporter plasmid (pAP1-luc), containing four tandem copies of the TPA-responsive element consensus motif TGACTCA coupled to the −36 to +37 rat prolactin minimal promoter (35Rincon M. Flavell R. EMBO J. 1994; 13: 4370-4381Crossref PubMed Scopus (245) Google Scholar), was cotransfected with pEFlacZ into K562 and KD30 cells. When indicated, cells were stimulated with 20 ng/ml phorbol 12-myristate 13-acetate (Sigma) during the last 12 h, and luciferase and β-galactosidase expression were measured as above. Transfections were by electroporation using a Bio-Rad gene pulser (Bio-Rad) with 5 × 106 cells essentially as described (28Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Analysis of surface P-glycoprotein expression was by flow cytometry using the fluorescently labeled monoclonal antibody UIC2 (Immunotech, Marseille, France), essentially as described (36Mechetner E.B. Schott B. Morse B.S. Stein W.D. Druley T. Davis K.A. Tsuruo T. Roninson I.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12908-12913Crossref PubMed Scopus (172) Google Scholar), using a Becton Dickinson Flow Cytometer (BD Biosciences). Live cells were detected by exclusion of propidium iodide. Drug-induced cells were monitored 1, 2, 3, and 4 days after drug addition. Where indicated, single cells were sorted into 96-well plates after UIC2 staining at a density of one cell per well by a FACS Vantage (BD Biosciences) and clonally expanded. Crude cell membrane fractions were prepared from 1 × 108 cells essentially as described (37Gerlach J.H. Bell D.R. Karakousis C. Slocum H.K. Kartner N. Rustum Y.M. Ling V. Baker R.M. J. Clin. Oncol. 1987; 5: 1452-1460Crossref PubMed Scopus (219) Google Scholar), with minor modifications. Briefly, cells were lysed with a hypotonic buffer (50 mm mannitol, 50 mmTris-HCl, pH 7.4, 2 mm EGTA) and centrifuged at a low speed (500 × g) to pellet nuclei and associated membranes such as endoplasmic reticulum and Golgi apparatus (plasma membrane-depleted fraction). The supernatant from the low speed fractionation was further centrifuged at 100,000 × gto obtain the plasma membrane-enriched fraction. Plasma membrane-enriched and -depleted fractions (200 μg) were separated by SDS-PAGE, and proteins were transferred electrophoretically to Immobilon membranes (Millipore, Watford, United Kingdom). Filters were incubated overnight at 4 °C with 0.1 μg/ml of the anti-P-glycoprotein monoclonal C219 (Cis-Bio International, Gif-Sur-Yvette, France) or 1:1000 anti-Na+/K+-ATPase α2 subunit, clone IID8, (Affinity Bioreagents Inc., Golden, CO) in 5 g/100 ml skimmed milk phosphate-buffered saline containing 0.1 g/100 ml Tween 20. Protein was visualized by enhanced chemiluminescence (Amersham Biosciences) after incubating with and horseradish peroxidase-conjugated goat and mouse IgG secondary antibody (1/1000) (Dako, Ely, United Kingdom). Incorporation of [35S]Met in nascent proteins was determined by trichloroacetic acid precipitable counts, following standard procedures (38Bonifacino J.S. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1998Google Scholar). Extracts of drug-induced K562, KC40, and MANN cells, sucrose gradient centrifugation, and RNA extraction followed standard procedures (39West M.J. Sullivan N.F. Willis A.E. Oncogene. 1995; 11: 2515-2524PubMed Google Scholar). Where indicated, buffers contained 20 mm EDTA. Isolated RNA was precipitated with 3m LiCl and resuspended in 10 μl of water. Detection ofMDR1 mRNA was by RT-PCR as described above. The myeloid leukemia-derived cell line K562 expresses very low levels of MDR1 mRNA, barely detectable by standard RT-PCR assays (Fig.1 A), although readily determined by a more sensitive method like real-time RT-PCR using poly(A)+ mRNA (data not shown). K562 cells responded to short-term exposure (drug induction) to several different cytotoxic drugs (doxorubicin, colchicine, colcemid, vinblastine, and cytarabine) by up-regulating MDR1 mRNA levels (Fig. 1 A). Real-time RT-PCR showed a 30- to 100-fold increase in MDR1mRNA levels in drug-induced cells compared with naı̈ve (not exposed to drug) K562 cells (data not shown). This effect was due to the cytotoxic drug, because the increase in MDR1 mRNA was both time- and dose-dependent (Fig. 1 B). To generate K562 sublines resistant to low levels of drugs, three cytotoxic drugs with different modes of action were used, colchicine (which binds tubulin and prevents mitosis), doxorubicin (a DNA intercalating agent), and cytarabine (a pyrimidine analogue). A one-step drug selection resulted in the generation of resistant pools of clones (see “Experimental Procedures”). These lines were called KC40, KD30, and KA25 (colchicine-, doxorubicin-, and cytarabine (araC)-resistant, respectively). MDR1 mRNA was substantially up-regulated in each of these lines (Fig. 1 A), shown by real-time RT-PCR to be between 2- and 5-fold greater than the levels found in the 3-day drug-induced cells (data not shown). Thus, both drug induction and selection for drug resistance result in substantial increases in steady-state MDR1 mRNA levels, independent of the mode of action of the drug. Gene amplification is a common mechanism for the up-regulation ofMDR1 mRNA in cell lines (4Gottesman M.M. Pastan I. Annu. Rev. Biochem. 1993; 62: 385-427Crossref PubMed Scopus (3547) Google Scholar) and is normally accompanied by rearrangements and deletions in the amplified locus. Genomic DNA from drug-resistant (KC40 and KD30) cells had the same MDR1gene copy number as the parental line K562, thus no detectable amplification or rearrangements of the locus had occurred (Fig.2 A). To determine whether MDR1 mRNA up-regulation was due to transcriptional activation (40Kantharidis P. El-Osta S. Silva M. Lee G. Hu X.F. Zalcberg J. Drug Resist. Updat. 2000; 3: 99-108Crossref PubMed Scopus (25) Google Scholar), we used two different approaches. We initially used a gene reporter assay in which an MDR1promoter fragment was placed upstream of the luciferase reporter gene (34Jin S. Scotto K.W. Mol. Cell. Biol. 1998; 18: 4377-4384Crossref PubMed Google Scholar). This reporter plasmid has been used previously to demonstrate transcriptional activation of MDR1 in, among others, human colon carcinoma SW620 cells (34Jin S. Scotto K.W. Mol. Cell. Biol. 1998; 18: 4377-4384Crossref PubMed Google Scholar). Luciferase expression was equivalent in naı̈ve (not drug-treated) and all of the drug-resistant K562 lines (Fig. 2 B), suggesting that there is no promoter up-regulation. As AP-1 plays a role in the transcriptional activation of MDR1 (41Daschner P.J. Ciolino H.P. Plouzek C.A. Yeh G.C. Breast Cancer Res. Treat. 1999; 53: 229-240Crossref PubMed Scopus (86) Google Scholar), and because K562 cells resistant to etoposide (another P-glycoprotein substrate) have up-regulated levels ofc-jun and c-fos mRNAs and increased AP-1 binding activity (42Ritke M.K. Yalowich J.C. Biochem. Pharmacol. 1993; 46: 2007-2020Crossref PubMed Scopus (47) Google Scholar, 43Ritke M.K. Bergoltz V.V. Allan W.P. Yalowich J.C. Biochem. Pharmacol. 1994; 48: 525-533Crossref PubMed Scopus (33) Google Scholar), it was necessary to exclude the possibility that the AP-1 pathway had been inactivated in these cells. To do this we used a reporter plasmid, pAP1-luc, in which the luciferase gene expression was driven by four human collagenase TPA-responsive elements and the minimal rat prolactin promoter (35Rincon M. Flavell R. EMBO J. 1994; 13: 4370-4381Crossref PubMed Scopus (245) Google Scholar). This TPA-responsive element has been used in numerous studies because of its high affinity for the AP-1 complex, and, as expected, transient transfection in Jurkat T-cells results in an increase of luciferase activity upon TPA activation (35Rincon M. Flavell R. EMBO J. 1994; 13: 4370-4381Crossref PubMed Scopus (245) Google Scholar), which increases MDR1 gene expression in several cell lines, including K562 cells (44Chaudhary P.M. Roninson I.B. Oncol. Res. 1992; 4: 281-290PubMed Google Scholar). Naı̈ve and drug-resistant cells transiently transfected with pAP1-luc produced similar levels of luciferase activity, both before and after TPA activation (Fig. 2 C). Thus, the pathway responsible of activating AP-1 is equally functional in both naı̈ve and drug-resistant K562 cells, and the lack of transcriptional activation of the MDR1 promoter in drug-resistant cells cannot be due to a lack of AP-1 functionality. To confirm that transcriptional activation of the MDR1promoter is not responsible for MDR1 mRNA up-regulation we also studied the MDR1 promoter in its native chromosomal context by nuclear run-on experiments. Despite the increase in steady-state MDR1 mRNA levels, transcription initiated from the MDR1 promoter was low compared with transcription from other control genes (e.g. GAPDH,β-actin, or ε-globin) and did not increase significantly in drug-resistant or drug-induced cells (data not shown). Thus, transcriptional activation does not appear to be responsible for the up-regulation of MDR1 mRNA in K562 cells following drug induction or upon selection for drug resistance. Because the increase in steady-stateMDR1 mRNA was not due to de novo mRNA synthesis, we asked whether changes in the rates of mRNA decay might be involved. To determine the half-lives of MDR1mRNA in naive, drug-induced and drug-resistant cells, we treated cells with actinomycin D to inhibit transcription (see “Experimental Procedures”). MDR1 mRNA from naı̈ve K562 cells had a very short half-life (approximately 1 h) determined by real-time RT-PCR (Fig. 3 A). In contrast, MDR1 mRNA half-life values for doxorubicin- and colchicine-resistant K562 cells were 12–16 h (Fig. 3 A). This was confirmed by Northern analysis (Fig. 3 B) and is in good agreement with the 14-h half-life reported for another independently derived doxorubicin-resistant K562 line (K562/ADR) (45Muller C. Goubin F. Ferrandis E. Cornil-Scharwtz I. Bailly J.D. Bordier C. Benard J. Sikic B.I. Laurent G. Mol. Pharmacol. 1995; 47: 51-56PubMed Google Scholar). Drug-induced K562 cells also had a long MDR1 mRNA half-life (Fig. 3A). The half-lives of other short-lived messages, such as Id2 and RAR-α, were confirmed to be 1–2 h as reported previously (29Ishiguro A. Spirin K.S. Shiohara M. Tobler A. Gombart A.F. Israel M.A. Norton J.D. Koeffler H.P. Blood. 1996; 87: 5225-5231Crossref PubMed Google Scholar, 30Kizaki M. Koeffler H.P. Lin C.W. Miller C.W. Leuk. Res. 1990; 14: 645-655Crossref PubMed Scopus (14) Google Scholar) in both naı̈ve and drug-resistant K562 cells (Fig. 3 C). Similarly, the half-life of a long-lived message (GAPDH) was also unchanged in drug-resistant cells (20–24 h in both naı̈ve K562 and KD30 cells) (Fig. 3 B). Thus, the stabilization of theMDR1 mRNA is specific and not a general phenomenon affecting other short-lived mRNAs. In conclusion, up-regulation ofMDR1 mRNA levels following exposure to drugs, after either a transient induction or drug selection, is primarily due to a specific increase in mRNA stability. Transient exposure to cytotoxic drugs (drug induction) led to an increase in MDR1 mRNA levels through mRNA stability. This observation led us to ask whether MDR1mRNA up-regulation was accompanied by an increase in P-glycoprotein expression. Cell surface expression of P-glycoprotein was measured by flow cytometry using the P-glycoprotein-specific monoclonal antibody UIC2 (36Mechetner E.B. Schott B. Morse B.S. Stein W.D. Druley T. Davis K.A. Tsuruo T. Roninson I.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12908-12913Crossref PubMed Scopus (172) Google Scholar). There was no significant increase in UIC2-positive cells following drug induction at any stage during the 4-day incubation period compared with naı̈ve K562 cells. In contrast, cells selected for resistance to the P-glycoprotein substrates colchicine (KC40 cells) or doxorubicin (KD30 cells) showed a large (10- to 100-fold) increase in UIC2 reactivity, indicating the presence of active P-glycoprotein on the cell surface. Cells selected for resistance to cytarabine (KA25 cells), which is not transported by P-glycoprotein, showed much lower levels of surface P-glycoprotein expression than the KC40 and KD30 cells, despite having similarMDR1 mRNA levels. Only 40% of cytarabine-resistant (KA25) cells expressed surface P-glycoprotein, consistent with the fact that resistance to cytarabine is known to be mediated by other P-glycoprotein-independent mechanisms (11Chaudhary P.M. Roninson I.B. J. Natl. Cancer Inst. 1993; 85: 632-639Crossref PubMed Scopus (360) Google Scholar) (Fig.4 A). Because the assay above detects only active P-glycoprotein in the plasma membrane, it was necessary to exclude the possibility that P-glycoprotein was expressed in drug-induced cells, but either inserted in the plasma membrane in an inactive form or accumulated intracellularly. Cell membranes from naı̈ve, drug-induced, and drug-resistant K562 cells were analyzed for P-glycoprotein expression by immunoblotting using the monoclonal antibody C219. A band of ∼190 kDa, corresponding to mature P-glycoprotein, was detected in the plasma membrane-enriched fraction from drug-resistant lines but not from the naı̈ve or drug-induced cells (Fig. 4 B). P-glycopr
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