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(CANCER RESEARCH 50. 1426-1430. March 1. 1990] Mechanisms of Multidrug Resistance in HL60 Cells: Detection of Resistanceassociated Proteins with Antibodies against Synthetic Peptides That Correspond to the Deduced Sequence of P-Glycoprotein1 David Marquardt, Sue McCrone, and Melvin S. Center2 Division of Biology, Kansas Slale University, Manhattan, Kansas 66506 ABSTRACT HL60 cells isolated for resistance to Adriamycin are multidrug resist ant and defective in the cellular accumulation of drug. These cells do not however overexpress mdr\ and do not contain detectable levels of Pglycoprotein. In the present study we have prepared antisera against synthetic peptides that correspond to various sequence domains of Pglycoprotein and have examined by Western blot analysis the reactivity of these antisera with proteins contained in membranes of III (ill \dr cells. All antisera are highly reactive with a M, 180,000 (plSO) Pglycoprotein contained in membranes of HL60 cells isolated for resistance to vincristine (HL60/Vinc). In contrast, of 13 antisera tested 12 do not react with any resistance-associated protein in the III 60 Adr isolate. One antiserum (ASP14) is however highly reactive with a M, 190,000 protein (pl90) contained in III.60. Vdr membranes. This protein is not detected in drug-sensitive cells. ASP14 also reacts with proteins p 195 and pSO contained in a second independent III.60/Adr isolate. Analysis of membrane subfractions shows that pl90 is located primarily in the endoplasmi reticulum with only low levels contained in plasma mem branes. Additional studies demonstrate that endoplasmic reticulum of III 6(1 Vdr cells contain a major V/, 190,000 protein that is capable of binding the photoaffinity agent 8-azido|a-"P|ATP. p 195 contained in a second 111.(.IIAdr isolate is also labeled with 8-azido|a-"P|ATP. These results thus demonstrate that antiserum against a specific Pglycoprotein sequence detects a pl90 (pl95) resistance-associated mem brane protein in two independent III 6(1 Adr isolates. pl90 (pl95) and P-glycoprotein thus contain a minor sequence homology and based on the specificity of ASP14 this occurs in a region which may be involved in nucleotide binding. Possibly this sequence is common to and essential for the functionality of proteins which contribute to resistance by reducing cellular drug levels. INTRODUCTION Experimental cell lines treated with cytotoxic drugs such a colchicine, Adriamycin, actinomycin D, or vincristine fre quently develop resistance to the selecting agent as well as resistance to structurally unrelated compounds (1, 2). This multidrug resistance in many isolates occurs as a result of reduced accumulation of drug that seems to be related to an enhanced drug efflux system (3, 4). Reduced drug levels are frequently related to overexpression of P-glycoprotein, a surface membrane phosphoglycoprotein with a molecular weight of 150,000-180,000 (5-8). A family of genes termed mdrcode for P-glycoprotein (9-12) and in human cell lines there appear to be two members of this family mdr\ (12) and mdr3 (10, 13). Only mdr\ has been found to be overexpressed in resistant cell lines (13), whereas expression of mdr3 sequences has been detected in human liver (13). Recently HL60 cells selected for resistance to Adriamycin have been isolated and characterized (14, 15). These cells are multidrug resistant and defective in the cellular accumulation Received 8/17/89; revised 11/8/89; accepted 11/27/89. 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. 1This investigation was supported by Research Grant CA-37585 from the National Cancer Institute. Department of Health and Human Services, and by a grant from Bristol-Myers. 2To whom requests for reprints should be addressed. of drug (14, 15). Despite this phenotype, isolates of HL60/Adr cells do not overexpress mdr\ ( 16) and do not contain detectable levels of P-glycoprotein (17-19). Analysis of these cells has revealed, however, certain membrane protein changes which may contribute to drug resistance. Thus it has been found that development of resistance is accompanied by a major increase in the phosphorylation levels of a M, 150,000 membrane pro tein which is contained in sensitive cells (18, 20). It has also been observed that membranes of HL60/Adr cells contain a resistance-associated M, 190,000 ATP-binding protein (16). In the present study we have extended these findings and have examined the possibility that proteins which contribute to drug resistance in HL60/Adr cells may have some sequence homol ogy with P-glycoprotein. Interest in this possibility was derived from the finding that both p 1901 (16) and P-glycoprotein (21) are capable of binding ATP and thus may contain similar sequences at the nucleotide-binding site. Thus in the present study we have prepared antisera against synthetic peptides which correspond to the deduced sequence of human P-glyco protein (12) and have examined the reactivity of this material with membrane proteins of HL60/Adr cells. Of several antipeptide sera studied one has been found which reacts with a M, 190,000-195,000 protein in two independent HL60/Adr iso lates. MATERIALS AND METHODS Cells. HL60 cells were isolated for resistance to Adriamycin (HL60/ Adr) or vincristine (HL60/Vinc) as described previously (15, 17). The HL60/Adr and HL60/Vinc isolates exhibit an 80- and 100-fold increase in resistance to the selecting agents, respectively. A second independent HL60/Adr isolate (14) exhibiting a 100-fold increase in resistance to Adriamycin was generously provided by Dr. Alex Hindenburg. Peptide Synthesis and Immunization. Peptides were synthesized ac cording to the deduced sequence of human P-glycoprotein (12) by the Marglin and Merrifield solid state method (22). The sequence of the peptides and the regions of P-glycoprotein to which these sequences correspond are given in Table 1. Synthetic peptides containing lysine at the amino terminus were conjugated by means of glutaraldehyde to keyhole limpet hemocyanin. The conjugated material (0.3-1.0 mg) was emulsified with Freund's complete adjuvant and thereafter injected s.c. into a New Zealand White rabbit. After 3 weeks a second injection of conjugated material in incomplete adjuvant was made and after an additional 2 weeks the rabbit was bled. Antisera were tested for reactiv ity against synthetic peptides by using a radioimmunoassay procedure essentially as described by Mumby et al. (23), except that the I25Ilabeled protein A was used to detect antibody binding to peptide. Immunoblots. Membrane proteins (50 Mg)were separated by electrophoresis in a 7.5% sodium dodecyl sulfate polyacrylamide gel (24) and the proteins were transferred to nitrocellulose paper as described by Towbin et al. (25). The paper was incubated in PNBT for 2 h at 37°C. The paper was thereafter incubated with antisera diluted in PNBT for 15 h at room temperature. After washing with PNBT the paper was incubated with I25l-labeled protein A (5 x 10' cpm/ml) for 2 h at room temperature. After extensive washing the paper was dried and iinmn 3The abbreviations used are: pl90. A/, 190,000; other proteins are similarly designated; 8-azido[a-"P]ATP, AzAT"P; PNBT, 0.02 M phosphate buffer (pH 7.3)-O.I5 M NaCl-1% bovine serum albumin-0.05% Tween 20. 1426 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1990 American Association for Cancer Research. DETECTION OF RESISTANCE-ASSOCIATED Fig. 1. Reactivity of antipcptidc sera with membrane proteins of sensitive and resistant cells. Membranes were prepared from sensitive cells (Lane I), HL60/Adr cells (Lane 2), or HL60/Vinc cells (Lane 3), and Western blot analysis was carried out as described in "Ma terials and Methods," using antisera ASPI (A). ASP9 (B), or ASP21 (C). The sequences of the peptides to which the corresponding antisera are directed are given in Table I. Ordinate. molecular weight in thousands. 1 180- 2 PROTEINS IN HL60/Adr CELLS B 2 3 3 I 2 3 f 1234 Table I Reactivity of antipeptide sera with proteins of drug-resistant HL60 cells reactivity p Pepiide P-glycoprotein'' 190+++++++++++ sequence*GEMTD1FANAGNLEDLMSFANAGNLEDLMSN1TSDINDTGFFMNLEEDLNTRLTDDVSKINEVVSWFDDPKNTTGALTSGQALKDKKELEGAGNVTFGEVVFNYPTRP no."129233811412117IS914612Peptide amino acids73-9079-9396-110171-185801-815880-8941034-10481049-10631143-11571158-11721173-11871201-12151266-1280Antisera 0 0 +++ " Peptide number corresponds to original laboratory designation. * Peptide sequence corresponds to the deduced mdr\ P-glycoprotein sequence (12). f Antisera reactivity with P-glycoprotein or P190 was determined with mem branes of HL60/Vinc or HL60/Adr cells, respectively, using Western blot analy sis. noreactive proteins were detected by autoradiography. Cell Membranes. Membranes were isolated from sensitive and re sistant HL60 cells as previously described (26). For certain experiments plasma membranes and cndoplasmic reticulum were isolated after centrifugation of a crude membrane fraction in a discontinuous sucrose density gradient (26). Labeling Membrane Proteins with AzAT'2P. Membrane proteins were labeled with the photoaffinity agent AzAT"P (8.6 Ci/mmol, purchased from 1CN) as described previously (16). Labeling conditions used for each experiment are given in the figure legends. Radioactively labeled proteins were analyzed after polyacrylamide gel electrophoresis and autoradiography. 50- RESULTS Analysis of Membrane Proteins of HL60/Adr Cells Reactive with Antisera to P-Glycoprotein Peptide Sequences. Antibodywas prepared against synthetic peptides which correspond to the deduced sequence of P-glycoprotein (12) and the reactivity of this material with membrane proteins of sensitive, HL60/ Adr and HL60/Vinc cells was examined by Western blot analy sis. The HL60/Vinc isolate has previously been shown to overexpress marl (16) and to contain high levels of a M, 180,000 P-glycoprotein (17, 20). The results of a screening assay with isolated membranes using 3 different antipeptide sera are shown in Fig. 1. All three sera are highly reactive with the M, 180,000 P-glycoprotein contained in the HL60/Vinc isolate (Fig. 1, Lane 3\ Table 1). Serum against peptide 1 (Table 1) also reacts with a M, 110,000 protein contained in these cells (Fig. IA, Lane 3). The nature of pi 10 and its involvement in drug resistance is not known at the present time. In contrast to the results obtained with HL60/Vinc cells the three antisera do not react with any protein contained in membranes of sensitive cells (Fig. 1, Lane J) or the HL60/Adr isolate (Fig. 1, Lane 2). Further studies with 10 additional antipeptide sera show that all are reactive with a single M, 180,000 protein contained in Fig. 2. Reactivity of ASPI 4 with membrane proteins of sensitive and resistant cells. Membranes were prepared from sensitive cells (Lane /), HL60/Adr cells (Lane 2). a second HL60/Adr isolate (14) (Lane J). and HL60/Vinc cells (¡Mne 4), and Western blot analysis was carried out as described in "Materials and Methods." using ASP 14. Ordinate, molecular weight in thousands. membranes of HL60/Vinc cells (Table 1). In contrast, of these 10 sera 9 do not react with any resistance-associated protein in HL60/Adr cells (Table 1). It has been found, however, that one antipeptide serum, ASP 14 (Table 1), is highly reactive with Pglycoprotein of HL60/Vinc cells (Fig. 2, Lane 4) and with a M, 190,000 protein contained in membranes of the HL60/Adr 1427 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1990 American Association for Cancer Research. DETECTION OF RESISTANCE-ASSOCIATED PROTEINS IN HL60/Adr CELLS 1234 190- 190- Fig. 3. p 190 distribution in membrane subfractions. Plasma membranes and endoplasmic reticulum were prepared from sensitive and HL60/Adr cells as described in "Materials and Methods." Membrane proteins were separated by polyacrylamide gel electrophoresis and Western blot analysis was carried out with the ASPI 4 antiserum (Table 1). Lanes I and 3, endoplasmic reticulum of sensitive and resistant cells, respectively. Lanes 2 and 4, plasma membranes of sensitive and resistant cells, respectively. Ordinate, molecular weight in thousands. isolate (Fig. 2, Lane 2). This antiserum also reacts with two resistance-associated proteins p 195 and p50 present in mem branes of a second independent Adriamycin-resistant isolate (14) (Fig. 2, Lane 3). The structural relationship between pl95 and p50 is not known at the present time, pi90 (pi95), p50, or p 180 are not detected in drug-sensitive cells with the ASP 14 serum (Fig. 2, Lane ]). The antiserum does, however, react with some low-molecular-weight proteins contained in both sensitive and resistant cells (Fig. 2). Experiments have also been carried out in which plasma membranes and endoplasmic reticulum were prepared from HL60/Adr cells and the presence of p 190 in these membrane fractions was examined by Western blot analysis with ASP 14. The results demonstrate that p 190 is located primarily in the endoplasmic reticulum (Fig. 3, Lane 3) with only barely detect able levels in the plasma membranes (Fig. 3, Lane 4). In parallel Fig. 4. AzAT32P labeling of membrane proteins. A crude membrane fraction from sensitive and HL60/Adr cells was separated into plasma membranes and endoplasmic reticulum (26), and proteins were labeled with AzAT3!P as described in "Materials and Methods." Incubations with AzAT3IP were for 2 min, followed by exposure of the samples to UV irradiation for 2 min. Radioactively labeled proteins were detected after polyacrylamide gel electrophoresis and autoradiography. Lanes 1 and 3, plasma membranes of sensitive and resistant cells, respec tively. Lanes 2 and -I. endoplasmic reticulum of sensitive and resistant cells, respectively. Ordinate, molecular weight in thousands. experiments with sensitive cells p 190 is not detectable in either the endoplasmic reticulum (Fig. 3, Lane 1) or plasma mem branes (Fig. 3, Lane 2). A. lower-molecular-weight protein with a molecular weight of 85,000 which is contained in endoplasmic reticulum of both sensitive and resistant cells has been found to be reactive with ASP 14 (Fig. 3, Lanes 1 and 3). Analysis of Membrane AzAT32P-binding Proteins. Previous studies have shown that membranes of HL60/Adr cells contain a M, 190,000 protein which is highly labeled with the photoaffinity agent AzAT32P (16). In view of this finding it was of interest to determine if this protein corresponds to p 190 which is reactive with ASP 14. To examine this, plasma membranes and endoplasmic reticulum were isolated from sensitive and 1428 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1990 American Association for Cancer Research. DETECTION OF RESISTANCE-ASSOCIATED PROTEINS IN HL60/Adr CELLS resistant cells and thereafter labeled with AzAT'2P as described in "Materials and Methods." The radioactively labeled proteins I 2 3 were analyzed after polyacrylamide gel electrophoresis. The results demonstrate that AzAT"P labels a resistance-associated M, 190,000 protein which is located exclusively in the endoplasmic of the HL60/Adr isolate (Fig. 4, Lane 4). With this labeling procedure p 190 is not detected in the plasma mem brane fraction of resistant cells (Fig. 4, Lane 3). There is essentially no detectable labeling of p 190 with AzAT'2P in either plasma membranes (Fig. 4, Lane 1) or endoplasmic reticulum (Fig. 4, Lane 2) in drug-sensitive HL60 cells. The results of this experiment and those of Fig. 3 thus demonstrate that the membrane distribution of AzAT32P-labeled p 190 cor responds exactly to that of pl90 reactive with ASP14. It is thus indicated that p 190 reactive with ASP 14 is an ATP-binding protein. AzAT32P-labeling experiments have also been carried out with membranes of a second independent HL60/Adr isolate (14). Membranes of these cells contain pl95 and p50 which are reactive with ASP 14 (Fig. 2). Incubation of membranes with AzAT32P results in the labeling of a resistance-associated pro i tein with a molecular weight of 195,000 (Fig. 5, Lane 1). A protein, with molecular weight of 50,000 is also labeled to a greater extent than a protein of similar molecular weight con tained in drug-sensitive cells (Fig. 5, Lanes 1 and 3). The increased labeling of p50 of resistant cells has been detected only under conditions in which incubations with AzAT32P are carried out for time periods of about 10-20 s. During longer incubation periods the levels of p50 labeling in sensitive and resistant cells are similar. Under the conditions of these exper iments p 190 of HL60/Adr membranes is also labeled with AzAT32P but the extent of labeling is somewhat less than that found for p 195 (Fig. 5, Lanes 1 and 2). The results of these studies thus suggest that the ASP14-reactive protein p 195 is capable of binding ATP. p50 which also reacts with this antiserum may also be an ATP-binding protein. Experiments have been carried out to immunoprecipitate AzAT12P-labeled p 190 with ASP 14. Thus far we have not detected immunoprecipitation of radioactively labeled protein. Further studies suggest however that the ASP 14 serum does not react with native proteins in immunoprecipitation experiments. DISCUSSION HL60 cells isolated for resistance to Adriamycin are defective in the cellular accumulation of drug and this occurs as a result of enhanced levels of an energy-dependent efflux system (14, 15). Recently a M, 190,000 resistance-associated ATP-binding protein has been detected in membranes of these cells, suggest ing the possibility that this protein functions in the drug efflux pathway of this isolate (16). Several lines of evidence suggest that p 190 is structurally distinct from P-glycoprotein. Thus, HL60/Adr cells do not overexpress mdr\ (16, 19) and the Pglycoprotein monoclonal antibody C219 (27) does not react with any protein in membranes of HL60/Adr cells (17, 18, 20). In the present study we have extended these findings and have examined in greater detail possible sequence homology of p 190 Fig. 5. AzAT3!P labeling of membrane proteins. Membranes were isolated and P-glycoprotein. To approach this problem we have pre from two independent HL60/Adr isolates (Lanes I and 2) and from sensitive pared antisera against peptides with correspond to the deduced cells (Lane 3), and proteins were labeled with AzAT32P as described in "Materials and Methods." Results shown in Lane 1 were obtained with an isolate prepared sequence of P-glycoprotein (12) and have examined the reactiv by liliali;! el al. (14). Incubations with Az.AT3!P were for 10 s, followed by ity of this material with membrane proteins in drug-resistant exposure of the samples to UV irradiation for 2 min. Radioactively labeled HL60 cells. Thirteen antipeptide sera have been prepared, all proteins were detected after poKan\l.imidc gel electrophoresis and autoradiography. Exposure to AzAT"P was for 10 s, since under these conditions we can of which are highly reactive with P-glycoprotein contained in detect labeling of a P50 protein contained in an HL60/Adr isolate (Lane I). HL60 cells isolated for resistance to vincristine. Screening Ordinate, molecular weight in thousands. assays using Western blot analysis show that 12 of these antisera are not reactive with any resistance-associated protein in 1429 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1990 American Association for Cancer Research. DETECTION OF RESISTANCE-ASSOCIATED HL60/Adr cells. One antipeptide serum, ASP14, was found however to be highly reactive with a M, 190,000 protein con tained in membranes of two independent HL60/Adr isolates. The basis of the slight difference in the molecular weights of the proteins is unknown but may be related to higher levels of glycosylation of p 195. It was also found in these studies that one HL60/Adr isolate contains a p50 protein which reacts with ASP 14. The nature of this protein and its structural and func tional relationship with pi90 (pi95) remains to be determined. The finding that ASP 14 reacts with resistance-associated p 190 (p 195) and P-glycoprotein suggests that these proteins share a common sequence contained in peptide 14. At the present time it is not known if the complete P-glycoprotein, peptide 14 sequence is contained in p 190 (p 195). It is indicated, however, from hybridization studies with mdrl (16) and studies with many antipeptide sera directed against a variety of se quence domains of P-glycoprotein (Table 1) that peptide 14 may be the only sequence shared by p 190 (p 195) and P-glyco protein. This raises the possibility that the sequence of peptide 14 is highly conserved in proteins which contribute to resistance by reducing cellular drug levels in an energy-requiring reaction. This would be consistent with the evidence which suggests that the peptide 14 sequence includes part of a nucleotide-binding region. Thus peptide 14 has limited sequence homology with nucleotide-binding sites detected in other proteins (28) and proteins reactive with ASP 14 such as P-glycoprotein and p 190 (p 195) are capable of binding the photoaffmity agent AzAT"P. P50, a protein reactive with ASP 14 and contained in a single HL60/Adr isolate, may also be an ATP-binding protein. These results taken together therefore suggest that ASP 14 is directed against part of a nucleotide-binding sequence and that this sequence may be highly conserved in proteins involved in multidrug resistance. This site may thus be of major importance to the function of these proteins. Previously we have shown that HL60 cells isolated for resist ance to Adriamycin do not contain amplified mdr\ or mdri and that these sequences are not overexpressed in the resistant cells (16). These results along with our immunological studies have provided strong evidence that p 190 (p 195) is encoded by a new multidrug-resistance gene which is not genetically related to mdr\ (12) or mdrl (10, 13). In view of this finding ASP14 may be of considerable value in analyzing multidrug-resistant cells. Thus the serum may be important in detecting resistanceassociated proteins in experimental cell isolates which do not contain P-glycoprotein but which exhibit reduced accumulation of drug (29). ASP 14 may also be of value in identifying new proteins which contribute to resistance in tumor cells from patients undergoing chemotherapy. Finally, this antipeptide serum should be useful as a probe in the isolation of a P190 complementary DNA. ACKNOWLEDGMENTS The authors wish to thank Dr. Alex Hindenburg for providing an HL60/Adr isolate for use in these studies. REFERENCES 1. Biedler, J. L., and Riehm, H. 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S., and Trent, J. M. Pharmaco logical and biological evidence for differing mechanisms of doxorubicin resistance in two human tumor cell lines. Cancer Res., 48: 2793-2797, 1988. 1430 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1990 American Association for Cancer Research. Mechanisms of Multidrug Resistance in HL60 Cells: Detection of Resistance-associated Proteins with Antibodies against Synthetic Peptides That Correspond to the Deduced Sequence of P-Glycoprotein David Marquardt, Sue McCrone and Melvin S. Center Cancer Res 1990;50:1426-1430. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/50/5/1426 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. 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