Unbiased Examination of Changes in Plasma Membrane Proteins in Drug Resistant Cancer Cells Amir M. Rahbar† and Catherine Fenselau*,†,‡ Department of Chemistry and Biochemistry, University of Maryland, College Park Maryland, and Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore Maryland Received July 26, 2005
In this study, an unbiased examination is made of the abundance changes between proteins found in the basolateral plasma membranes of a drug susceptible parental MCF-7 breast cancer cell line and a cell line selected from the parent line for resistance to the anticancer drug mitoxantrone. Plasma membrane proteins were differentially labeled metabolically, enriched using the colloidal silica pellicle method, and characterized by tandem mass spectrometry. Fifteen proteins were identified with significant (>2) changes, including receptors, adhesion proteins, proteins involved in amino acid uptake, and proteins involved in glucose uptake. From 40 µg of membrane proteins, 3227 unique peptides and 540 proteins were identified. Keywords: plasma membrane • quantitative proteomics • metabolic labeling • drug resistance • MCF-7 cells • mitoxantrone
Introduction When initial cancer treatments do not fully eradicate the cancerous mass, it is not uncommon for these remaining tumor cells to be selected for resistance to the drug used for treatment, and to exhibit resistance to other anti-cancer drugs as well.1 This multidrug resistance is, perhaps, the major impediment in the treatment of cancer today.2 To control acquired multidrug resistance, the mechanisms that confer this drug resistance must be understood.3 Over 50% of commercially available drugs target membrane proteins.4 Membrane proteins are difficult to study, due to their hydrophobicity. Conditions required for the solubilization of membrane proteins are usually incompatible with enrichment, fractionation, or enzymatic digestion.5 Membrane proteins often precipitate in isoelectric focusing experiments, which excludes 2D gels as a separation method for membrane proteins.5,6 Recently, this laboratory reported the incorporation of Jacobson’s pellicle method into a proteomics strategy, as a method to enrich the basolateral plasma membrane 20-fold, based on Western blot analysis.7 This technique, previously described by Jacobson,8,9 allowed the identification of 366 proteins, about 50% of which had previously been reported to be plasma membrane proteins. Recently, other groups have reported the enrichment of plasma membrane proteins by alkylation with biotinylated reagents, followed by affinity chromatography.10-14 Plasma membrane proteins have also been challenging to quantitate. Neither 2-D gel separation for densitometry, or * To whom correspondence should be addressed. Tel: (301) 405-8616. Fax: (301) 405-8615. E-mail:
[email protected]. † Department of Chemistry and Biochemistry, University of Maryland. ‡ Greenebaum Cancer Center, University of Maryland School of Medicine.
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conversion to peptides for chemical derivatization is reliable. This laboratory has previously modified the method first introduced by Chait and co-workers15 for metabolic labeling of proteins, to provide incorporation of both 13C6-arginine and 13C -lysine, and to work in cultured human MCF-7 cells.16 In 6 the present study, this differential labeling technique is used in combination with the colloidal silica enrichment and the proteomic analysis strategy, we previously developed to quantitate changes in abundances of basolateral plasma membrane proteins from MCF-7 breast cancer cells selected during culture in increasing concentrations of mitoxantrone.17
Materials and Methods Materials. Criterion precast gels (13.3 × 8.7 cm, 4-15%), Laemmli sample buffer, 10× PBS, 10× Tris/Glycine/SDS Buffer, 10× Tris/Glycine Buffer, Biosafe Coomassie stain, nitrocellulose, DC Protein Assay, prestained protein broad range standards, and filter paper were purchased from Bio-Rad (Hercules, CA). DTT, EDTA disodium salt, fetal calf serum, imidazole, iodoacetamide, LUDOX-CL cationic colloidal silica, MES, MgCl2, mouse R-human Na/K ATPase primary antibody, NaCl, Na2CO3, Nycodenz, Penicillin-Streptomycin solution, poly(acrylic acid) (100,000 typical molecular weight), protease inhibitor cocktail, ProteoQwest Colorimetric Western Blotting Kit, and trifluoroacetic acid were purchased from Sigma Aldrich (St. Louis, MO). RPMI 1640 medium was purchased from Invitrogen (Carlsbad, CA). Improved Minimal Essential Medium was purchased from American Type Culture Collection (Manassas, VA). Modified porcine trypsin was purchased from Promega (Madison, WI). MEM (Eagle) Media with Earle’s Salts without L-lysine or L-arginine, and Dialyzed Fetal Bovine Serum was purchased from Atlanta Biologicals (Lawrenceville, GA). Carbon-13 (13C) labeled L-lysine and L-arginine isotopes at 98% purity was purchased from Cambridge Isotope Laboratories 10.1021/pr0502370 CCC: $30.25
2005 American Chemical Society
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(Andover, MA). Acetic acid, acetonitrile, CaCl2, formic acid, methanol, and NH4HCO3 were purchased from Fisher (Pittsburgh, PA). Equipment. The Mini-PROTEAN 3 electrophoresis system, mini trans-blot cell, Criterion precast gel system, and GS-800 densitometer were purchased from Bio-Rad (Hercules, CA). Nanospray emitters were purchased from New Objectives (Woburn, MA). Mass spectra were obtained using an Applied Biosystems Qstar Pulsar i (Foster City, CA) equipped with a nanospray ion source from Protana (Odense, Denmark). LCMS was performed online with the LC Packings Ultimate Nano LC System (Sunnyvale, CA). LC-MS head with Liquid Junction was purchased from Proxeon (Odense, Denmark). Cell Culture and Metabolic Labeling. The human breast cancer mitoxantrone resistant MCF-7 cell line (MXR MCF-7), and the drug susceptible parental MCF-7 cell line (MCF-7) were gifts from Dr. K. H. Cowan (The Eppley Institute, University of Nebraska Medical Center, Omaha, Nebraska). The drug resistant line was developed directly from the parental line.17 Cell cultures were grown in 150 cm2 cell culture flasks using Improved Minimal Essential Medium containing 5% fetal calf serum and antibiotics at 37 °C and 5% CO2.18 Metabolically labeled drug susceptible MCF-7 cells were grown using media specially prepared in-house as previously described.16 Briefly, MCF-7 cells were grown in media containing 13C labeled L-lysine and L-arginine as the predominant source of biologically usable lysine and arginine. MXR MCF-7 cells were grown in the same manner, using unlabeled Larginine and L-lysine in the cell culture media instead of the isotopically labeled amino acids. Preparation of the Basolateral Plasma Membrane Fraction from Cell Culture Monolayers. Basolateral plasma membranes from MCF-7 and MXR MCF-7 cells growing as monolayers were isolated using a modified cationic colloidal silica plasma membrane isolation procedure as previously described.7-9 Briefly, cell culture monolayers were coated with a layer of positively charged cationic colloidal silica, followed by a layer of poly(acrylic acid) while still attached to the cell culture flasks, so that all washes and cell coating solutions were added directly to the flasks with the cells still attached and decanted from the flasks to remove. A hypotonic lysis buffer (2.5 mM imidazole, pH 7, supplemented with protease inhibitor cocktail from Sigma Aldrich) was added to the culture flasks with the coated cells still attached. The cells were allowed to swell and lysis was achieved by pipeting the lysis buffer in each flask up and down, or using a needle and syringe to apply shearing force. Following cell lysis, the basolateral plasma membranes remain attached to the cell culture flasks. These plasma membranes are then washed sequentially with 5 M NaCl, PBS with 10 mM EDTA, and 100 mM Na2CO3, while still attached to the flasks to remove contamination from soluble proteins and increase the plasma membrane enrichment. The membranes are scraped from the bottom of the cell culture flasks using a cell scraper and pelleted by centrifugation at 14 000 × g in an SW28 rotor for 20 min. The pelleted membranes were solubilized directly in SDS-PAGE loading buffer and the protein concentration determined using the Bio-Rad DC Protein Assay. One-Dimensional SDS-PAGE Analysis. A 20-µg portion of plasma membrane protein from MXR MCF-7 cells and 20 µg of plasma membrane protein from metabolically labeled drug susceptible MCF-7 cells were mixed together and loaded onto 4-15% gels and run according to manufacturer’s specifications
using the Bio-Rad Criterion precast gel system. Following electrophoresis, the gels were stained using Bio-Rad Biosafe Coomassie stain and the stained gels were scanned using a GS800 densitometer from Bio-Rad (Hercules, CA). Mass Spectrometry. The SDS-PAGE gel was excised into 28 gel bands and tryptic digestion was performed on each gel slices.19 After extraction from the gel bands, the tryptic peptides were placed in 0.1% formic acid (FA) in preparation for nanoLC-MS/MS analysis. The tryptic peptides were analyzed by online nanoLC-MS/MS. Reversed-phase conditions were A: 97.5% H2O/2.5% ACN/0.1%FA, B: 97.5% ACN/2.5% H2O/ 0.1%FA with a 60 min gradient from 5%-35%B on a PepMap 75 µm I.D., 15 cm, 3 µm, 100 Å column from LC Packings (Sunnyvale, CA). Ions of mass/charge ratio in the range of 400 to 1400 Da were identified in the initial survey scan, and the monoisotopic peaks of doubly and triply charged ions were selected for tandem mass spectrometry analysis. Each 10 s analysis cycle consisted of a 1 s survey scan, followed by three 3 s MS/MS scans of the three most intense ions. Dynamic exclusion was applied to reduce redundancy. The cycle was repeated throughout the chromatographic run. The ion spray voltage was set at 2600 V. Protein Identification. Each protein was identified based on sequences from two or more peptides using the integrated Qstar software Analyst QS with Bioanalyst and ProID with a minimum confidence level of 99.4%. Peptides were also manually sequenced from the tandem mass spectrometry data. The ABI integrated software Analyst QS v1.0 SP8 with Bioanalystv1.1and ProID 1.1 was used for the acquisition and analysis of all mass spectrometry data. The database used was the Swiss-Prot/TrEMBL. The ProID search parameters were as follow: MS mass tolerance 0.3 Da; MS/MS tolerance 0.3 Da; monoisotopic peak used for the identification; modifications allowed were methionine oxidation, carboxamidomethylation of cysteine, and up to one missed cleavage. Each of the peptides identified and reported was a unique identification. Often peptides were identified multiple times, however the peptide was only counted once. Protein Quantitation. Ratios of unlabeled/labeled monoisotopic peak areas were calculated to determine protein abundance changes between the two cell lines. Ratios g 2 or e 0.5, which correspond to a 100% abundance difference between the 2 cell lines, were considered biologically significant and subsequently reported. Replicate experiments were performed using membrane mixtures from three different pairs of harvests, i.e., from both unlabeled MXR MCF-7 cells and labeled drug susceptible MCF-7 cells. Protein ratios from the replicate experiments were averaged and provided experimental uncertainties.
Results For protein identifications, 40 µg of basolateral plasma membrane proteins isolated from the MXR MCF-7 line was fractionated on an SDS-PAGE gel (Figure 1, lane 2). Peptides recovered from in-gel digests of excised gel bands were resuspended in the mobile phase A and analyzed by LC-MS/ MS. In addition to protein identification using the Applied Biosystems integrated software, tandem mass spectra were manually interpreted and the peptide sequence tags were used to validate manually the identity of each of the proteins identified. A total of 540 distinct proteins was identified from 3227 unique peptide identifications. Table 1 in the Supporting Journal of Proteome Research • Vol. 4, No. 6, 2005 2149
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Figure 3 shows spectra of the five labeled and unlabeled peptide pairs used to calculate the relative abundances of the 4F2 cell-surface antigen (an amino acid transporter) from susceptible and drug resistant MCF-7 cells. The ratio was calculated for each individual peptide pair and the values are averaged in the final results. Ratios ranged from 4.1 to 4.7, with and average of 4.4 ( 0.3. Table 1 lists all of the proteins whose relative abundance changes exceeded 2-fold. The proteins whose names are in red have higher abundances in the mitoxantrone resistant MCR-7 cell line, while proteins listed in blue have higher abundances in the drug susceptible MCR-7 parental cell line. These changes range from a thirteen-fold reduced abundance (integrin alpha3) to an 8-fold increase in the abundance of the large neutal amino acid transporter small subunit 1. Two other transmembrane proteins, transferrin receptor and breast cancer resistance protein (an ATP-dependent transporter) are abundant in the resistant cell line, but not detectable in the control line.
Discussion
Figure 1. SDS-PAGE gel of proteins isolated from basolateral plasma membranes from MXR and drug susceptible MCF-7 cell cultures. Lane 1 - Molecular weight standards. Lane 2-40 µg plasma membrane proteins from MXR MCF-7 cells. Lane 3 mixture of 20 µg plasma membrane proteins from MXR MCF-7 cells and 20 µg plasma membrane proteins from the drug susceptible MCF-7 cells.
Information shows the list of all proteins identified from the basolateral plasma membrane fraction isolated from the MXR MCF-7 cell line along with their subcellular location and the number of peptides used to make the identification. Figure 2 shows a graphical representation of the subcellular locations of each of the proteins as defined by the Swiss-Prot annotation and Gene Ontology assignments. It should be noted that 1643 peptides, 51% of the total, are assigned by this conservative approach as originating from proteins in the plasma membrane. Several papers have recently reported identification of proteins in the plasma membrane that are assigned in the databases to other regions of the cell,10-12 and the issue of regiospecificity is being reassessed as proteomics experiments provide more information. Plasma membranes from the two cell lines were isolated and the proteins extracted as described in the previous section. The protein concentrations of these fractions were determined and the plasma membrane proteins from the two samples were mixed at a 1:1 ratio. For protein quantitation, a mixture of 20 µg of basolateral plasma membrane proteins isolated from the MXR MCF-7 cell line and 20 µg of basolateral plasma membrane proteins isolated from the drug susceptible MCF-7 cell line was separated on an SDS-PAGE gel (Figure 1, lane 3). The gel bands were excised and subjected to tryptic digestion and the peptides were recovered and analyzed by LC-MS as before. Isotopomeric peptide pairs coeluted and protein ratios were measured from the areas of monoisotopic peptide peaks. Pairs of labeled and unlabeled peptides were compared for all the proteins identified. Abundance ratios were determined and the proteins for which changes in abundance were more than 2-fold are reported. 2150
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All of the proteins showing abundance changes have been previously identified in the literature except for the hypothetical protein DKFZp686D0452. This protein is predicted to be a member of the integrin family of proteins and has 96% sequence homology with the integrin beta-1 subunit. The proteins summarized in Table 1 can be classified into four different groups. Cell adhesion proteins include integrin alpha2, integrin alpha 3, and hypothetical protein DKFZp686D0452 (beta integrin). All three of these proteins, which have reduced abundances in the resistant cell line, are associated with cell adhesion and may be associated with metastasis. It has also been proposed recently,20 based on statistical analyses, that adhesion proteins may play a role in acquired drug resistance. Transport proteins quantitated in Table 1 are the breast cancer resistance protein (BCRP), facilitated glucose transporter, member 1, 4F2 cell-surface antigen heavy chain and large neutral amino acids transporter small subunit 1. 4F2 Antigen acts in a dimer with the large neutral transporter to facilitate amino acid uptake by the cells,21 while the decrease in GLUT-1, and the increase in stomatin are both associated with decreased glucose uptake.22,23 Signal transduction proteins and receptors compose the third group of proteins, and include guanine nucleotide-binding protein alpha-13 subunit, guanine nucleotide-binding protein G(S) alpha subunit, erythrocyte band 7, ephrin type B receptor 4, dihydropyridine receptor alpha 2, and tumor-associated calcium signal transducer 2. Not all of the functions of these transducers have been clearly worked out. In the fourth category, transferrin receptor protein 1 and clathrin heavy chain 1 are proteins involved in receptor mediated endocytosis. The goal of this study was to identify the differences in abundances of plasma membrane proteins in the two cell lines and to use these data to elucidate possible mechanisms by which changes in plasma membrane proteins contribute to drug resistance in the mitoxantrone-resistant MCF-7 cell line. These cells are approximately 4000-fold resistant to the cytotoxic effects of mitoxantrone, and cross resistant to several other antitumor agents as well.17 One of the major protein changes observed in the present study is the presence of the breast cancer resistance protein (BCRP), also called the ATP-
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Figure 2. Subcellular distribution of proteins identified in the MXR MCF-7 basolateral plasma membrane fraction.
Figure 3. Peptide pairs from the 4F2 cell surface antigen protein analyzed from the labeling experiments.
binding cassette protein ABCG2, in the MXR MCF-7 cell line but not at detectable levels in the drug susceptible cell line.
BCRP was discovered by Doyle, Ross, and co-workers in the multidrug resistant breast cancer cell line MCF-7/AdrVp,24 Journal of Proteome Research • Vol. 4, No. 6, 2005 2151
research articles Table 1. Protein Abundance Changes in the Basolateral Membranes of MCF-7 Cells and Mitoxantrone-resistant MCF-7 Cells
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Acknowledgment. The authors wish to acknowledge The National Science Foundation (NSF Grant CHE-9634238) and the National Institutes of Health (GM 21248) for funding of this research. The authors would like to thank Drs. Frances Ligler and Michael Lassman from the U.S. Naval Research Laboratory for the use of their facilities and Andrei Chertov for the growth and maintenance of the cell lines used in these experiments. Supporting Information Available: List of all proteins identified from the basolateral plasma membrane fraction isolated from the MXR MCF-7 cell line along with their subcellular location and the number of peptides used to make the identification. This material is available free of charge via the Internet at http://pubs.acs.org. References
*Average of measurements made on three separate sets of harvests.
and subsequently, it was also shown to be overexpressed in MCF-7 cells resistant to mitoxantrone.25 Thus, its identification and characterization in the present study are consistent with the earlier work, and confirm the effectiveness of the strategy involving colloidal silica and metabolic labeling for comparative proteomics. A second protein, transferrin receptor protein 1, is also found to be abundant in the MXR MCF-7 cell line, but is not detected in the drug susceptible cell line. Transferrin receptor protein 1 mediates endocytosis of transferrin into the cell26,27 and is the major mechanism for iron uptake by cells.26 Increased abundance of transferrin receptor protein 1 has been previously reported in cancer cells in culture and also in human pancreatic cancer tissue.28-30 This has led to the evaluation of transferrin conjugates for drug delivery.31 Two reports have linked increased numbers of transferrin receptors with drug resistance in cultured cancer cells.28,32 Antibody labeling and cell sorting were used in a strategy highly complementary to the unbiased proteomic approach of the present work. It is not obvious why drug resistant cancer cells should need more iron than drug susceptible cells, or if the transferrin receptor contributes to resistance by another mechanism. It has been suggested that the cellular requirement for iron-III is increased to facilitate rapid growth in diseased cells.29,33 The Guanine nucleotide-binding protein, alpha-13 subunit, was found here to have almost a 12-fold decrease in abundance in the MXR MCF-7 cell line. This protein family has not been previously associated with drug resistance, however it has been previously identified as an activator of apoptosis.34,35 Blocking of the apoptotic pathway is one way that drug resistance can be achieved, so this may be another mechanism used to achieve drug resistance by the MXR MCF-7 cell line. Of the 15 proteins exhibiting significant abundance changes, BCRP has been most directly associated with drug resistance previously. On the basis of the detection of increased abundances of guanine-nucleotide binding protein and transferrin receptor in the plasma membrane, the present study suggests two additional mechanisms of drug resistance that may operate in the mitoxantrone drug resistant MCF-7 breast cancer cell line. 2152
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(1) Galmarini, C. M.; Galmarini, F. C. Curr. Opin. Investig. Drugs 2003, 4, 1416-1421. (2) Thomas, H.; Coley, H. M. Cancer Control 2003, 10, 159-165. (3) Gottesman, M. M.; Fojo, T.; Bates, S. E. Nat. Rev. Cancer 2002, 2, 48-58. (4) Wu, C. C.; Yates, J. R., III Nat. Biotechnol. 2003, 21, 262-267. (5) Blonder, J.; Goshe, M. B.; Moore, R. J.; Pasa-Tolic, L.; Masselon, C. D.; Lipton, M. S.; Smith, R. D. J. Proteome Res. 2002, 1, 351360. (6) Santoni, V.; Molloy, M.; Rabilloud, T. Electrophoresis 2000, 21, 1054-1070. (7) Rahbar, A. M.; Fenselau, C. J. Proteome Res. 2004, 3, 1267-1277. (8) Chaney, L. K.; Jacobson, B. S. J. Biol. Chem. 1983, 258, 1006210072. (9) Stolz, D. B.; Jacobson, B. S. J. Cell Sci. 1992, 103 (Pt 1), 39-51. (10) Zhao, Y.; Zhang, W.; Kho, Y. Anal. Chem. 2004, 76, 1817-1823. (11) Jang, J. H.; Hanash, S. Proteomics 2003, 3, 1947-1954. (12) Shin, B. K.; Wang, H.; Yim, A. M.; Le Naour, F.; Brichory, F.; Jang, J. H.; Zhao, R.; Puravs, E.; Tra, J.; Michael, C. W.; Misek, D. E.; Hanash, S. M. J. Biol. Chem. 2003, 278, 7607-7616. (13) Peirce, M. J.; Wait, R.; Begum, S.; Saklatvala, J.; Cope, A. P. Mol. Cell Proteomics 2004, 3, 56-65. (14) Peirce, M. J.; Begum, S.; Saklatvala, J.; Cope, A. P.; Wait, R. Proteomics 2005, 5, 2417-2421. (15) Oda, J.; Huang, K.; Cross, F. R.; Cowburn, D.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6591-6596. (16) Gehrmann, M. L.; Hathout, Y.; Fenselau, C. J. Proteome Res. 2004, 3, 1063-1068. (17) Nakagawa, M.; Schneider, E.; Dixon, K. H.; Horton, J.; Kelley, K.; Morrow, C.; Cowan, K. H. Cancer Res. 1992, 52, 6175-6181. (18) Hathout, Y.; Riordan, K.; Gehrmann, M.; Fenselau, C. J. Proteome Res. 2002, 1, 435-442. (19) Jensen, O. N.; Wilm, M.; Shevchenko, A.; Mann, M. Methods Mol. Biol. 1999, 112, 513-530. (20) Stein, W. D.; Litman, T.; Fojo, T.; Bates, S. E. Int. J. Cancer 2005, 113, 861-865. (21) Storey, B. T.; Fugere, C.; Lesieur-Brooks, A.; Vaslet, C.; Thompson, N. L. Int. J. Cancer 2005. (22) Zhang, J. Z.; Hayashi, H.; Ebina, Y.; Prohaska, R.; Ismail-Beigi, F. Arch. Biochem. Biophys. 1999, 372, 173-178. (23) Zhang, J. Z.; Abbud, W.; Prohaska, R.; Ismail-Beigi, F. Am. J. Physiol. Cell Physiol. 2001, 280, C1277-1283. (24) Doyle, L. A.; Yang, W.; Abruzzo, L. V.; Krogmann, T.; Gao, Y.; Rishi, A. K.; Ross, D. D. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1566515670. (25) Doyle, L. A.; Ross, D. D. Oncogene 2003, 22, 7340-7358. (26) Qian, Z. M.; Li, H.; Sun, H.; Ho, K. Pharmacol Rev. 2002, 54, 561587. (27) Aisen, P. Int. J. Biochem. Cell Biol. 2004, 36, 2137-2143. (28) Barabas, K.; Faulk, W. P. Biochem. Biophys. Res. Commun. 1993, 197, 702-708. (29) Muller, C. I.; Miller, C. W.; Kawabata, H.; McKenna, R. J., Jr.; Marchevsky, A. M.; Koeffler, H. P. Oncol. Rep. 2005, 14, 299303. (30) Ryschich, E.; Huszty, G.; Knaebel, H. P.; Hartel, M.; Buchler, M. W.; Schmidt, J. Eur. J. Cancer 2004, 40, 1418-1422. (31) Li, H.; Qian, Z. M. Med. Res. Rev. 2002, 22, 225-250. (32) Savada, D.; Phillips, T.; Lin, C.; Kane, S. E. Cancer Lett. 2002, 179, 151-156.
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