Highly Altered Protein Expression Profile in the Adriamycin Resistant

MCF-7: parental cell line; MelR MCF-7: melphalan resistant MCF-7; MCF-7/MX: .... K. H. Cowan, P. Gutierrez, and J. A. Moscow for MCF-7 cell lines. We ...
0 downloads 0 Views 257KB Size
Download PDF
Highly Altered Protein Expression Profile in the Adriamycin Resistant MCF-7 Cell Line Marion L. Gehrmann, Catherine Fenselau, and Yetrib Hathout* Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742 Received July 28, 2003

The protein expression pattern in the cytosol fraction of the adriamycin resistant MCF-7 cell line (MCF7/ADR) was compared to that of the parental MCF-7 cell line using two-dimensional gel electrophoresis and mass spectrometry. Twenty proteins with altered abundances were identified and studied in MCF7/ADR. Both up regulation and down regulation are characterized. The most striking differences were found for proteins that were uniquely expressed in this cell line and not detectable in the parental MCF-7 cell line. These proteins include annexin I, the neuronal ubiquitin carboxyl hydrolase isoenzyme L-1 (also known as PGP9.5), glutathione-S-transferase pi class, nicotinamide N-methyltransferase, and interleukin-18 precursor. On the other hand, catechol-O-methyltransferase was expressed in the parental cell line, but was not detected in the adriamycin resistant cell line. This protein expression pattern was unique to MCF-7/ADR and not observed in MCF-7 cell lines selected for resistant to etoposide, mitoxantrone or melphalan. Keywords: comparative proteomics • adriamycin • MCF-7 • ubiquitin carboxyl hydrolase • glutathione S-transferase • annexins • chemoresistance

Introduction The adriamycin resistant MCF-7 (MCF-7/ADR) cell line was first selected by Batist et al.1 in 1986 after exposing an MCF-7 cell line to increasing concentrations of the drug adriamycin (also known as doxorubicin). The cells that survived drug treatment were isolated and were found to be 200 times more resistant to adriamycin than the parental cell line. MCF-7/ADR cells were also found to be cross-resistant to a range of other anti-neoplastic agents from the Vinca alkaloids and anthracyclines.1,2 Such multidrug resistance (MDR) is believed to be partly due to overexpression of the membrane glycoprotein termed P170 or P-glycoprotein. This protein is a transmembrane efflux pump that uses two ATP molecules to transport one drug molecule.3,4 Its overproduction in cancer cells is highly correlated with development of the multidrug resistance (MDR) phenotype.5,6 However, treatment of MCF7/ADR with verapamil, a potent inhibitor of P-glycoprotein did not restore full sensitivity of these cells to adriamycin,7 suggesting that other mechanisms might be involved in the development of resistance to the drug. Indeed, several cytosolic proteins were found by early investigators to be markedly altered in their expression or activity in MCF-7/ADR cells. These changes included alterations in phase I enzymes with a marked decrease in the activity of aryl hydrocarbon hydroxylase8 and in phase II drug-metabolizing enzymes, with a marked increase in glutathione S-transferase activity,1 and a moderate increase * To whom correspondence should be addressed. Present address: Children’s National Medical Center, Center for Genetic Medicine, 111 Michigan Ave. NW, Washington, DC 20010. Tel: (202) 884-3136. Fax: (202) 884-6012. E-mail: [email protected]. 10.1021/pr0340577 CCC: $27.50

 2004 American Chemical Society

in other drug conjugating enzymes such as glucuronyltransferase and sulfotransferase.8 Even though no direct evidence of adriamycin detoxification by GST was found in the MCF7/ADR cells,9 a good correlation was reported between the level of expression of pi class GST and the level of resistance to the drug.1 GST pi was reported to possess an intrinsic peroxidase activity and was proposed to detoxify free radical byproducts generated by adriamycin, rather than detoxify the drug by conjugation with glutathione.9 Other proteins involved in the pentose phosphate pathway, e.g., glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione reductase, and glutathione peroxidase, have also been found to be altered in their abundance and activity in the MCF-7/ ADR.10 These alterations suggested that resistance of MCF-7/ ADR to adriamycin might be a multifactorial event, involving several pathways. Although the MCF-7/ADR cell line was isolated from an adriamycin treated MCF-7 cell line, its nature and origin are the subjects of ongoing debates.11,12 Chromosomal karyotyping and DNA fingerprinting revealed a significant divergence between the MCF-7/ADR cell line and the parental MCF-7 cell line.2,13 In addition, the MCF-7/ADR cells have a full caspase-3 activity which is not found in the parental MCF-7 cells.11,12 These differences have led to a call for a name change of the cell line from MCF-7/ADR to NCI/ADR-RES cell line.13 In the present study, a proteomics approach is used to examine the overall expression pattern of cytosolic protein in this MCF-7/ADR cell line. To this end, two-dimensional gel electrophoresis and computerized image analysis were used to compare the cytosolic proteome of MCF-7/ADR cell line to that of the parental MCF-7 cell line and to those of other drug Journal of Proteome Research 2004, 3, 403-409

403

Published on Web 12/06/2003

research articles resistant MCF-7 cell lines. Proteins of interest were identified by mass spectrometry. The present study was expected to confirm the previously characterized alterations, and also to search for additionally altered protein abundances, which may led to a better characterization of this MCF-7/ADR cell line and to better understanding of its adriamycin resistant phenotype.

Experimental Section Cell Culture and Preparation of the Cytosolic Fraction. The parental MCF-7, etoposide resistant MCF-7 (MCF-7/VP)14 and mitoxantrone resistant MCF-7 (MCF-7/MX)15 cell lines were obtained from Dr. Kenneth H. Cowan (Eppley Institute, University of Nebraska Medical Center). Melphalan-resistant MCF-7 cell line (MelR MCF-7)16 was obtained from J. A. Moscow (Department of Pediatrics, University of Kentucky College of Medicine, Lexington, KY). Adriamycin resistant MCF-7 cell line (MCF-7/ADR)1 was gift from Dr. Peter Gutierrez (Marlene and Stewart Greenebaum Cancer center, University of Maryland school of Medicine). All these cell lines were maintained in culture in our laboratory in Improved Minimal Essential Medium (ATCC, Manassas, VA) containing 10% fetal calf serum (Sigma Co., St. Louis, MO) and 1.2% of a stabilized solution of penicillin (5000 units) /streptomycin (5 mg/mL) (Sigma Co., St Louis, MO). Cell growth was carried out at 37 °C under 5% CO2-95% air atmosphere. Confluent cells in 150 cm2 flasks (Corning, Inc., Corning, NY) were washed twice with 15 mL of 10 mM phosphate buffer saline solution (PBS) and detached by adding 5 mL of trypsin (cell culture grade, Sigma Co, St. Louis, MO) followed by an incubation at 37 °C for 2-3 min. To stop the activity of the residual trypsin, a 15-mL volume of serum-supplemented medium was added, and the cells were pelleted by centrifugation at 500g for 10 min at 10-12 °C. The cell pellets were washed twice with 5 volumes of ice-cold 10 mM PBS, followed by one wash with 5 volumes of ice-cold 10 mM NaCl solution. The cytosolic proteins were then extracted from the cells following a previously described method 17 using digitonin extraction buffer (10 mM PIPES, 0.015% digitonin, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl2, 5 mM EDTA, and 1 mM protease inhibitor (PMSF), pH 6.8) with gentle shaking at 4 °C for 10 min. The cytosolic fraction was separated from the rest of cell pellet by centrifugation at 500g for 10 min. The supernatant containing cytosolic protein was transferred to a clean tube and centrifuged at 8000g for 20 min. To remove any residual microscopic debris, the supernatant was collected again and further centrifuged at 140000g for 2 h. Protein concentration was determined in the final supernatant using the BioRad protein assay dye reagent, following the manufacturer’s instructions. The sample was then stored in aliquots of 500 µL in micro centrifuge tubes at -80 °C. Two-Dimensional Gel Electrophoresis. About 200 µg of total cytosolic proteins were separated by two-dimensional gel electrophoresis (2-DG) following a previously described procedure.18 After protein separation, the gel slabs were fixed for 30 min in a solution of methanol/water/acetic acid (45:50:5, v/v/v) followed by three washes for 5 min in deionized water. Staining was preformed with Bio-Safe colloidal Coomassie blue G-250 (BioRad, Hercules, CA) for 1 h, followed by ample rinsing with deionized water until the desired contrast was obtained. To confirm reproducibility, cytosolic fractions were prepared from three independent cell growths for each cell lines, and gels of these independently prepared cytosolic fractions were compared. 404

Journal of Proteome Research • Vol. 3, No. 3, 2004

Gehrmann et al.

Analysis of Gel Images. The stained gels were scanned with a GS-800 densitometer (BioRad, Hercules, CA), and the images were saved as TIFF files. Z3 Software (Compugen Ltd., Tel Aviv, Israel) was used to compare raw gel images. The two images to be compared were layered, with semiautomatic matching of spots and manually anchoring spots that are believed to be similar in the two gels. Then the volumes of each protein in each pair of spots in the two images was measured and registered. The relative expression (RE) of a protein in one gel image is compared to its RE in the second gel image to provide a numerical value representing the differential expression (DE) of that protein. This operation was repeated for up to three pairs of gels (three gels from a drug resistant MCF-7 cell line and three gels from the parental MCF-7 cell lines, each gel is obtained from an independent cell culture) to exclude any error in matching and to judge the significance of the DE. Identification of Gel Separated Proteins. Spots were excised with the tip of a clean polypropylene pipet and transferred to a micro centrifuge tube containing 100 µL of deionized water. Tryptic digestion was preformed as described in detail by Jensen et al.19 the peptides were extracted then dried by vacuum centrifugation. The dried peptides were redissolved in 10 µL of 0.1% TFA, and desalted using C18 ZipTip micropipet tips (Millipore Co., Bedford, MA) following the manufacturer’s user guide. The eluted peptides were again dried by vacuum centrifugation and redissolved in a small volume (5 to 10 µL) of water/methanol/acetic acid (50:48:2, v/v/v) for nanospray mass spectrometry analysis on a hybrid quadrupole time-offlight instrument (QStar/Pulsar, Applied Biosystems, Foster City, CA). Routinely, a capillary tip (Protana, Odense, Denmark) was loaded with 1-2 µL of the peptide solution and mounted into the nanospray ion source. Spray voltage was set at 0.9 kV, and collision energy was ramped from 15 to 50 eV, depending on the nature of the selected ion. Typically, doubly and sometimes triply charged ions were selected for MS/MS analysis. Protein identification was performed with an interfaced script of the Mascot search engine, which uses the raw MS/MS data to search the NCBI protein database. Usually, protein identification is considered accurate when the MS/MS results from three or more peptides in a given sample identify the same protein.

Results and Discussion The expression patterns of proteins in the cytosol of the four drug resistant MCF-7 cell lines (MelR MCF-7, MCF-7/Vp, MCF7/MX, and MCF-7/ADR) were compared to that of the parental MCF-7 cells using 2-DG image analysis software as described in the Experimental section. Differential protein expression data were obtained from independent comparison of at least three pairs of gels obtained from three independent cell growths (three from the drug resistant cell line and three from the parental MCF-7 cell line). The three gels from the three independent cell growths of the parental MCF-7 cell lines were also compared with each other to check variability that can occur from sample to sample preparation and from gel to gel. An example of the differential expression data is presented in Figure 1. For instance, triosephosphate isomerase (Figure 1A) was found not to be differentially expressed among the 5 cell lines studied. A little variability (10%) was observed when comparing its relative expression in samples prepared from three independent cultures of the parental MCF-7 cells. Peroxiredoxin 2 was found significantly down-expressed in MCF-

Protein Expression in Adriamycin Resistant MCF-7

research articles Sinha et al.20 reported that although MCF-7/ADR cells presented lower drug accumulation than the parental MCF-7 cells, they also produced much less hydroxyl radical than MCF-7 following treatment with adriamycin. This lower freeradical formation in MCF-7/ADR was not due to the poor drug accumulation in the cells or changes in the adriamycin metabolizing enzymes, but rather correlated to a high activity of glutathione peroxidase.20 Batist et al.1 further demonstrated that glutathione peroxidase and glutathione S-transferase activities were markedly increased by 12 and 45-fold, respectively, in MCF-7/ADR compared to the parental MCF-7 cell line. The observed overexpression of glutathione S-transferase is consistent with the 45-fold increase in the activity of glutathione S-transferase reported by Batist et al.1 Indeed, the relative intensity of the spot corresponding to this protein was very high in this resistant cell line and almost invisible in the MCF-7 cell line (Figure 2, Table 1). Earlier workers correlated the high activity of GST by antibody and other characteristics to the overexpression of an anionic isoenzyme similar to the human placenta GST class-pi isoenzyme.1 In the present study, the up-regulated GST was firmly identified as the pi class isoenzyme, based on MS/MS microsequencing experiments and matching the microsequences against a current library of known human glutathione-S-transferases. The sequence coverage obtained was about 46% of the human GST class-pi enzyme. The spot corresponding to the selenium-dependent glutathione peroxidase (GHSPx1), which was suggested to be responsible for the high activity of peroxidase in MCF-7/ADR 20,21 was not identified in the present study. Attempts to locate GHSPx1 in the 2-DG map using its theoretical molecular mass and pI of 21899 Da and 6.15 did not reveal any differentially expressed spot with these coordinates. Thus, its differential activity reported earlier in these cells could not be verified or confirmed here.

Figure 1. Differential expression of three proteins among the five cell lines used in this study. Each bar represents the average and the standard deviation of the differential expression of the protein after comparison of three pair of gels (three gels from a drug resistant MCF-7 cell line were compared independently to three gels from the parental MCF-7). Each gel is obtained from and independent cell culture. The relative expression of each protein was also verified between three gels obtained from three independent cultures of the parental MCF-7 cell line. The horizontal line represents the reference line for a nondifferentially expressed protein. MCF-7: parental cell line; MelR: melphalan resistant MCF-7; MX: mitoxantrone resistant MCF-7; VP-16: etoposide resistant MCF-7; ADR: adriamycin resistant MCF-7.

7/ADR, while unchanged in the other MCF-7 cell lines (Figure 1B). Other proteins, such as annexin V, were found overexpressed in the MCF-7/ADR cells, while remaining unchanged in MCF-7/Vp, MCF-7/MX, and MelR MCF-7 (Figure 1C). Among the four drug resistant cell lines studied, MCF-7/ADR was found to be the most divergent, based on its cytosolic protein expression pattern. The 2-DG maps of the cytosolic proteins prepared from the parental MCF-7 cell line and the MCF-7/ADR cell line are shown in Figure 2. A number of proteins were found to be differentially expressed and are indicated by arrows in the figure and listed in Table 1 with their differential expression. This expression pattern was reproducible from gel to gel, and from cell batch to cell batch across the six month period of the study.

Other enzymes with peroxidase activity were identified in the 2-DG map and were found differentially expressed at the protein level in MCF-7/ADR. Abundances of peroxiredoxin-1 (PDX1) and peroxiredoxin-6 (PDX6), thioredoxin-dependent peroxidases, were found to be moderately increased by almost a factor of 2 in MCF-7/ADR. Surprisingly, the abundance of peroxiredoxin-2 (PDX2), an isoenzyme belonging to the same thioredoxin-dependent peroxidase family, was found markedly decreased, by almost a factor of 7, in MCF-7/ADR relative to MCF-7. All of these enzymes might be expected to be upregulated in the adriamycin resistant cell line, because they are expected to protect cell from superoxides that can be produced following adriamycin metabolism.22 Thioredoxin, which is an enzyme that reactivates oxidized peroxiredoxins, remained unchanged between the two cell lines. The question remains by what means these cells still exhibit high peroxidase activity. One enzyme that may contribute is the overexpressed GST-pi. Normally, GST-pi is involved in the conjugation of xenobiotics to glutathione. In the absence of evidence for conjugation of doxorubicin to glutathione,9 it is suggested that this GST-pi found in MCF-7/ADR may possess an intrinsic peroxidase activity and may be involved in detoxification of organic peroxides generated by adriamycin metabolism. This suggestion is somewhat plausible because this protein has been reported to possess an unusual intrinsic organic peroxidase activity.1 Levels of other GST classes identified in this study, such us GST mu-3 and GST omega-1, remained unchanged between the two cell lines. Journal of Proteome Research • Vol. 3, No. 3, 2004 405

research articles

Gehrmann et al.

Figure 2. Two-dimensional gel electropherograms of cytosolic fractions from MCF-7 (left) and MCF-7/ADR (right). Arrows indicate the proteins that are differentially expressed in MCF-7/ADR relative to the parental cell line MCF-7. The proteins are indicated by their abbreviated nomenclature and are reported in Table 1 with their differential expression. Table 1. Some Differentially Expressed Proteins Found in MCF-7/ADR spota

protein name

Swiss-Prot accession no.

differential expressionb (MCF-7/ADR)/(MCF-7)

function

G6PD ANX5 LDH-B ANX1 ANX3 14-3-3 σ NNMT UCH-L1 PDX6 COMT GST-pi IL-18 PDX2 PDX1 PEBP CYPA TRX S100C CACY UBIQ

glucose-6-phosphate dehydrogensae annexin V L-lactate dehydrogenase B chain annexin I annexin III 14-3-3 protein sigma nicotinamide N-methyltransferase ubiquitin carboxyl hydrolase-L1 peroxiredoxine-6 catechol-O-methyltransferase glutathione S-transferase pi interleukin-18 precursor peroxiredoxin-2 peroxiredoxin-1 phosphatidylethanolamine binding protein cyclophilin A thioredoxin calgizzarin calcyclin ubiquitin

P11413 P08758 P07195 P04083 P12429 P31947 P40261 P09936 P30041 P21964 P09211 Q14116 P32119 Q06830 P30086 P05092 P10599 P31949 P06703 P02248

0.24 ( 0.08 7.50 ( 1.3 6.20 ( 0.6 nd in MCF-7 nd in MCF-7 2.70 ( 0.3 nd in MCF-7 nd in MCF-7 1.50 ( 0.1 nd in MCF-7/ADR nd in MCF-7 nd in MCF-7 0.17 ( 0.06 1.70 ( 0.06 0.30 ( 0.06 0.40 ( 0.10 1.50 ( 0.20 3.90 ( 0.05 4.70 ( 1.00 1.80 ( 0.30

metabolism Ca2+ binding metabolism Ca2+ binding Ca2+ binding cell cycle detoxification protein recycling detoxification detoxification detoxification signaling detoxification detoxification kinase inhibitor chaperon redox cycle Ca2+ binding Ca2+ binding protein recycling

a See Figure 2 for spot name. b Differential expression: the ratio of the relative expression of a spot in the comparative image (MCF7/ADR) to its relative expression in the reference image (MCF-7). The values represent the average and the standard deviation of three experiments. nd: not detected.

Alteration in the pentose phosphate pathway reported earlier by Yeh et al.10 was also confirmed here. In this previous study, the Vmax activity for glucose-6-phosphate dehydrogenase (G6PD) was found to be 50-fold lower in MCF-7/ADR relative to the parental cell line.10 This change is quite important and should be reflected at the protein expression level. Indeed, using comparative gel images we found that G6PD expression was 4 to 5 times lower in MCF-7/ADR, compared to the parental cell line MCF-7 (Figure 2, Table 1). As the earlier workers have pointed out, G6PD is a key enzyme in the pentose phosphate pathway and a major source of NADPH. The 6-phosphoglyconate dehydrogenase, another NADPH producing enzyme acting downstream of G6PD in the pentose pathway was also reported to be moderately less active in MCF-7/ADR than in MCF-7.10 NADPH is normally used by glutathione reductase to convert oxidized glutathione (GSSG) to reduced glutathione. Therefore, these NADPH producing enzymes should be rather up-regulated in MCF-7/ADR to produce more NADPH and protect cells from oxidative damage. But this was not the case, 406

Journal of Proteome Research • Vol. 3, No. 3, 2004

as demonstrated in either the previous10 or the present study. Overall, the decrease in the expression of the NADPH producing enzymes (e.g., G6PD) and PDX2 in MCF-7/ADR remains somewhat surprising and needs more detailed studies in order to explain this unexpected phenomenon. In addition to the differentially expressed proteins discussed above, several other proteins were found remarkably altered in their expression in MCF-7/ADR. The most striking changes found in MCF-7/ADR were detected in the acidic part of the gel slab (Figure 3). In addition to GST-pi discussed above, three other spots in this section were observed only in MCF-7/ADR and not in the parental cell line MCF-7. These spots were identified as ubiquitin carboxyl hydrolase isoenzyme L1 (UCHL1), also known as PGP9.5; nicotinamide N-methyltransferase (NNMT); and interleukin-18 precursor (IL-18). Catechol-Omethyltransferase was detected in the parental line, but was found to be strongly decreased and not detectable in MCF-7/ ADR (Figure 3). Other proteins were found either increased or decreased in MCF-7/ADR relative to MCF-7. The increased

research articles

Protein Expression in Adriamycin Resistant MCF-7

Figure 3. Close-up image of the acidic part of the 2-DG maps of MCF-7 (left) and MCF-7/ADR (right). Black circles: proteins that are up-regulated in MCF-7/ADR; Yelllow circles: proteins that are down-regulated in MCF-7/ADR; Red circles: proteins that are uniquely expressed in MCF-7/ADR. ANX5 and ANX3: annexin V and III respectively; COMT: catechol-O-methyltransferase; GSTpi: glutathione S-transferase pi class; IL18: interleukine-18 precursor; NNMT: N-nicotinamide methyltransferase; PDX2: peroxiredoxine-2; UCH-L1: ubiquitin carboxyl hydrolase L1 also known as PGP9.5.

proteins included annexins III and V and lactate dehydrogenase B (LDH-B) and the decreased proteins include adenine phosphoribosyltransferase (APRT) and PDX2 discussed above. MCF-7/ADR cells have markedly increased expression of annexin isoforms relative to MCF-7 cells. Annexin V was increased by a factor of 7, whereas annexin III and I (see Figure 2) were expressed only in MCF-7/ADR. The annexins are a Ca2+ binding protein family and their function is not well understood in cancer cells. However, they have been found overexpressed in a number of cancer cells (for a review see ref 23). Sinha et al.24 also reported a differential overexpression of annexin I in a gastric carcinoma cell line resistant to daunorubicin. However, a rational correlation with drug-resistance remains to be established. L-Lactate dehydrogenase B subunit (LDH-B), also known as LDH-H, was found to be up-regulated by a factor of 7 in MCF-7/ADR relative to MCF-7 (Figure 3, Table 1). This protein is a key enzyme in anaerobic cellular metabolism it catalyses the transformation of pyruvate to lactate. Its up-regulation has been reported as a consequence of exposing rat heart cells to doxorubicin25 and it was suggested that this is indicative of oxidative stress. Another striking change found in MCF-7/ADR is the overexpression of ubiquitin carboxyl hydrolase L1 (UCH-L1) (Figure 3). This protein has been reported to be predominately expressed in neuronal tissue26 and to possess both deubiquinating and ubiquitinating characteristics.27,28 Other studies have shown that the increased expression of this protein is strongly associated with the pathological stage of solid tumors of lung,29 pancreas,30 and colon.31 This is the first time that overexpression of UCH-L1 has been detected in MCF-7 cell lines. Among the four resistant lines under study in this laboratory, only the adriamycin resistant cell line strongly expressed this protein. It was not detected in MCF-7 lines selected for resistance to melphalan, etoposide or mitoxantrone (Figure 4). The contributions of this protein to resistance to adriamycin await further investigation.

Figure 4. Close-up image of the 2-DG maps of the different drug resistant MCF-7 cell lines. The red arrow shows the area of the spot corresponding to ubiquitin carboxyl hydrolase isoenzym L1 (PGP9.5). MCF-7: parental cell line; MelR MCF-7: melphalan resistant MCF-7; MCF-7/MX: mitoxantrone resistant MCF-7; MCF7/VP: etoposide resistant MCF-7; MCF-7/ADR: adriamycin resistant MCF-7.

Interleukin-18 (IL-18) was found uniquely expressed in MCF7/ADR and not in the parental MCF-7 cell line (Figure 3). This protein was recently found to be up-regulated in certain human tumors with metastatic characteristic.32 But again, as in the case of UCH-L1, the expression of this IL-18 in MCF-7/ADR and its correlation with resistance to adriamycin need more careful investigation. Finally, two methyl transferases were identified as altered, however in opposite directions. Like UCH-L1, nicotinamide N-methyltransferase is significantly overexpressed in MCF-7/ ADR, though not in the cytosols of three other drug resistant MCF-7 lines (not shown). This protein is believed to catalyze the N-methylation of nicotinamide pyridines and it may be involved in the biotransformation of many drugs and xenobiotics.33 The expression of catechol-O-methyltransferase was dramatically attenuated in MCF-7/ADR compared to MCF-7 (Figure 3). This enzyme is believed to be involved in estrogen metabolism.34 Its absence in MCF-7/ADR may be related to the fact that these cells are estrogen receptor negative35 and not to the fact that these cells are resistant to adriamycin. However, this speculation needs to be supported by more elaborated experiment.

Conclusions The comparative proteomics study presented here reveals that several proteins with different functions are altered in their expression in MCF-7/ADR cells. Whether each of these alterations is related to resistance to adriamycin remains unclear. For instance the increased expression of GST-pi was unique to MCF-7/ADR and was not observed in any of the other drug Journal of Proteome Research • Vol. 3, No. 3, 2004 407

research articles resistant cell lines studied, even in the MCF-7 cell line resistant to mitoxantrone. The structures and supposed mechanisms of mitoxantrone and adriamycin are closely related. They are metabolized the same way in the cells. So, one might expect that MCF-7/Mx should also have a high level of GST-pi. But it is not the case, as revealed by comparative 2-DG maps (Figure 4). It is really hard and not straightforward to correlate the alteration of the expression of every single protein found in MCF-7/ADR to resistance to adriamycin. This unique protein expression pattern of MCF-7/ADR may be just a phenotype of this cell line and has no direct contribution to the resistance of these cells to adriamycin except may be for some proteins. The divergence of MCF-7/ADR from the other drug resistant cell lines derived from MCF-7 is not completely surprising, since this cell line has a very different chromosomal karyotype than the parental MCF-7 cells.2,36 In addition, DNA fingerprint studies, clustered this cell line far apart from their parental MCF-7 cell line.37 Whether these MCF-7/ADR cells are a result of transformation of MCF-7 cells by adriamycin or of a selection of a subclone inherently present among MCF-7 cells36 remain to be clarified. A proteome phenotyping study, using other adriamycin resistant cancer cell lines, could help answering such a question. The next step is to include examination of the cytosolic proteome of other adriamycin resistant cell lines and see if there are similarities in their protein expression patterns. This may lead to better characterization of this MCF-7/ADR cell line now named NCI-ADR-RES.13

Acknowledgment. This work was supported by the National Institute of Health (GM 21248). We thank Profs. K. H. Cowan, P. Gutierrez, and J. A. Moscow for MCF-7 cell lines. We also thank O. Glister and V. Carroll for maintaining cell cultures. References (1) Batist, G.; Tulpule, A.; Sinha, B. K.; Katki, A. G.; Myers, C. E.; Cowan, K. H. J. Biol. Chem. 1986, 261, 15 544-15 549. (2) Fairchild, C. R.; Ivy, S. P.; Kao-Shan, C.-S.; Wang-Peng, J.; Rosen, N.; Israel, M. A.; Melera, P. W.; Cowan, K. H.; Golsmith, M. E. Isolation of amplified and overexpressed DNA sequences from adriamycin-resistant human breast cancer cells. Cancer Res. 1987, 47, 5141-5148. (3) Senior, A. E.; Bhagat, S. P-glycoprotein shows strong catalytic cooperativity between the two nucleotide sites. Biochemistry 1998, 37, 831-836. (4) Gottesman, M. M.; Fojo, T.; Bates, S. E. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat. Rev. Cancer 2002, 2, 48-58. (5) Simon, S. M.; Shindler, M. Cell biological mechanism of multidrug resistance in tumors. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 34973504. (6) Moscow, J. A.; Cowan, K. H. Multidrug resistance. J. Natl. Cancer. Inst. 1988, 80, 14-20. (7) Kramer, R. A.; Zakher, J.; Kim, G. Role of glutathione redox cycle in acquired and de novo multidrug resistance. Science 1988, 241, 694-697. (8) Cowan, K. H.; Batist, G.; Tulpule, A.; Sinha, B. K.; Myers, C. E. Similar biochemical changes associated with multidrug resistance in human breast cancer cells and carcinogen-induced resistance to xenobiotics in rats. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 93289332. (9) Gaudiano, G.; Koch, T. H.; Le Bello, M.; Nuccetelli, M.; Ravagnan, G.; Serafino, A.; Sinibaldi-Vallebona, P. Lack of glutathione conjugation to adriamycin in human breast cancer MCF-7/DOX cells. Biochem. Pharmacol. 2000, 60, 1915-1923. (10) Yeh, C. C.; Ochipinti, S. J.; Cowan, K. H.; Chabner, B. A.; Myers, C. E. Adriamycin resistance in human tumor cells associated with marked alterations in the regulation of the hexose monophosphate shunt and its response to oxidant stress. Cancer Res. 1987, 47, 5994-5999.

408

Journal of Proteome Research • Vol. 3, No. 3, 2004

Gehrmann et al. (11) Pirinia, F.; Breuleux, M.; Schneider, E.; Hochmeister, M.; Bates, S. E.; Marti, A.; Hotz, M. A.; Betticher, D. C.; Norner, M. Uncertain identity of doxorubicin-resistant MCF-7 cell lines expressing mutated p 53. J. Natl. Cancer Inst. 2000, 92, 1535-1536. (12) Mehta, K.; Devarajan, E.; Chen, J.; Multani, A.; Pthak, S. Multidrugresistant MCF-7 cells: an identity crisis. J. Natl. Cancer Inst. 2002, 94, 1652-1653. (13) Scudiero, D. A.; Monks, A.; Sausville, V. A. Cell line in the NCI anticancer screen [Letter]. J. Natl. Cancer Inst. 1998, 90, 862. (14) Schneider, E.; Horton, J. K.; Yang, C.-H.; Nakagawa, M.; Cowan, K. H. Multidrug resistance-associated protein gene overexpression and reduced drug sensitivity of topoisomerase II in human breast carcinoma MCF7 cell line selected for etoposide resistance. Cancer Res. 1994, 54, 152-158. (15) Nakagawa, M.; Schneider, E.; Dixon, K. H.; Horton, J.; Kelley, K.; Morrow, C.; Cowan, K. H. Reduced intracellular drug accumulation in the absence of P-glycoprotein (mdr1) overexpression in mitoxantrone-resistant human MCF-7 breast cancer cells. Cancer Res. 1992, 52, 6175-6181. (16) Moscow, J. A.; Swanson, C. A.; Cowan, K. H.; Decreased melphalan accumulation in human breast cancer cell line selected fro resistance to melphalan Br. J. Cancer 1993, 68, 732-737. (17) Ramsby, M. L.; Makowski, G. L. Differential detergent fractionation of eukaryotic cells. In Methods in Molecular Biology; Link, A. J.; Ed.; Humana Press: Totowa, NJ, 1999; Vol. 112, pp 53-66. (18) Hathout, Y.; Riordan, K.; Gehrmann, M.; Fenselau, C. Differential protein expression in the cytosol fraction of an MCF-7 breast cancer cell line selected for resistance toward melphalan. J. Proteome Res. 2002, 1, 435-442. (19) Jensen, O. N.; Wilm, M.; Shevchenko, A.; Mann, M. Sample preparation methods for mass spectrometry peptide mapping directly from 2-DE gels. In Methods in Molecular Biology; Link, A. J., Ed.; Humana Press: Totowa, NJ, 1999; Vol. 112, pp 513530. (20) Sinha, B. K.; Katki, A. G.; Batist, G.; Cowan, K. H.; Myers, C. E. Differential formation of hydroxyl radicals by adriamycin in sensitive and resistant MCF-7 human breast tumor cells: implication for mechanism of action. Biochemistry 1987, 26, 37763781. (21) Sinha, B. K.; Mimnaugh, E. G.; Rajagopalan, S.; Myers, C. E. Adriamycin activation and oxygen free radical formation in human breast tumor cells: protective role of glutathione peroxidase in adriamycin. Cancer Res. 1989, 49, 3844-3848. (22) Doroshow, J. H. Effect of anthracycline antibiotics on oxygen radical formation in rat heart. Cancer Res. 1983, 43, 460-472. (23) Gerke, V.; Moss, S. E. Annexins: from structure to function. Physiol. Rev. 2002, 82, 331-371. (24) Sinha, P.; Hu¨tter, G.; Ko¨ttgen, E.; Dietel, M.; Schadendorf, D.; Lage, H. Increased expression of annexin I and thioredoxin detected by two-dimensional gel electrophoresis of drug resistant human stomach cancer cells. J. Biochem. Biophys. Methods 1998, 37, 105-116. (25) Howrad, P. H.; Payne, S.; Wong, L.; Gonzalez, B.; Lewis, W. Lactate dehydrogenase activity in cultured neonatal rat heart cells exposed to doxorubicin. Exp. Mol. Pathol. 1988, 48, 311-316. (26) Wilkinson, K. D.; Lee, K.; Deshpande, S.; Duerksen-Hughes, P.; Boss, J. M.; Pohl, J. The neuron-specific protein PGP9.5 is a ubiquitin carboxyl-terminal hydrolase. Science 1989, 246, 670673. (27) Larsen, C. N.; Krantz, B. A.; Wilkinson, K. D. Substrate specificity of deubiquitinating enzymes: ubiquitin C-terminal hydrolases. Biochemistry 1998, 37, 3358-3368 (28) Liu, Y.; Fallon, L.; Lashuel, H. A.; Lie, Z.; Jr Lansbury, P. T. The UCH-L1 gene encodes two opposing enzymatic activities that affect R-synuclein degradation and parkinson’s disease susceptibility. Cell 2002, 111, 209-218. (29) Hibi, K.; Westra, W. H.; Borges, M.; Goodman, S.; Sidransky, D.; Jen, J. PGP9.5 as a candidate tumor marker for nonsmall-cell lung cancer. Am. J. Pathol. 1999, 155, 711-715. (30) Tezel, E.; Hibi, K, Nagasaka, T.; Nakao, A. PGP9.5 as prognostic factor in pancreatic cancer. Clin. Cancer Res. 2000, 6, 47644767. (31) Yamazaki, T.; Hibi, K.; Takase, T.; Tezel, E.; Nakayama, H.; Kasai, Y.; Ito, K.; Akiyama, S.; Nagasaka, T.; Nakao, A. PGP9.5 as a marker for invasive colorectal cancer. Clin. Cancer Res. 2001, 8, 192195. (32) Jiang, D.; Ying, W.; Lu, Y.; Wan, J.; Zhai, Y.; Liu, W.; Zhu, Y.; Qiu, Z.; Qian, X.; He, F. Identification of metastasis-associated proteins by proteomics analysis and functional exploration of interleukin18 in metastasis. Proteomics 2003, 3, 724-737.

research articles

Protein Expression in Adriamycin Resistant MCF-7 (33) Aksoy, S.; Szunlanski, C. L.; Weinshilbaum, R. M. Human liver nicotinamide N-methyltransferase. J. Biol. Chem. 1994, 269, 14 835-14 840. (34) Dawling, S.; Roodi, N.; Mernaugh, R. L.; Wang, X.; Parl, F. F. Catechol-O-methyltransferase (COMT)-mediated metabolism of catechol-estrogen: comparison of wild-type and variant COMT isoforms. Cancer Reas. 2001, 61, 6716-6722. (35) Vikers, P. J.; Dickson, R. B.; Shoemaker, R.; Cowan, K. H. A multidrug-reistant MCF-7 human breast cnacer cell line which exhibits cross-resistance to antiestrogens and hormone-independent tumor growth in vivo. Mol. Endocrinol. 1988, 2, 886892.

(36) Devarajan, E.; Chen, J.; Multani, A. S.; Ptahak, S.; Sahin, A. A.; Mehta, K. Human breast cancer MCF-7 cell line contains inherently drug-resistant subclones with distinct genotypic and phenotypic features. Int. J. Oncol. 2002, 20, 913-920. (37) Ross, D. T.; Scerf, U.; Eisen, M. B.; Perou, C. M.; Rees, C.; Spellman, P.; Iyer, V.; Jeffrey, S. S.; Van de Rijin, M.; Waltham, M.; Pergamenschikov, A.; Lee, J. C. F.; Lashkari, D.; Shalon, D.; Myers, T. G.; Weinstein, J. N.; Botstein, D.; Brown, P. O. Systematic variation in gene expression patterns in human cancer cell lines. Nat. Genet. 2000, 24, 227-235.

PR0340577

Journal of Proteome Research • Vol. 3, No. 3, 2004 409