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Proteome Analysis of Vinca Alkaloid Response and Resistance in Acute Lymphoblastic Leukemia Reveals Novel Cytoskeletal Alterations

2003; Elsevier BV; Volume: 278; Issue: 46 Linguagem: Inglês

10.1074/jbc.m303378200

1083-351X

Nicole M. Verrills, Bradley J. Walsh, Gary S. Cobon, Peter G. Hains, Maria Kavallaris,

Microtubule and mitosis dynamics

Vinca alkaloids are used widely in the treatment of both childhood and adult cancers. Their cellular target is the β-tubulin subunit of α/β-tubulin heterodimers, and they act to inhibit cell division by disrupting microtubule dynamics. Despite the effectiveness of these agents, drug resistance is a major clinical problem. To identify the underlying mechanisms behind vinca alkaloid resistance, we have performed high resolution differential proteome analysis. Treatment of drug-sensitive human leukemia cells (CCRF-CEM) with vincristine identified numerous proteins involved in the cellular response to vincristine. In addition, differential protein expression was analyzed in leukemia cell lines selected for resistance to vincristine (CEM/VCR R) and vinblastine (CEM/VLB100). This combined proteomic approach identified 10 proteins altered in both vinca alkaloid response and resistance: β-tubulin, α-tubulin, actin, heat shock protein 90β, 14-3-3τ, 14-3-3ϵ, L-plastin, lamin B1, heterogeneous nuclear ribonuclear protein-F, and heterogeneous nuclear ribonuclear protein-K. Several of these proteins have not previously been associated with drug resistance and are thus novel targets for elucidation of resistance mechanisms. In addition, seven of these proteins are associated with the tubulin and/or actin cytoskeletons. This study provides novel insights into the interrelationship between the microtubule and microfilament systems in vinca alkaloid resistance. Vinca alkaloids are used widely in the treatment of both childhood and adult cancers. Their cellular target is the β-tubulin subunit of α/β-tubulin heterodimers, and they act to inhibit cell division by disrupting microtubule dynamics. Despite the effectiveness of these agents, drug resistance is a major clinical problem. To identify the underlying mechanisms behind vinca alkaloid resistance, we have performed high resolution differential proteome analysis. Treatment of drug-sensitive human leukemia cells (CCRF-CEM) with vincristine identified numerous proteins involved in the cellular response to vincristine. In addition, differential protein expression was analyzed in leukemia cell lines selected for resistance to vincristine (CEM/VCR R) and vinblastine (CEM/VLB100). This combined proteomic approach identified 10 proteins altered in both vinca alkaloid response and resistance: β-tubulin, α-tubulin, actin, heat shock protein 90β, 14-3-3τ, 14-3-3ϵ, L-plastin, lamin B1, heterogeneous nuclear ribonuclear protein-F, and heterogeneous nuclear ribonuclear protein-K. Several of these proteins have not previously been associated with drug resistance and are thus novel targets for elucidation of resistance mechanisms. In addition, seven of these proteins are associated with the tubulin and/or actin cytoskeletons. This study provides novel insights into the interrelationship between the microtubule and microfilament systems in vinca alkaloid resistance. Cancer is the most common cause of death from disease in children in developed countries, and the most frequent childhood malignancy is acute lymphoblastic leukemia (ALL). 1The abbreviations used are: ALL, acute lymphoblastic leukemia; DE, differential expression; IPG, immobilized pH gradient; mAb, monoclonal antibody; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MDR, multidrug resistance; MS, mass spectrometry; MS/MS, tandem MS; PMF, peptide mass fingerprinting; VCR, vincristine; VLB, vinblastine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TCTP, translationally controlled tumor protein; hnRNP, heterogeneous nuclear ribonuclear protein; MAP, microtubule-associated protein.1The abbreviations used are: ALL, acute lymphoblastic leukemia; DE, differential expression; IPG, immobilized pH gradient; mAb, monoclonal antibody; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MDR, multidrug resistance; MS, mass spectrometry; MS/MS, tandem MS; PMF, peptide mass fingerprinting; VCR, vincristine; VLB, vinblastine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TCTP, translationally controlled tumor protein; hnRNP, heterogeneous nuclear ribonuclear protein; MAP, microtubule-associated protein. With current treatment regimes, the majority of patients will be long term survivors, however, almost one-third of ALL patients relapse and most of those die due to the development of drug resistance. Vinca alkaloids, such as vincristine (VCR) and vinblastine (VLB), are natural product drugs used extensively in the treatment of ALL (1Pui C.H. Evans W.E. N. Engl. J. Med. 1998; 339: 605-615Crossref PubMed Scopus (799) Google Scholar). These agents target the β-tubulin subunit of α/β-tubulin heterodimers, inhibiting the addition of heterodimers onto growing microtubules and, hence, resulting in depolymerization of microtubules (2Jordan M.A. Wilson L. Curr. Opin. Cell Biol. 1998; 10: 123-130Crossref PubMed Scopus (597) Google Scholar). Microtubules are dynamic structures that are constantly growing and shortening (3Mitchison T.J. Kirschner M. Nature. 1984; 312: 237-242Crossref PubMed Scopus (2269) Google Scholar), and microtubule dynamics play an important role in many cellular events, including signal transduction, intracellular transport, cellular organization, and cell division. As such, the tubulin/microtubule system remains an important target for anticancer therapy (4Jordan A. Hadfield J.A. Lawrence N.J. McGown A.T. Med. Res. Rev. 1998; 18: 259-296Crossref PubMed Scopus (612) Google Scholar). The development of resistance to chemotherapy agents poses a major clinical problem. Many cells develop resistance not only to the selecting agent but also exhibit cross-resistance to other structurally unrelated compounds. This classic multidrug resistance (MDR) phenotype is often characterized by overexpression of the transmembrane efflux pump P-glycoprotein (5Roninson I.B. Biochem. Pharmacol. 1992; 43: 95-102Crossref PubMed Scopus (180) Google Scholar) or by expression of multidrug resistance-associated proteins (6Kavallaris M. Anticancer Drugs. 1997; 8: 17-25Crossref PubMed Scopus (62) Google Scholar, 7Ishikawa T. Kuo M.T. Furuta K. Suzuki M. Clin. Chem. Lab. Med. 2000; 38: 893-897Crossref PubMed Scopus (45) Google Scholar, 8Suzuki T. Nishio K. Tanabe S. Curr. Drug. Metab. 2001; 2: 367-377Crossref PubMed Scopus (62) Google Scholar). However, classic MDR is not the only mechanism of resistance to vinca alkaloids. Alterations to the drug target, tubulin, and tubulin-associated proteins, have been associated with vinca alkaloid resistance (Reviewed in Ref. 9Drukman S. Kavallaris M. Int. J. Oncol. 2002; 21: 621-628PubMed Google Scholar). Our laboratory has identified multiple microtubule changes in vincristine- (CEM/VCR R) and vinblastine- (CEM/VLB100) resistant leukemia cells (10Kavallaris M. Tait A.S. Walsh B.J. He L. Horwitz S.B. Norris M.D. Haber M. Cancer Res. 2001; 61: 5803-5809PubMed Google Scholar). As tubulin is the target for vinca alkaloids, the observed changes are hypothesized to favor more stable microtubules or affect the microtubule dynamics such that vinca alkaloid effectiveness is decreased. Coordinated interaction of microtubules and other cytoskeletal proteins is crucial for many cellular processes, including cell division; thus we proposed that other cytoskeletal elements are highly likely to be altered in these cells. To identify protein changes associated with vinca alkaloid resistance, the expression of cellular proteins in the drug-sensitive and drug-resistant cell lines was analyzed using a differential proteomic approach. In addition, to further decipher the mechanisms of resistance to VCR in ALL, we must improve our understanding of the mechanism of action and cellular response to this agent. To this aim, we have analyzed protein expression changes in leukemia cells treated with VCR. 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We and others have utilized isoelectric focusing and two-dimensional gel electrophoresis (2D-PAGE) to identify altered tubulin proteins in antimicrotubule drug-resistant cells (23Cabral F. Abraham I. Gottesman M.M. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4388-4391Crossref PubMed Scopus (75) Google Scholar, 24Boggs B. Cabral F. Mol. Cell. Biol. 1987; 7: 2700-2707Crossref PubMed Scopus (41) Google Scholar, 25Burkhart C.A. Kavallaris M. Band Horwitz S. Biochim. Biophys. Acta. 2001; 1471: O1-O9PubMed Google Scholar, 26Verdier-Pinard P. Wang F. Martello L. Burd B. Orr G.A. Horwitz S.B. Biochemistry. 2003; 42: 5349-5357Crossref PubMed Scopus (93) Google Scholar); however, no studies to date have analyzed global protein changes in resistance to the vinca alkaloids VCR and VLB. In the present study, changes in the expression of numerous proteins were identified in vinca alkaloid-resistant cells. Of these, 15 proteins are associated with the tubulin and/or actin cytoskeleton. These proteins are all potential targets for involvement in the resistance phenotype of tubulin-targeted anticancer agents. To highlight those proteins most likely to play a direct involvement in the resistance phenotype we have combined the analysis of drug-sensitive and -resistant cell lines, with protein expression changes in vinca alkaloid-treated cells. This novel approach has identified 10 proteins, which are involved in both drug response and drug resistance, and are hence potential targets for improved treatment of relapsed disease, and thus worthy of further characterization. Of these proteins, seven are associated with the tubulin and/or actin cytoskeletons. Cell Culture—Human T-cell acute lymphoblastic leukemia cells, CCRF-CEM, and drug-resistant sublines, CEM/VCR R (vincristine-selected), and CEM/VLB100 (vinblastine-selected), were maintained in RPMI 1640 containing 10% fetal calf serum as suspension cultures. The CEM/VCR R are 22,600-fold (27Haber M. Norris M.D. Kavallaris M. Bell D.R. Davey R.A. White L. Stewart B.W. Cancer Res. 1989; 49: 5281-5287PubMed Google Scholar) and CEM/VLB100 are 200- to 800-fold (28Beck W.T. Cirtain M.C. Look A.T. Ashmun R.A. Cancer Res. 1986; 46: 778-784PubMed Google Scholar) resistant to VCR and VLB, respectively. Mid-log phase cells were harvested for protein analysis by centrifugation at 1500 rpm for 5 min and washed three times in phosphate-buffered saline. Vincristine Treatment of CEM Cells—CCRF-CEM cells were seeded at 3 × 105 cells/ml 24 h before adding drug so as to ensure cells were in mid-log phase of cell growth. Cells were treated with 0, 2, 4, or 8 nm VCR for 24 h. Cells from each treatment were counted using the trypan blue exclusion assay (29Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Siedman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., Canada1988Google Scholar), then harvested for protein analysis as described above. Two-dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE)— Cell pellets were resuspended in lysis buffer (7 m urea, 2 m thiourea, 2% CHAPS, 1% sulfobetaine-3–10, 1% amidosulfobetaine-14, 2 mm tributylphosphine, 65 mm dithiothreitol, 1% carrier ampholyte (pH range 3–10), 1% carrier ampholyte (pH range 4–6), 0.01% bromphenol blue) to a final concentration of 1 mg/ml as determined by amino acid analysis (30Cordwell S.J. Wilkins M.R. Cerpa-Poljak A. Gooley A.A. Duncan M. Williams K.L. Humphery-Smith I. Electrophoresis. 1995; 16: 438-443Crossref PubMed Scopus (120) Google Scholar). Cells were lysed by pulse sonication twice for 10 s on ice. Endonuclease (1 unit/μg of protein; Sigma) was added and incubated at room temperature for 30 min. Protein extracts were centrifuged at 18,000 × g for 12 min, and the supernatant was collected. Narrow range immobilized pH gradient (IPG) strips, pH 4.5–5.5 (Amersham Biosciences, Uppsala, Sweden), were rehydrated in 500 μl of lysis buffer. Protein (100 μg for analytical and 500 μg for preparative gels) was cup-loaded and isoelectric focused for 150,000 Vh on a Multiphor II apparatus (Amersham Biosciences). Alternatively, 60 μg of protein in 500 μl of lysis buffer was in-gel-rehydrated in pH 4–7 IPGs and focused for 80,000 Vh. Second dimension SDS-PAGE was performed using 8–18% T polyacrylamide gels as previously described (31Verrills N.M. Harry J.H. Walsh B.J. Hains P.G. Robinson E.S. Electrophoresis. 2000; 21: 3810-3822Crossref PubMed Scopus (12) Google Scholar). Analytical gels were stained with SYPRO Ruby® (Bio-Rad) or transferred to nitrocellulose (see below). Preparative gels were stained with colloidal Coomassie Blue G250. Levels of protein expression were determined on SYPRO Ruby®-stained gels using the Z3 Image Analysis program (Compugen, Israel). Expression values (q value) were obtained from at least three independent two-dimensional gels, and differences in protein expression between the control CCRF-CEM cells and VCR-treated CEM cells, the CEM/VCR R or CEM/VLB100 cell lines, were determined by dividing the q value of the test sample by the control CCRF-CEM sample. Student's t tests were used to determine statistical significance (p < 0.05). Immunoblotting—For specific protein detection analytical two-dimensional gels were transferred to nitrocellulose using standard methods. Total α-tubulin (Sigma clone DM1A) and class I β-tubulin were detected using monoclonal antibodies as previously described (10Kavallaris M. Tait A.S. Walsh B.J. He L. Horwitz S.B. Norris M.D. Haber M. Cancer Res. 2001; 61: 5803-5809PubMed Google Scholar). The class I β-tubulin Ab was kindly provided by Dr. R. Luduena, University of Texas, San Antonio, TX. The peptide used to raise this antibody is CEEAEEEA, corresponding to the C-terminal sequence of class I β-tubulin except for the C-terminal cysteine (32Roach M.C. Boucher V.L. Walss C. Ravdin P.M. Luduena R.F. Cell Motil. Cytoskeleton. 1998; 39: 273-285Crossref PubMed Scopus (69) Google Scholar). Acetylated α-tubulin was detected using a mAb (Sigma clone 6–11 B-1) at 1:1000 dilution. MALDI-TOF Mass Spectrometry Protein Identification—Spots were excised from Coomassie Blue-stained preparative gels, washed twice in 25 mm NH4HCO3, 50% acetonitrile, spun dry, and in-gel trypsin-digested in 10 ng/μl trypsin (Promega) in 25 mm NH4HCO3 for 16 h at 37 °C. Peptides were extracted from the gel with 50% (v/v) acetonitrile, 1% (v/v) trifluoroacetic acid solution. A 1-μl aliquot was spotted onto a sample plate with 1 μl of matrix (α-cyano-4-hydroxycinnamic acid, 8 mg/ml in 50% v/v acetonitrile, 1% v/v trifluoroacetic acid). Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry acquisition was performed on a TofSpec 2E mass spectrometer (Micromass, Manchester, UK) set to reflectron mode. Known trypsin auto-cleavage peptide masses (842.51 and 2211.10 Da) were used for a two-point internal calibration for each spectrum. Monoisotopic peptide masses were searched against the theoretical peptide masses of all human proteins in the Swiss-Prot and TrEMBL protein databases (www.expasy.org) using the MassLynx search program (Micromass, Manchester, UK). A minimum number of four peptides was required for a positive identification with a peptide mass tolerance of ±50 ppm and allowing for 1 missed cleavage. ESI-TOF Tandem Mass Spectrometry—For exact identification of class I β-tubulin, upon analysis of MALDI-TOF mass spectra, a peptide at 1301.7 Da was selected for amino acid sequencing by ESI-TOF MS/MS. After in-gel trypsin digestion, the peptides were purified using a porous R2 resin column (33Larsen M.R. Larsen P.M. Fey S.J. Roepstorff P. Electrophoresis. 2001; 22: 566-575Crossref PubMed Scopus (33) Google Scholar). The sample was then analyzed by ESI-TOF MS/MS using a Micromass Q-TOF MS, and data were manually acquired using borosilicate capillaries for nanospray acquisition. Data was acquired over the m/z range 400–1800 Da to select peptides for MS/MS analysis. After peptides were selected, the MS was switched to MS/MS mode, and data were collected over the m/z range 50–2000 Da with variable collision energy settings. The peptide sequence was compared with the human β-tubulin sequences in the Swiss-Prot database. Cellular Response to Vincristine—VCR is an antimitotic agent that induces mitotic arrest and cell death (34Jordan M.A. Himes R.H. Wilson L. Cancer Res. 1985; 45: 2741-2747PubMed Google Scholar). To elucidate the intermediate proteins involved in the cellular response to VCR, mid-log phase CCRF-CEM leukemia cells were treated with increasing concentrations of the drug for 24 h. The percentage of viable cells before and after treatment was determined by trypan blue exclusion (Fig. 1). As the concentration of VCR increases, the percentage of viable cells decreases. At the highest VCR concentration (8 nm) the percentage of viable cells is reduced to 58.2%. The VCR-treated cells display chromosome condensation indicative of mitotic arrest and at the higher concentrations show membrane blebbing and cell death (data not shown). To analyze the protein expression after VCR treatment, cellular proteins were separated by 2D-PAGE using pH 4–7 and narrow range pH 4.5–5.5 IPGs in the first dimension. Protein expression changes between the control and drug-treated cells were determined using the Z3 image analysis program. Differentially expressed proteins were excised from Coomassie Blue-stained gels and analyzed by MALDI-TOF mass spectrometry for protein identification. Proteins displaying a significant change in response to VCR treatment are listed in Table I (and Supplementary Fig. 1).Table IProtein changes in response to 24-h vincristine treatmentSpot no.aSpot numbers are shown on Supplementary Fig. 1.Acc. no.bSwiss-Prot accession number, accessible at www.expasy.org.Protein identificationProtein expressioncThe expression value is the "q" value determined by using the Z3 image analysis program and is the mean ± S.D. of three individual experiments. The ratio is the differential expression (DE) ratio, calculated by dividing the mean expression value of the protein in the VCR-treated cells by that of the control untreated cell line, CCRF-CEM. p values were determined by Student's t tests for each protein.CEM2 nm VCR4 nm VCR8 nm VCRq ± S.D.q ± S.D.Ratiop valueq ± S.D.Ratiop valueq ± S.D.Ratiop value1P54727RAD232274 ± 7911018 ± 3390.450.0267307 ± 3550.140.0039158 ± 1820.070.00202P08238HSP90 β (fragment, first half)0 ± 03P0886540 S ribosomal protein SA6357 ± 9225147 ± 9270.810.11373106 ± 9770.490.00292219 ± 4340.350.00024P0886540 S ribosomal protein SA1348 ± 6922695 ± 4452.000.01692384 ± 3771.770.03912114 ± 3651.570.09785P0886540 S ribosomal protein SA37 ± 43280 ± 1177.680.0079743 ± 21620.360.0007754 ± 10220.66<0.00016P0886540 S ribosomal protein SA0 ± 0116 ± 820.0300362 ± 810.0001723 ± 1990.00037P05209α-tubulin 1dThere are six known isotypes of α-tubulin with varying C-terminal sequences (26). The nomenclature used here is according to the Swiss-Prot entry; i.e. protein name: α-tubulin 1; gene name: TUBA1; C-terminal sequence: MAALEKDYEEVGVDSVEGEGEEEGEEEY. Protein name: α-tubulin 4; gene name: TUBA4; C-terminal sequence: MAALEKDYEEVGIDSYEDEDEGEE.1600 ± 1111591 ± 3950.990.9646556 ± 1400.35<0.0001545 ± 450.34<0.00018Q07244HNRNP K1665 ± 1981270 ± 2910.760.0658508 ± 2390.310.0003414 ± 1400.25<0.00019No ID1041 ± 177685 ± 2350.660.0521804 ± 3800.770.3030677 ± 600.650.008110Q07244HNRNP K1921 ± 5931409 ± 950.730.13881178 ± 5320.610.1114390 ± 2110.200.002811Q07244HNRNP K3998 ± 12173386 ± 7950.850.43191597 ± 7680.400.01571051 ± 2340.260.003112Q02790p591872 ± 2991293 ± 3640.690.0493906 ± 2190.480.0020650 ± 2470.350.000713P05218Class I β-tubulin235 ± 631000 ± 3104.270.00292394 ± 87010.210.00263155 ± 82213.460.000414 eProteins shown in italics did not significantly change with VCR treatment.Q99778Protein disulfide isomerase1867 ± 4632009 ± 3471.080.64092126 ± 8111.140.59992190 ± 6251.170.437715No ID1016 ± 108727 ± 1170.720.0108163 ± 2280.160.0005133 ± 1530.13<0.000116No ID737 ± 140508 ± 1830.690.0942346 ± 1740.470.0127287 ± 1490.390.004517P02570; P02571Actin (fragment-middle)0 ± 00 ± 0814 ± 54<0.00011426 ± 6880.006118P02570; P02571Actin (fragment-middle)0 ± 0118 ± 1380.1403405 ± 2240.0111928 ± 3660.002319P02570; P02571Actin (fragment-second half)0 ± 041 ± 470.13421093 ± 93<0.0001836 ± 2120.000220P20700Lamin B1 (fragment-first half)242 ± 43696 ± 1892.870.00341886 ± 6737.790.00281873 ± 5707.730.001321Q9UKK9ADP sugar pyrophosphatase1424 ± 105903 ± 1160.630.0006363 ± 4190.250.0027195 ± 2270.14<0.000122P02570; P02571Actin (fragment-first half)158 ± 77698 ± 1424.430.00051790 ± 38711.370.00022043 ± 71212.970.001923P02570; P02571Actin (fragment-first half)403 ± 2301476 ± 1053.660.00013092 ± 8077.670.00074138 ± 96410.270.000324No ID1099 ± 98938 ± 1240.850.0877758 ± 2050.690.0240379 ± 1050.35<0.000125No ID0 ± 00 ± 0630 ± 116<0.0001961 ± 2750.000426P3194614-3-3β204 ± 9848 ± 2864.160.00412327 ± 33311.42<0.00012702 ± 39813.26<0.000127No ID305 ± 151353 ± 651.160.5784773 ± 742.540.0014943 ± 3023.100.009128P2734814-3-3τ239 ± 105604 ± 1922.530.01561644 ± 2926.890.00011665 ± 1226.97<0.000129P13693TCTP3490 ± 4483446 ± 10070.990.93833651 ± 10621.050.79003224 ± 8730.920.606230P13693TCTP596 ± 432979 ± 1251.640.13951645 ± 4032.760.01201414 ± 3252.370.023231P4265514-3-3ϵ2866 ± 1582779 ± 5790.970.78152199 ± 6670.770.09971885 ± 3470.660.002132P2734814-3-3τ2295 ± 2091985 ± 2220.870.08871692 ± 3100.740.01821196 ± 2250.520.000433P4265514-3-3ϵ79 ± 34840 ± 50010.630.02302703 ± 28734.22<0.00012693 ± 73834.090.000434P52597HNRNP F1322 ± 2521052 ± 3010.800.2182967 ± 2910.730.1148677 ± 2620.510.012135P13796L-plastin4830 ± 7963741 ± 15310.770.25392227 ± 6580.460.0024949 ± 3450.200.000136P20700Lamin B1(full-length)3858 ± 6732701 ± 3680.700.02352434 ± 12520.630.09201041 ± 4120.270.000437P5503626 S proteasome1838 ± 1781984 ± 3261.080.46171098 ± 1080.600.00041317 ± 2310.720.011838P05209α-tubulin 1563 ± 161841 ± 3011.490.15561590 ± 1422.82<0.00012558 ± 7014.540.001439P05215α-tubulin 4442 ± 2261005 ± 2752.270.01952965 ± 5216.710.00012728 ± 2656.17<0.0001a Spot numbers are shown on Supplementary Fig. 1.b Swiss-Prot accession number, accessible at www.expasy.org.c The expression value is the "q" value determined by using the Z3 image analysis program and is the mean ± S.D. of three individual experiments. The ratio is the differential expression (DE) ratio, calculated by dividing the mean expression value of the protein in the VCR-treated cells by that of the control untreated cell line, CCRF-CEM. p values were determined by Student's t tests for each protein.d There are six known isotypes of α-tubulin with varying C-terminal sequences (26Verdier-Pinard P. Wang F. Martello L. Burd B. Orr G.A. Horwitz S.B. Biochemistry. 2003; 42: 5349-5357Crossref PubMed Scopus (93) Google Scholar). The nomenclature used here is according to the Swiss-Prot entry; i.e. protein name: α-tubulin 1; gene name: TUBA1; C-terminal sequence: MAALEKDYEEVGVDSVEGEGEEEGEEEY. Protein name: α-tubulin 4; gene name: TUBA4; C-terminal sequence: MAALEKDYEEVGIDSYEDEDEGEE.e Proteins shown in italics did not significantly change with VCR treatment. Open table in a new tab Altered Tubulin Proteins—Modifications to the drug target were observed in response to VCR treatment. Expression of a class I β-tubulin protein increased as the VCR concentration increased (Fig. 2). This protein spot is a more basic isoform 2In this context, isoform refers to isoelectric variants of known proteins. (pI ∼ 5.05) than the highly expressed major class I β-tubulin (pI ∼ 4.8) in these cells, suggesting this protein has been modified. The C-terminal region of α- and β-tubulin can undergo numerous post-translational modifications, including phosphorylation, polyglutamylation, polyglycylation, and detyrosination (35Rosenbaum J. Curr. Biol. 2000; 10: R801-R803Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The expected mass of the C-terminal tryptic peptide of class I β-tubulin is 6290.51 Da and contains 20 positively charged amino acids. A large peptide of this acidic nature is not amenable to the negative ion MALDI-TOF MS utilized in this study (36Rao S. Aberg F. Nieves E. Band Horwitz S. Orr G.A. Biochemistry. 2001; 40: 2096-2103Crossref PubMed Scopus (32) Google Scholar), and indeed this peptide was not observed in the mass spectra. Although β-tubulin isotypes are characterized by their C-terminal sequences (37Luduena R.F. Int. Rev. Cytol. 1998; 178: 207-275Crossref PubMed Google Scholar), there are a number of other amino acid differences between each isotype. The class I β-tubulin proteins were all identified by the presence of peptide masses 1301.71 Da (representing amino acids 47–62 only possible in class I); 1631.9 Da (representing amino acids 63–77 with methionine oxidized, only possible in class I and IV); and 1885.99 Da (representing amino acids 363–379 with methionine oxidized, only possible in class I and IV). For further confirmation, the peptide at 1301.71 Da was sequenced by ESI-TOF MS/MS and matched to that of class I β-tubulin (data not shown). Of 23 peptides identified in each class I β-tubulin protein spot, no changes in peptide mass were observed between the major intrinsic 3In this context, intrinsic refers to protein species expressed in parental drug-sensitive CCRF-CEM cells. class I β-tubulin protein and the more basic isoelectric variant induced with VCR treatment. Although MS identified the induced protein as class I β-tubulin, a mAb directed toward the C terminus of this tubulin isotype did not react with the modified protein (data not shown). Thus it is likely that the modification of the basic class I β-tubulin induced in response to VCR treatment is due to a modification at the C terminus. β-Tubulin forms a heterodimer with α-tubulin prior to assembly of microtubules. Modifications to α-tubulin were also observed in response to VCR. At least two more basic isoforms of α-tubulin 1 and one more basic isoform of α-tubulin 4, were induced by VCR treatment (Fig. 3A and Table I). The identification of these protein spots as α-tubulin was obtained by MALDI-TOF MS and was confirmed by immunoblotting with a total α-tubulin antibody (Fig. 3B). The intrinsic α-tubulin 1 and 4 proteins showed no significant change in response to VCR treatment (Fig. 3A; dashed arrows). Two proteins known to associate and/or bind to tubulin were also altered in response to VCR (Table I). FKBP59 (p59) associates with mitotic microtubules, and this protein showed a dose-response decrease in expression (Fig. 4A). The translationally controlled tumor protein (TCTP) is a calcium-binding protein that was recently identified as a microtubule-stabilizing protein (38Gachet Y. Tournier S. Lee M. Lazaris-Karatzas A. Poulton T. Bommer U.-A. J. Cell Sci. 1999; 112: 1257-1271Crossref PubMed Google Scholar, 39Yarm F.R. Mol. Cell. Biol. 2002; 22: 6209-6221Crossref PubMed Scopus (220) Google Scholar). Two isoforms of this protein are expressed in these cells, and the more acidic isoform (indicated by an arrow) increases in response to VCR (Fig. 4B). Protein Cleavage—Specific protein cleavage by caspases occurs during apoptosis (reviewed in Refs. 40Budihardjo I. Oliver H. Lutter M. Luo X. Wang X. Annu. Rev. Cell Dev. Biol. 1999; 15: 269-290Crossref PubMed Scopus (2240) Google Scholar and 41Takahashi A. Int. J. Hematol. 1999; 70: 226-232PubMed Google Scholar). At least 10 different protein spots were identified as cleaved actin polypeptides. Analysis of the peptide sequence coverage indicates that four proteins represent the N-terminal region, four others represent the C-terminal region, and two spots are the middle portion of the mature actin polypeptide. The four protein spots covering the N-terminal region of actin are shown in Fig. 5A. The tryptic peptides from these spots cover the first half of the actin protein (Fig. 5A, panel ii). Additionally, each polypeptide was analyzed by Edman sequencing (data not shown). Spots 1 and 2 were N-