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Originally published In Press as doi:10.1074/jbc.M303378200 on August 29, 2003

J. Biol. Chem., Vol. 278, Issue 46, 45082-45093, November 14, 2003
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Proteome Analysis of Vinca Alkaloid Response and Resistance in Acute Lymphoblastic Leukemia Reveals Novel Cytoskeletal Alterations*

Nicole M. Verrills{ddagger}§, Bradley J. Walsh§, Gary S. Cobon§, Peter G. Hains§, and Maria Kavallaris{ddagger}||

From the {ddagger}Children's Cancer Institute Australia for Medical Research, High St. (P. O. Box 81), Randwick, New South Wales 2031 and the §Australian Proteome Analysis Facility, Macquarie University, Sydney, New South Wales 2109, Australia

Received for publication, April 2, 2003 , and in revised form, August 20, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Vinca alkaloids are used widely in the treatment of both childhood and adult cancers. Their cellular target is the {beta}-tubulin subunit of {alpha}/{beta}-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: {beta}-tubulin, {alpha}-tubulin, actin, heat shock protein 90{beta}, 14-3-3{tau}, 14-3-3{epsilon}, 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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).1 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 (1). These agents target the {beta}-tubulin subunit of {alpha}/{beta}-tubulin heterodimers, inhibiting the addition of heterodimers onto growing microtubules and, hence, resulting in depolymerization of microtubules (2). Microtubules are dynamic structures that are constantly growing and shortening (3), 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 (4).

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 (5) or by expression of multidrug resistance-associated proteins (6–8). 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. 9). Our laboratory has identified multiple microtubule changes in vincristine- (CEM/VCR R) and vinblastine- (CEM/VLB100) resistant leukemia cells (10). 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.

Protein expression profiling has been used successfully to identify proteins associated with cellular processes such as apoptosis and necrosis (11–17) and in the analysis of resistance to cytotoxic drugs such as daunorubicin, mitoxantrone, vindesine, cisplatin, fotemustine, and etoposide (18–22). We and others have utilized isoelectric focusing and two-dimensional gel electrophoresis (2D-PAGE) to identify altered tubulin proteins in antimicrotubule drug-resistant cells (23–26); 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 (27) and CEM/VLB100 are 200- to 800-fold (28) 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 x 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 (29), 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 (30). 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 x 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 (31). 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 {alpha}-tubulin (Sigma clone DM1A) and class I {beta}-tubulin were detected using monoclonal antibodies as previously described (10). The class I {beta}-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 {beta}-tubulin except for the C-terminal cysteine (32). Acetylated {alpha}-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 ({alpha}-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 {beta}-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 (33). 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 {beta}-tubulin sequences in the Swiss-Prot database.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cellular Response to Vincristine—VCR is an antimitotic agent that induces mitotic arrest and cell death (34). 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).



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  		FIG. 1.
Vincristine treatment of CCRF-CEM leukemia cells induces cell death. CCRF-CEM human T-cell leukemia cells were treated with 0, 2 nM, 4 nM, and 8 nM VCR for 24 h. The percentage of viable cells was calculated after VCR treatment using trypan blue exclusion.

 


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  		TABLE I
Protein changes in response to 24-h vincristine treatment

 
Altered Tubulin Proteins—Modifications to the drug target were observed in response to VCR treatment. Expression of a class I {beta}-tubulin protein increased as the VCR concentration increased (Fig. 2). This protein spot is a more basic isoform2 (pI ~ 5.05) than the highly expressed major class I {beta}-tubulin (pI ~ 4.8) in these cells, suggesting this protein has been modified. The C-terminal region of {alpha}- and {beta}-tubulin can undergo numerous post-translational modifications, including phosphorylation, polyglutamylation, polyglycylation, and detyrosination (35). The expected mass of the C-terminal tryptic peptide of class I {beta}-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 (36), and indeed this peptide was not observed in the mass spectra. Although {beta}-tubulin isotypes are characterized by their C-terminal sequences (37), there are a number of other amino acid differences between each isotype. The class I {beta}-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 {beta}-tubulin (data not shown). Of 23 peptides identified in each class I {beta}-tubulin protein spot, no changes in peptide mass were observed between the major intrinsic3 class I {beta}-tubulin protein and the more basic isoelectric variant induced with VCR treatment. Although MS identified the induced protein as class I {beta}-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 {beta}-tubulin induced in response to VCR treatment is due to a modification at the C terminus.



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  		FIG. 2.
Vincristine induces modified class I {beta}-tubulin. A, CCRF-CEM cellular proteins were extracted after 24-h VCR treatment, separated by 2D-PAGE using pH 4.5–5.5 IPGs in the first dimension and 8–18%T SDS-PAGE and stained with SYPRO Ruby. Protein spots were identified using MALDI-TOF MS peptide mass fingerprinting. Arrow indicates a class I {beta}-tubulin protein that increases in expression with increasing VCR concentration. This protein is a lower molecular weight and more basic isoelectric variant than the highly expressed intrinsic class I {beta}-tubulin expressed in these cells. B, the expression value (q value) of the basic isoelectric variant class I {beta}-tubulin protein spot was determined using the Z3 image analysis program. Bars, S.E. of four individual experiments; **, p < 0.005.

 

{beta}-Tubulin forms a heterodimer with {alpha}-tubulin prior to assembly of microtubules. Modifications to {alpha}-tubulin were also observed in response to VCR. At least two more basic isoforms of {alpha}-tubulin 1 and one more basic isoform of {alpha}-tubulin 4, were induced by VCR treatment (Fig. 3A and Table I). The identification of these protein spots as {alpha}-tubulin was obtained by MALDI-TOF MS and was confirmed by immunoblotting with a total {alpha}-tubulin antibody (Fig. 3B). The intrinsic {alpha}-tubulin 1 and 4 proteins showed no significant change in response to VCR treatment (Fig. 3A; dashed arrows).



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  		FIG. 3.
Vincristine induces modified {alpha}-tubulin. A, cellular proteins were separated on pH 4–7 IPGs in the first dimension and 8–18%T SDS-PAGE. Dashed arrows indicate the intrinsic {alpha}-tubulin isoforms expressed in these cells. Filled arrows indicate the more basic {alpha}-tubulin isoforms induced upon VCR treatment. B, two-dimensional gels were transferred to nitrocellulose and probed with a total {alpha}-tubulin antibody.

 
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 (38, 39). 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).



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  		FIG. 4.
Vincristine treatment induces altered expression of tubulin-binding proteins. A, with increasing VCR concentration, the expression of p59 is down-regulated and B, The expression of the translationally controlled tumor protein (TCTP) is up-regulated. Expression levels of each protein were determined as described for Fig. 2. Bars, S.E. for four individual experiments. *, p < 0.05; **, p < 0.005.

 
Protein Cleavage—Specific protein cleavage by caspases occurs during apoptosis (reviewed in Refs. 40 and 41). 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-terminally blocked, indicating that these polypeptides begin at the N terminus where acetylation of the N-terminal methionine causes blockage of Edman chemistry. The N terminus of spot 3 begins at Ala19, thus this protein has undergone both N- and C-terminal cleavage.



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  		FIG. 5.
Vincristine treatment induces protein cleavage. CCRF-CEM cells were treated with various concentrations of vincristine (0–8 nM) for 24 h prior to separating and identifying total cellular proteins as described for Fig. 2. As the VCR concentration increased numerous actin cleavage products increased in intensity (spots 1–4, A(i)). Similarly, increased cleavage of lamin B1 (spot 1, B(i)) and HSP90{beta} (spots 1–3, C(i)) occurred with VCR treatment. Tryptic peptides from each of the protein spots numbered were analyzed by MALDI-TOF MS and peptide mass fingerprinting. The peptides identified from the four actin polypeptides that increase in a dose-response manner to VCR treatment are shown. The location of each peptide is shaded in gray on the full-length actin polypeptide sequence (A(ii)). The location of the lamin B1 peptides identified by MS are shaded in gray on the full-length lamin B1 polypeptide sequence (B(ii)). The location of the HSP90{beta} peptides identified by MS are shaded in gray on the full-length HSP90{beta} polypeptide sequence (C(ii)).

 
Low molecular weight fragments of two other proteins were identified in response to VCR treatment. Lamin B1 (Fig. 5B), a component of the nuclear lamina, and HSP90{beta} (Fig. 5C), a heat shock protein. The peptides identified from these protein spots both correspond to the N-terminal region of the polypeptide for lamin B1 and HSP90{beta}, respectively (Fig. 5, B (panel ii) and C (panel ii)).

Other Protein Changes—Numerous proteins showed altered expression in response to VCR treatment (Table I). The 40 S ribosomal protein SA (also called laminin binding receptor precursor) displayed altered expression of various isoelectric isoforms. With increasing VCR concentration, the major isoform of this protein is down-regulated. Concurrently, a more basic isoform, and two other minor, lower molecular weight isoforms, are up-regulated (Table I and Supplementary Fig. 1). The RAD23, 26 S proteasome subunit, and ADP sugar pyrophosphatase all display a dose-response decrease in expression with increasing VCR concentration (Table I and Supplementary Fig. 1).

Differential Cytoskeletal Protein Expression in Vinca Alkaloid-resistant Cell Lines—To characterize protein expression changes in cells resistant to vinca alkaloids, cellular protein expression of the CCRF-CEM cells was compared with the VCR-resistant CEM/VCR R cell line, and the VLB-resistant CEM/VLB100 cells. Image analysis identified 46 proteins displaying significant changes in expression in the VCR R and/or the VLB100 cells. Of these proteins, 42 were positively identified using MALDI-TOF MS peptide mass fingerprinting. In addition, a further 51 proteins were identified that did not change in expression in the resistant cell lines (Supplementary Table I and Supplementary Fig. 2). Differential expression analysis identified 15 proteins known to be associated with the microtubule and/or actin cytoskeletons (Table II). Seven proteins are associated with the microtubule cytoskeleton, including class II {beta}-tubulin, which is highly down-regulated in the VCR R and to a lesser extent in the VLB100 cells (Fig. 6). Altered expression of the intermediate family proteins lamin B1 and vimentin was also observed in the vinca alkaloid-resistant cells (Table II).


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  		TABLE II
Differentially expressed cytoskeletal proteins in vinca alkaloid-resistant cells

 



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  		FIG. 6.
Class II {beta}-tubulin is down-regulated in vinca alkaloid-resistant leukemia cells. Cellular proteins from untreated CCRF-CEM, CEM/VCR R, and CEM/VLB100 cells were separated by 2D-PAGE and identified as described for Fig. 2. A, arrow indicates class II {beta}-tubulin, which is down-regulated in both the VCR R and VLB100 cells. B, expression levels of each protein was determined as described for Fig. 2. Bars, S.E. for three individual experiments. *, p < 0.05; **, p < 0.005.

 
Proteins Altered in Both Drug Resistance and Drug Response—To identify protein changes common to both vinca alkaloid resistance and the cellular response to vinca alkaloid treatment, data from the VCR-treated and -resistant cell lines were combined (Table III). Ten proteins are altered in both drug response and in at least one of the resistant cell lines.


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  		TABLE III
Summary of protein changes in response to vincristine and in vinca alkaloid-resistant cells

 
The 14-3-3 family of proteins consist of at least seven different isotypes in humans, and are involved in cell signaling (42). In response to VCR treatment, two isotypes, 14-3-3{tau} and 14-3-3{epsilon}, were down-regulated (Fig. 7). These same two isotypes are differentially expressed in the VCR R cells, with 14-3-3{epsilon} up-regulated and 14-3-3{tau} down-regulated. In addition, VCR treatment induced the expression of three modified isoforms of 14-3-3{tau}, {epsilon}, and {beta}.



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  		FIG. 7.
14-3-3 proteins are altered in vinca alkaloid response and resistance. Two-dimensional gel region showing expression of 14-3-3 protein isoforms. CCRF-CEM cellular proteins from control and 24-h VCR treated cells, and CEM/VCR R and CEM/VLB100 cellular proteins were separated by 2D-PAGE and proteins identified as described for Fig. 2A. Expression levels of each isoform were determined as described for Fig 2B. Five distinct 14-3-3 protein spots showed altered expression after VCR treatment or in the resistant cell lines. 14-3-3{epsilon} (spot 1) decreases with VCR treatment and is up-regulated in the VCR R cells. 14-3-3{tau} (spot 2) also decreases with VCR treatment and is down-regulated in the VCR R cells. 14-3-3 protein spots 3–5 are all up-regulated in response to VCR treatment. Bars, S.E. for at least three individual experiments. *, p < 0.05; **, p < 0.005.

 
The relative expression of four other proteins that altered in both drug response and drug resistance are shown in Fig. 8. Heterogeneous nuclear ribonuclear protein (hnRNP) F was down-regulated in response to VCR treatment and is highly up-regulated in the VCR R cells. At least two isoforms of hnRNP K are expressed in these cells. Both isoforms decrease in expression in response to VCR and are also down-regulated in the VCR R and VLB100 cells. L-plastin showed down-regulation in response to VCR treatment and in the VLB100 cells. Cleavage of lamin B1 was shown to occur in response to VCR (Fig. 5), and the full-length lamin B1 protein concurrently decreases in expression (Fig. 8). This full-length protein is up-regulated in both the VCR R and VLB100 cells.



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  		FIG. 8.
Proteins altered in both vinca alkaloid response and resistance. Cellular proteins from untreated CCRF-CEM and 24-h VCR-treated cells, and CEM/VCR R and CEM/VLB100 cellular proteins were separated by 2D-PAGE and proteins identified as described for Fig. 2. Two-dimensional gel regions are shown with arrows indicating expression of: A, heterogeneous nuclear ribonuclear protein F (hnRNP-F); B, two isoforms of heterogeneous nuclear ribonuclear protein K (hnRNP K); C, L-plastin; and D, lamin B1 in untreated CCRF-CEM cells and after 8 nM VCR treatment for 24 h. The relative expression level of each protein pointed to after VCR treatment and in the VCR R and VLB100 cells is shown below each panel. Bars, S.E. for at least three individual experiments. *, p < 0.05; **, p < 0.005.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Proteome analysis of acute lymphoblastic leukemia cells treated with the commonly used vinca alkaloid, VCR, coupled with protein expression profiling of ALL cells resistant to both VCR and VLB, has identified unique changes in protein expression. Of these changes, ten proteins were altered in both the cellular response to drug treatment and in drug-resistant cells (Table III), and hence are potential targets for improved treatment of resistant disease. With the exception of the heterogeneous nuclear ribonuclear proteins and lamin B, these proteins are all associated with the microtubule and/or microfilament cytoskeleton.

Tubulin and Tubulin-binding Proteins—The cellular target for VCR is {beta}-tubulin. Various modifications to tubulin proteins were observed in response to VCR treatment (Fig. 2). Expression of a class I {beta}-tubulin protein increased immensely in cells treated with VCR. This protein is a more basic isoelectric variant than the highly expressed intrinsic class I {beta}-tubulin expressed in these cells. Class I {beta}-tubulin is the major isotype expressed in these cells and mutations in this gene have previously been associated with resistance to VCR in vitro (10). In addition, potential post-translational modifications have also been associated with VCR resistance (10). The more basic isoelectric point of this induced protein compared with the major class I {beta}-tubulin expressed in these cells suggests a modification to the {beta}-tubulin protein. Numerous post-translational modifications of {beta}-tubulin have been reported, including phosphorylation, polyglutamylation, and polyglycylation (35). These modifications all occur at the highly acidic and sequence divergent C-terminal region of the polypeptide. The C-terminal peptide of this modified protein could not be detected by MALDI-TOF or ESI-TOF MS (data not shown), but because no other peptide changes could be discerned between the intrinsic and more basic proteins, we suggest that a modification to the C-terminal region is the most likely cause for the increased isoelectric point. In support of this, a mAb directed to the C-terminal peptide of class I {beta}-tubulin did not react with the modified isoform (data not shown). Deglutamylation of {beta}-tubulin could result in the more basic isoelectric point observed for this protein, however the exact nature of this modification remains to be determined. Interestingly, this basic isoform of class I {beta}-tubulin is also down-regulated in the VCR R and VLB100 cells, suggesting that this modified protein may play a role in both vinca alkaloid response and resistance.

Increased expression of modified {alpha}-tubulin was observed in response to VCR treatment (Fig. 3). Microtubules are assembled from heterodimers of {alpha}/{beta}-tubulin, and like {beta}-tubulin, there are isotypes of {alpha}-tubulin that vary in their carboxyl-terminal region and can undergo numerous post-translational modifications (26, 35). Acetylation of {alpha}-tubulin has been associated with more stable microtubules (43). All of the induced {alpha}-tubulin isoforms were equally acetylated (data not shown), suggesting that acetylation is not affected. Polyglutamylation of {alpha}-tubulin is a post-translational modification that modulates the affinity of tubulin for microtubule-associated proteins (MAPs) (44) and is required for centriole formation, an organelle with the most stable microtubules (45). Deglutamylation could give rise to the altered isoforms of {alpha}-tubulin, and investigation of this potential modification is currently underway. The intrinsic {alpha}-tubulin 4 is also down-regulated in the VCR R and VLB100 cells.

In addition to tubulin alterations in response to vinca alkaloid treatment, altered expression of class II {beta}-tubulin was observed in both vinca alkaloid-resistant cells lines (Fig. 6 and Table II). The VLB100 cells have around 2-fold reduction (DE ratio 0.48) in this protein, whereas the VCR R cells massively down-regulate this isoform (DE ratio 0.02). A recent study showed that tubulin dimers consisting of class II {beta}-tubulin are the most sensitive to vinblastine compared with dimers made from other {beta}-tubulin isotypes (46), and resistance to the microtubule-stabilizing agent, paclitaxel, has previously been associated with increased expression of class II {beta}-tubulin (47, 48). Thus down-regulation of this isotype is likely to be contributing to resistance to vinca alkaloids.

Changes in tubulin-binding proteins have previously been associated with resistance to antimicrotubule agents (9). Members of the heat shock protein family, HSP70 and HSP90 are tubulin-binding proteins. Full-length HSP90{alpha} is down-regulated in the VCR R cells (DE ratio 0.38), and HSP90{beta} is down-regulated in the VLB100 cells (DE ratio 0.31). HSP90 binds to tubulin and inhibits tubulin polymerization (49), thus down-regulation of HSP90 in the resistant cells would result in more stable microtubules, again counteracting the effects of microtubule-destabilizing drugs. Interestingly, HSP90 binds to FKBP59 (p59) and the translationally controlled tumor protein (TCTP) both of which were altered in response to VCR (Fig. 4). TCTP displayed a dose-response increase in expression with VCR treatment. This protein was originally identified as a tumor-related protein (50) and has subsequently been found in a variety of normal cells. TCTP is a tubulin-binding protein, and overexpression of this protein leads to stabilization of microtubules (38). TCTP expression is slightly increased, although not statistically significant, in the VCR R (DE 1.27) and VLB100 (DE 1.41) cell lines. TCTP was recently described to be phosphorylated at Ser-46 and Thr-64, through polo-like kinases, and this phosphorylation decreases the microtubule-stabilizing activity of TCTP (39). Interestingly, the more acidic isoform of TCTP expressed in the CEM cells was up-regulated in response to VCR (Fig. 4). This isoform is likely to be the phosphoform of TCTP, which would result in decreased microtubule stability, as observed with vinca alkaloid treatment. FKBP59 shows a dose-response decrease with VCR treatment. FKBP59 exists in receptor-HSP90 complexes and binds to the glucocorticoid receptor (51). In addition, FKBP59 binds to cytoplasmic dynein and colocalizes with microtubules (52). Recent work suggests that these protein-protein interactions enable FKBP59 to participate in the receptor translocation along microtubules (53). Cleavage of HSP90 was also observed in response to VCR (Fig. 4). Proteolysis of HSP90 has been described in cells undergoing ionizing-radiation-induced apoptosis (15). Thus the cleavage observed herein may be due to apoptosis, however, because HSP90 is a tubulin-binding protein, it is also possible that cleavage of this protein is a more direct result of VCR action. The role of these tubulin-binding proteins in VCR response is under investigation.

HSP70 is another tubulin-binding protein that displays altered expression in the vinca alkaloid-resistant cell lines. Two members of the HSP70 family, the glucose-regulated stress protein 78 and heat shock cognate 71-kDa protein (HSC70), are both up-regulated in the VLB100 cells. HSP70 attaches to tubulin at the carboxyl terminus, in a region also recognized by MAPs (54), and a modified form of heat shock cognate 71-kDa protein was previously identified in cells resistant to tubulin-acting agents (55). It has been suggested that HSP70 may prevent microtubule formation by acting as an antagonist of MAPs, which are able to promote tubulin assembly and stabilize microtubules (56). In addition to the direct interaction with tubulin, HSP70 can affect microtubules through association with the microtubule-associated protein tau (57, 58). HSP70 proteins have been associated with resistance to apoptosis (59, 60), and increased expression of HSP70 was demonstrated in breast cancer patients after induction chemotherapy, where expression levels of both HSP27 and HSP70 correlated strongly with reduced disease-free survival (61).

Proteins Associated with the Actin Cytoskeleton—Caspase cleavage of specific proteins is a well characterized event during apoptosis (reviewed in Refs. 40, 41), and VCR is known to induce apoptosis in leukemia cells (62). Actin is a major cytoskeletal protein involved in cell structure and morphology, intracellular transport, and cell signaling. Actin is known to be cleaved during apoptosis and is a likely target for caspase-3 activity (63). Other studies indicate that alterations in the actin cytoskeleton is an early event in apoptosis, with cytoskeletal disruption in apoptotic cells promoting damage of the mitochondrial membrane resulting in enhanced release of the cytochrome c necessary for activation of caspase-9 and hence initiation of the caspase cascade (64). In addition, disruption of the actin cytoskeleton can induce apoptosis, with actin depolymerization triggering clustering and activation of FasL, resulting in subsequent caspase-8-mediated apoptosis (65). Alterations in actin proteins were also observed in vinca alkaloid-resistant cells. Non-muscle cells express two isotypes of actin, {beta} and {gamma}. The VLB100 cells down-regulate intrinsic {gamma}-actin, however, they also express an altered more basic isoform of this protein not present in the parental drug-sensitive CEM cells, which will be reported on elsewhere.4 Altered expression of actin-binding proteins was also observed in vinca alkaloid response and resistance. L-plastin is down-regulated in response to VCR treatment and is also down-regulated in the VLB100 cells (Fig. 8). L- and T-plastins are actin-bundling proteins involved in cytoskeletal reorganization in signal transduction pathways through phosphorylation by protein kinase C (66). Increased T-plastin expression has been associated with resistance to cisplatin (67) and UV-irradiation (68), however, this is the first report of altered L-plastin expression associated with drug resistance. The ubiquitous tropomodulin U-Tmod is another actin-binding protein that is down-regulated in both the VCR R and VLB100 cells (Table II). High levels of tropomodulin have been shown to inhibit tropomyosin binding to actin filaments (69), thereby affecting normal regulation of actin monomer dynamics and maintenance of thin filament length (70, 71). Thus down-regulation of tropomodulin in the VCR R and VLB100 cells may affect actin filament dynamics. The actin-binding protein myosin displays differential expression in the vinca alkaloid-resistant cell lines. Decreased expression was observed for one isoform of myosin regulatory light chain 2 in the VCR R cells and increased expression of two isoforms in the VLB100 cells. Myosin regulates actin dynamics, and the organization of the cytoskeleton depends entirely on the interaction of its dynamic components, particularly microtubules and actin microfilaments. Thus changes in myosin are likely to affect the dynamics of the cytoskeleton. That numerous microfilament-associated proteins are altered in both vinca alkaloid response and resistance suggests that the actin cytoskeleton may be involved with the resistance phenotype.

The 14-3-3 family of proteins consists of seven isotypes in humans, and play critical roles in cell signaling events that control progress through the cell cycle, transcriptional alterations in response to environmental cues, and programmed cell death (42). Differential phosphorylation of 14-3-3 proteins regulates their interactions with specific ligands, suggesting that these proteins are phospho-dependent signaling chaperones (42). In response to VCR treatment, two isotypes of 14-3-3 ({tau} and {epsilon}) showed down-regulation of the intrinsic proteins, and up-regulation of modified isoforms (Fig. 7). This suggests a change in the post-translational modification of these proteins, most likely phosphorylation, in response to VCR. Whether these changes are a direct result of VCR action or part of the apoptosis pathway remains to be determined. However, ionizing radiation of human prostate cancer cells induced down-regulation of intrinsic 14-3-3 isoforms and up-regulation of altered isoelectric point and molecular weight isoforms as determined by 2D-PAGE (16). This suggests that alterations in this family of proteins may be involved in a general role in apoptosis. 14-3-3{beta} expression was also greatly induced in response to VCR treatment (Fig. 7). Both 14-3-3{beta} and 14-3-3{zeta} associate with the microtubule-stabilizing protein tau and are effectors of tau phosphorylation thereby regulating microtubule-tau interactions and microtubule dynamics (72). In addition, 14-3-3{tau} was down-regulated and 14-3-3{epsilon} up-regulated in the VCR R cells (Table I). 14-3-3{zeta}, {tau}, and {epsilon} bind to phosphorylated cofilin, thereby regulating actin dynamics (73). Thus altered expression of 14-3-3 proteins may affect both the actin and tubulin cytoskeletal systems in these cells.

This study has identified a number of cytoskeletal proteins altered in both response and resistance to vinca alkaloids. Although these agents target microtubules, microtubules are a component of the cytoskeleton, and these results suggest a close inter-relationship between the actin and microtubule cytoskeletal systems. Furthermore, many of the proteins identified herein are known to interact with both cytoskeletal systems. HSP70 and HSP90, in addition to binding tubulin, are also associated with the actin cytoskeleton (56). HSP90 is thought to cross-link actin filaments (74), whereas HSP70 induces actin polymerization from monomers in vitro (75) and thus may stabilize actin filaments directly. In addition, through its association with the TCP-1 complex, HSP70 has the potential to modulate actin formation and dynamics in an indirect way (56). Although the direct role of many of these cytoskeletal proteins in antimicrotubule drug resistance is still unclear, these results highlight the inter-relationship between the two cytoskeletal systems, and further investigation of the proteins associated with both systems is warranted.

Other Protein Changes—Other proteins altered in both drug response and drug resistance include members of the heterogeneous nuclear ribonuclear protein (hnRNP) protein family and lamin B. Approximately 30 hnRNPs have been identified to date, all of which contain RNA binding motifs and auxiliary domains proposed to meditate protein-protein interactions (76). Numerous isoforms are generated by alternative pre-mRNA splicing and post-translational modifications, including serine and threonine phosphorylation and arginine methylation. Two isoforms of hnRNP K were down-regulated in both VCR response and in the VCR R and VLB100 cell lines (Fig. 8). hnRNP-K is thought to be involved in regulation of gene expression, through chromatin remodeling and regulation of pre-mRNA splicing (76–78). In addition to gene regulation, a number of hnRNP isotypes, including hnRNP K, are involved in apoptosis (11, 63, 79–81). Cleavage of hnRNP K has recently been demonstrated in Fas-induced apoptosis (11). It is possible that the decrease in hnRNP K in response to VCR is due to caspase cleavage. Another member of the hnRNP family, hnRNP F, is overexpressed in the VCR R cells, and showed down-regulation in response to VCR (Fig. 8). No association between this isotype and drug resistance has previously been reported. The expression changes in hnRNPs may therefore represent a novel pathway involved in vinca alkaloid resistance. In addition, a subset of hnRNP proteins have recently been shown to bind cytoplasmic and nuclear actin (82, 83), thus it is possible that the hnRNP changes are also associated with the cytoskeleton.

In response to VCR, lamin B1 displayed down-regulation of the full-length protein (Fig. 8) and up-regulation of a lamin B1 fragment, consisting only of the first half of the protein (Fig. 5). Lamins are type V intermediate filament proteins that constitute the nuclear lamina (84). Both lamin A and B are early targets for caspase degradation, before detectable DNA cleavage or chromatin condensation (85, 86). Expression of an uncleavable mutant form of lamin in cultured cells was shown to result in inhibition of chromatin condensation and a delay in DNA cleavage (87), suggesting that lamin degradation facilitates nuclease activation during apoptosis. The full-length Lamin B1 protein is also up-regulated in the VCR R and VLB100 cells (Table II and Fig. 8). Altered expression of this protein has not previously been associated with drug resistance, however, it is possible that increased expression of lamin B may act to inhibit or delay the onset of apoptosis.

In summary, proteomic analysis of human leukemia cells has identified 10 individual proteins with altered expression in vinca alkaloid resistance and in response to vinca alkaloid treatment. Many of these proteins are directly associated with microtubules. Although validation that these proteins are directly involved in resistance is required, the changes in tubulin-binding proteins are predicted to favor more stable microtubules, hence reducing the efficacy of vinca alkaloids. Several other proteins are associated with the actin cytoskeleton, highlighting the interactions between the two cytoskeletal systems. Whether the actin cytoskeletal changes are directly involved in the resistance phenotype or are compensation for microtubule alterations is unclear. Further characterization of the altered proteins is now under way to elucidate their role in drug resistance. This study has identified novel protein changes associated with drug response and resistance to vinca alkaloids in childhood leukemia, and these proteins are potential targets for cancer therapy.


   FOOTNOTES
 
* This work was supported in part by the Children's Cancer Institute Australia for Medical Research, which is affiliated with the University of New South Wales and Sydney Children's Hospital, and by grants from the National Health and Medical Research Council, New South Wales Cancer Council, and Cure Cancer Australia Foundation. This research has been facilitated by access to the Australian Proteome Analysis Facility established under the Australian Governments' Major National Research Facilities Program. 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. Back

The on-line version of this article (available at http://www.jbc.org) contains Figs. S1 and S2 and Table S1. Back

A recipient of an Australian Postgraduate Award and an Australian Proteome Analysis Facility Supplementary Award. Back

|| To whom correspondence should be addressed. Tel.: 61-2-9382-1823; Fax: 61-2-9382-1850; E-mail: m.kavallaris{at}unsw.edu.au.

1 The 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. Back

2 In this context, isoform refers to isoelectric variants of known proteins. Back

3 In this context, intrinsic refers to protein species expressed in parental drug-sensitive CCRF-CEM cells. Back

4 N. M. Verrills, M. Liu, M. Ivery, P. Gunning, and M. Kavallaris, submitted for publication. Back



   REFERENCES
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 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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