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[Cancer Research 63, 6928-6934, October 15, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Proteomic Profiling Drug-Induced Apoptosis in Non-Small Cell Lung Carcinoma

Identification of RS/DJ-1 and RhoGDI{alpha}1

Jeffrey P. MacKeigan, Casey M. Clements, John D. Lich, R. Marshall Pope, Yaacov Hod and Jenny P-Y. Ting2

Lineberger Comprehensive Cancer Center [J. P. M., C. M. C., J. D. L., J. P-Y. T.] and Departments of Microbiology and Immunology [J. P. M., C. M. C., J. D. L., J. P-Y. T.] and Biochemistry [R. M. P.], University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, and Prostate Cancer Research Center, Department of Urology, State University of New York, Stony Brook, New York 11794 [Y. H.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The growing knowledge of the tight connection between apoptosis and cancer has lead to an explosion of research revolving around apoptotic induction with chemotherapeutic agents and small molecule inhibitors. The chemotherapeutic agent paclitaxel (Taxol) activates mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase and, combined with MEK inhibition, synergistically enhances apoptosis. Here we implement a proteomic approach using two-dimensional gels coupled with mass spectrometry to identify proteins altered with this coordinated combination treatment. We found that the combined treatment of paclitaxel and MEK inhibitor uniquely altered the proteins RS/DJ-1 (RNA-binding regulatory subunit/DJ-1 PARK7) and RhoGDI{alpha} (Rho GDP-dissociation inhibitor {alpha}). Functional proteomic analysis by exogenous expression or short interfering RNA targeting confirmed a role in survival and apoptosis for these proteins. Analysis of primary lung tumors with matched adjacent normal tissue confirmed RS/DJ-1 overexpression in non-small cell lung carcinoma. This study shows the power of proteomic profiling coupled with functional analysis for the discovery of novel molecular targets and potential cancer cell-specific biomarkers.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Programmed cell death or apoptosis is characterized by distinct morphology of cell shrinkage, nuclear condensation, DNA fragmentation, and plasma membrane blebbing (1 , 2) . The growing knowledge of apoptosis and tight connection with cancer has lead to an explosion of research revolving around inducing apoptosis in cancer cells with chemotherapeutic agents, small molecule inhibitors, and biologic approaches. Paradoxically, recent discoveries indicate that chemotherapeutic agents also activate survival signals and compromise tumor cytotoxic effects. Chemotherapy-induced activation of cell survival signaling cascades has been demonstrated for anticancer treatments, such as ionizing radiation, topoisomerase targeting, and microtubule inhibitor drugs. Specifically, chemotherapy-induced activation of nuclear factor {kappa}B leads to the expression of antiapoptotic genes, including Bcl-2 and inhibitors of apoptosis family members, and inhibition of nuclear factor {kappa}B activity significantly enhances apoptosis (3, 4, 5) . Additionally, the important cancer chemotherapeutic agent paclitaxel activates cell survival pathways, such as the Raf-MEK3 -ERK pathway (6 , 7) . In the ERK MAP kinase cascade, activated Raf-1 initiates the signaling cascade through MEK, which in turn phosphorylates a second serine-threonine kinase, ERK. ERK phosphorylates additional kinases and specific transcription factors important in cell proliferation and survival. Thus, inhibiting these survival signals in combination with conventional chemotherapeutic agents is a rational approach to combat cancer.

The MAP kinase members play important roles in cell survival and death. Reports demonstrate that a delicate balance between JNK and ERK activation exists in determining neuronal apoptosis or cell survival (8 , 9) . The balance between JNK and ERK activation may likewise be important in cancer. Thus, tilting the balance to apoptosis by activating cell death signals and inhibiting survival signals may be crucial in determining the fate of a cancer cell. Paclitaxel induces MEK/ERK, whereas the combination of paclitaxel and MEK inhibition shifts this balance and enhances apoptosis dramatically over each agent alone (6 , 10) . The use of selective small molecule inhibitors that block this cascade confirms the MAP kinase cascade as a valuable molecular target in cancer (11, 12, 13, 14, 15, 16) .

The elucidation of survival and apoptotic proteins activated or down-regulated in cancer cells is of paramount importance to improve cancer therapy. We use this coordinated combinatorial approach of paclitaxel and MEK inhibition to profile thousands of proteins by two-dimensional gel electrophoresis and mass spectrometry. Using two-dimensional gel electrophoresis to profile proteins that are unique to only the combination treatment, we identified RS/DJ-1 (RNA-binding regulatory subunit/DJ-1) and RhoGDI{alpha} (Rho GDP-dissociation inhibitor {alpha}). Furthermore, we show that RS/DJ-1 functions to protect cells from apoptosis and that reducing RhoGDI{alpha} levels through siRNA induces apoptosis. These findings associate both RS/DJ-1 and RhoGDI{alpha} with the apoptotic response in cancer cells. Real-time RT-PCR analyses and protein levels of matched primary NSCLC and corresponding normal lung tissue confirmed overexpression of RS/DJ-1. Overall, this study shows the power of proteomic profiling coupled with functional proteomic analysis for the discovery of novel molecular targets and potential cancer cell-specific biomarkers.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
The human NSCLC H157 cells were cultured as described previously (6) . For selected experiments with transiently transfected cells, H157 cells (5 x 103 cells/well) were grown in 96-well plates, and 100 ng of RS/DJ-1 were introduced using FuGENE 6 (Roche Biochemicals) transfection reagent. After transfection for 24 h, the cells were incubated with the indicated concentrations of paclitaxel with or without the MEK inhibitor U0126. Stock solutions of paclitaxel (Sigma) and the MEK inhibitor U0126 (Promega) were dissolved in Me2SO (Sigma) and used as described previously (6) .

Two-Dimensional Gel Electrophoresis and Image Analysis.
Cells were treated with drugs for 360 min. After three washes in ice-cold PBS, cells were lysed in 7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, 2% DTT, and O.5% carrier ampholytes (pH 4–7) with a variety of inhibitors (17) . Samples were centrifuged (200,000 x g) for 1 h at 22°C, and total protein loading was measured by Bradford assay. Total protein (200 µg) was loaded on 18-cm immobilized pH gradient strips (pH 4–7) for two-dimensional gel electrophoresis with focusing for 58,000 V-h. The second dimension SDS-PAGE gels (8–16%; 19 x 18 cm) were stained with silver nitrate or fluorescent Sypro Ruby stain, imaged, and analyzed with Progenesis 2-D analysis software for spot detection, quantitation, and matching.

In-Gel Enzymatic Digestion and Mass Spectrometry.
To identify differentially expressed proteins, protein spots were excised from the gels, in-gel enzymatic digestion was performed with trypsin, and protein spots were processed for mass spectrometric analysis. Briefly, lyophilized tryptic digests were reconstituted in 5 µl of 0.2% formic acid. Of these, 0.5 µl was recrystallized with an equal volume of {alpha}-cyano-4-hydroxycinnamic acid prepared by diluting a saturated solution 1:5 in a mixture of ethanol, water, and 88% formic acid (45:43.6:11.4). Samples were analyzed by MALDI-TOF on a Reflex III instrument (Brüker Franzen) operated in the reflection mode using 20 kV accelerating potential and a 150 ns extraction delay. Autoproteolytic digest fragments of porcine trypsin (Promega) were used as internal calibrants to construct a mass scale. Data processing and database searches were performed using Mascot (Matrix Science) and the Profound algorithm (Proteometrics) software packages.

Real-Time RT-PCR.
Quantification of gene expression was performed via quantitative real-time RT-PCR using an ABI PRISM 7900 (Perkin-Elmer Applied Biosystems) system. cDNA was made from total RNA extracted from frozen normal and lung tumor tissues. The primers and probes for RS/DJ-1, RhoGDI{alpha}, and 18S rRNA were designed to span exon-intron junctions [RS/DJ-1, 5'-CCATATGATGTGGTGGTTCTAC-3' (forward primer), 5'-ACTTCCACAACCTATTTCATGAG-3' (reverse primer), and 5'-6FAM-ACCTGCACAGATGGCGGCTATCA-TAMRA-3' (probe); RhoGDI{alpha}, 5'-GCCTGCGAAAGTACAAGGAG-3' (forward primer), 5'-CGACTGCTTCTTGAAGCTCTC-3' (reverse primer), and 5'-6FAM-CGTGGCCGTTTCCGCAGACC-TAMRA-3' (probe); 18S rRNA, 5'-GCTGCTGGCACCAGACTT-3' (forward primer); 5'-CGGCTACCACATCCAAGG-3' (reverse primer); and 5'-6TET-CAAATTACCCACTCCCGACCCG-TAMRA-3' (probe)]. Each sample was normalized on the basis of 18S rRNA expression. Standard curves for RS/DJ-1, RhoGDI{alpha}, and 18S were generated using serial dilution, containing 3,012,500, 200,833, 13,389, and 893 copies of template containing plasmids. The normalized amounts of RS/DJ-1 and RhoGDI{alpha} mRNA were determined by dividing the amount of respective mRNA by the amount of 18S rRNA for each matched sample.

siRNA Nucleic Acids.
The target-specific siRNA duplexes were selected from target sequences with an AA(N19) motif from the complete coding region of RhoGDI{alpha}. The 21-nucleotide complementary siRNAs were from bp positions 97–117 of RhoGDI{alpha} mRNA. The selected siRNAs were BLAST searched against the human genome sequence to ensure only one gene was targeted, whereas the control (nonsilencing) siRNA used has no known overlap, and the nonspecific siRNA silences an irrelevant mRNA target. The siRNAs were chemically synthesized and annealed by Xeragon with >97% purity. For siRNA introduction in 24-well plates, we transiently transfected H157 cells with 3 µl Oligofectamine (Life Technologies, Inc.) and 3 µl of 20 µM siRNA solution. Cells were assayed for silencing 48–96 h after transfection.

Measurement of DNA Fragmentation.
Quantitation of apoptotic cell death was determined by Cell Death ELISA (Roche Biochemicals) that measures cytoplasmic histone-DNA fragments produced during apoptosis (6) . The enrichment of histone-DNA fragment-treated cells is expressed as fold increase in absorbance as compared with control (DMSO-treated) cells.

Immunoblot Analysis.
Frozen tissue samples were solubilized in two-dimensional gel electrophoresis lysis buffer using a Dounce homogenizer, and crude homogenate was centrifuged for 15 min at 4°C. The protein concentration of the supernatant proteins was determined by Bradford and Coomassie Blue staining, and equivalent cell lysate protein amounts (20 µg) were resolved by 15% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, probed with anti-RhoGDI{alpha} monoclonal antibody (Santa Cruz Biotechnology), anti-RS/DJ-1 antibody (18) , or anti-actin antibody (Santa Cruz Biotechnology).

Cell Cycle Analysis.
Adherent and detached cells were collected with trypsin, resuspended at 2 x 106 cells/ml, and fixed in ice-cold 70% ethanol. Each sample was resuspended in propidium iodide (PI) stain buffer (0.1% Triton X-100, 200 µg of DNase-free RNase A, 20 µg of PI, and PBS) for 30 min, and samples were analyzed using a FACScan (Becton Dickinson) and ModFit LT (Verity Software).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Proteomic Profiling Events Preceding Drug-Induced Apoptosis.
Previously, we have shown that treatment of cells with paclitaxel activates cell survival ERK1/2 cascades and that additional inhibition of MEK/ERK enhanced apoptosis in breast, ovarian, and lung carcinoma cell lines (6) . Other reports found enhanced apoptosis with paclitaxel and MEK inhibitors and noted the importance of the order of addition (10 , 19) . Concurrent treatment or paclitaxel addition before MEK inhibition treatment is important to induce enhanced apoptosis over each agent alone. To reaffirm these results, we used concurrent treatment of paclitaxel and U0126 in H157 NSCLC cells. Using an ELISA that measures DNA-histone fragments, the combination of paclitaxel and MEK inhibition enhanced apoptosis compared with control-treated cells (Fig. 1a)Citation .



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  		Fig. 1. Proteomic profiling of enhanced apoptosis by two-dimensional gel electrophoresis. a, H157 lung carcinoma cells were treated concurrently with 250 nM paclitaxel in the absence or presence of 10 µM U0126 for 24 h. Apoptosis was measured by ELISA that detects DNA-histone fragmentation, and the data are expressed as fold increase in absorbance as compared with control treated cells. b, the master expression profile map indicates the regions outlined. c, close-up sections of specific two-dimensional gel regions for each drug treatment. The outlined regions show proteins differentially regulated. Proteins indicated with arrows and database numbers are differentially regulated upon treatment with paclitaxel, paclitaxel + U0126, or U0126. These results are representative of at least four independent experiments.

 
To analyze the underlying mechanisms and identify downstream mediators that are unique to the combined effects of paclitaxel and U0126, we initiated a proteomic analysis to identify target-specific proteins important for enhanced apoptosis over nonspecific targets. Using high-resolution two-dimensional gel electrophoresis, we determined the protein expression profile of H157 NSCLC cells. An expression profile map from control-treated cells was used to generate a master expression profile map of H157 cells. This master pattern of protein expression was used to identify differences after treatment with paclitaxel, U0126, or a combination of paclitaxel and U0126. The goal was to identify and functionally characterize crucial proteins that lie upstream of the actual event of drug-induced apoptosis.

The proteins were first separated according to their isoelectric point (pI) on 18-cm immobilized pH gradients strips (pH 4–7) and then separated by their molecular mass on 18-cm, 8–16% gradient gels. Our high-resolution two-dimensional gel electrophoresis gels are highly reproducible and yield characteristic two-dimensional gel electrophoresis protein patterns. Protein spots were resolved after each treatment (control, paclitaxel, MEK inhibitor, and paclitaxel + MEK inhibitor) in the pI 4–7 and molecular mass range 10–200 kDa. Using these isoelectric focusing and separation conditions, we were able to most effectively separate proteins under 100 kDa. Resolved proteins were visualized using either silver stain or SYPRO Ruby fluorescent dye. Computer-assisted spot detection of fluorescent dye-stained gels was used to measure spot volume for all protein spots in each drug treatment. The normalized spot volume represents the fluorescence signal intensity integrated over the area of each spot minus background intensity that surrounds the spot and standardized to the total volume of every spot in the gel. More than 2,000 spots were consistently detected on each gel, and a vast majority of protein spots remained unchanged using the criteria of >30,000 spot volume and spots with equal radius. The consistently detected protein spots with <50,000 spot volume were excluded from the analysis because we could not precisely confirm expression changes by visual inspection.

Identification of RS/DJ-1 and RhoGDI{alpha} as Novel Targets.
We treated H157 cells for 6 h to examine and characterize changes in the proteome that precede the induction of apoptosis. Selected regions from each treatment were expanded to illustrate the different protein expression profiles after the combination drug treatment (Fig. 1, b and c)Citation . Treatment-dependent differences in expression are highlighted with arrows and identified with numbers from our database. A number of protein spots show intensity changes, reflecting increased or decreased amount of protein present. Protein spots 441 and 491 appear exclusively in cells treated singly with 250 nM paclitaxel (Fig. 1c)Citation , whereas spots 535 and 584 are absent in cells treated with paclitaxel but present in the other three groups. The normalized spot volume of 535 and 584 is equivalent in control-, combination-, and U0126-treated cells. Protein spots 1038 and 1070 increased >2.5-fold in paclitaxel and paclitaxel plus U0126 treatment groups, but not in control- or U0126-treated cells. Interestingly, spots 1002 and 1053 are uniquely down-regulated in only the combination treatment.

This analysis has characterized 19 differentially expressed spots that changed >2.5-fold in magnitude upon drug treatment. Other changes that are <2.5-fold have been observed; however, they are too numerous to warrant a more detailed discussion. Among the proteins that were altered by >2.5-fold, paclitaxel affected the expression of more proteins than U0126 treatments (Fig. 2)Citation . Paclitaxel altered the expression of 14 proteins, U0126 altered the expression of 6 proteins, and the combined treatment altered the expression of 12 proteins. Of these changes in expression pattern, two are common to paclitaxel-treated cells regardless of the presence of U0126, one is a result of U0126 treatment regardless of the presence of paclitaxel, and five are altered in common for all three treatments.



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  		Fig. 2. Protein profiling in response to each drug treatment. Schematic diagram of the two-dimensional gel electrophoresis protein expression patterns after 6 h of treatment. Normalized spot volumes that increased >2.5-fold (filled shapes: paclitaxel, {bullet}; both U0126-containing treatments, {diamondsuit}; both paclitaxel-containing treatments, *; all drug treatments, {blacksquare}) or decreased by >2.5-fold (open shapes: paclitaxel, {circ}; all drug treatments, {square}; unique to only the combination of paclitaxel and U0126, {triangleup}) are displayed with database numbers followed by apparent pI and molecular mass in parentheses.

 
To enhance an understanding of the mechanism underlying combination paclitaxel and U0126 treatment, we focused on identifying changes in protein spots that were unique to only the combination treatment. Spot matching between gels followed by comparative analysis of spot intensities revealed that no protein spots were consistently new or increased >2.5-fold, and four spots consistently decreased 2.5-fold upon treatment. A schematic two-dimensional gel electrophoresis gel (Fig. 2)Citation shows the position of proteins displaying altered expression after treatment unique only to paclitaxel (441, 491, 533, 535, 584, 867, and 1134) or the combination (963, 1002, 1053, and 1197). Paclitaxel and paclitaxel plus U0126 treatment groups increased spot 1038 and 1070. All three treatments increased spot volume (615, 932, and 1318) and decreased in two protein spots (900 and 1276), whereas spot 689 was present and increased in treatments containing the MEK inhibitor U0126.

Of the protein spots with differential expression profiles unique only to the combination treatment with enhanced apoptosis, we identified spot 1053 as RS/DJ-1 and spot 963 as RhoGDI{alpha} by MALDI-TOF mass spectrometry. Mass peptide fingerprinting was obtained by following procedures described previously (20) , and the MALDI mass spectrum for RS/DJ-1 and RhoGDI{alpha} is shown in Fig. 3, a and bCitation , respectively. The protein coverage of peptides for RS/DJ-1 covered 53% of the protein sequence (Fig. 3a)Citation , whereas the protein coverage for RhoGDI{alpha} encompassed 39% of the protein (Fig. 3b)Citation . On average, 11 peptide masses were matched for each protein. The remaining down-regulated spots (spots 1002 and 1197) were unsuccessfully identified as a result of ambiguous spectra or spots that did not contain adequate material to obtain accurate peptide masses.



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  		Fig. 3. Peptide mass spectrum of the differentially expressed RS/DJ-1 protein and RhoGDI{alpha}. Spots were excised and subjected to in-gel digestion with trypsin. An aliquot of the supernatant containing tryptic peptides was analyzed by MALDI-TOF, which resulted in the peptide mass fingerprint and identification of RS/DJ-1 (a) and RhoGDI{alpha} (b). The tryptic fragment masses were submitted and matched for identification. The amino acid sequences below the peptide masses were deduced after database searching. The designated amino acid sequences for RS/DJ-1 and RhoGDI{alpha} are shown with the matched peptides to amino acids designated in bold.

 
RS/DJ-1 and RhoGDI{alpha} Expression in Primary Lung Carcinomas.
To examine RS/DJ-1 and RhoGDI{alpha} as candidates for therapeutic targets and potential markers for NSCLC, we measured expression in primary human malignant NSCLC tumor samples as compared with normal matched control tissue. The normal match controls were taken from histologically nontransformed tissues that were adjacent to the primary tumor. The tissue specimens were assayed for expression by real-time RT-PCR. The results are normalized to the level of 18S rRNA as a housekeeping gene. RS/DJ-1 is overexpressed in six of seven cases analyzed. RS/DJ-1 is overexpressed in NSCLC by an average of 7.7-fold as compared with adjacent matched control tissue (Fig. 4a)Citation . RhoGDI{alpha} mRNA is increased in fewer tumors, and the degree of overexpression is also less (Fig. 4b)Citation . To determine whether overexpression of RS/DJ-1 mRNA results in increased protein, protein levels of NSCLC tumors and normal adjacent lung tissue were examined by Western blot using anti-RS/DJ-1 antibody (Fig. 4c)Citation . The three cases examined with overexpressed mRNA showed higher RS/DJ-1 protein levels in tumors as compared with normal tissue.



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  		Fig. 4. RS/DJ-1 overexpression in NSCLC. a, overexpression of RS/DJ-1 mRNA in six of seven primary NSCLC tissues as compared with normal adjacent lung tissue. Measurements were performed for each sample by real-time PCR analysis. b, increased expression of RhoGDI{alpha} in four of seven primary NSCLC tumor tissues. Expression is presented as fold increase compared with normal adjacent lung tissue. The mRNA levels were calculated from four experiments, each performed in triplicate. Error bars represent SD. c, RS/DJ-1 protein is increased in primary NSCLC. Total protein lysates were prepared from three different matched samples of tumor (T) and adjacent normal tissue (N). Equal amounts of protein as determined by Bradford and Coomassie Blue staining were resolved by SDS-PAGE and immunoblotted for RS/DJ-1. These results are representative of five independent experiments.

 
Functional Proteomic Analysis of RS/DJ-1 and RhoGDI{alpha} in Apoptosis.
RS/DJ-1 was first identified as a novel oncogene that weakly transforms NIH3T3 cells when applied alone and dramatically enhances transformation in cooperation with Ras. These results implicated its role in cellular proliferation (21) . Additional studies demonstrate that RS/DJ-1 is involved in the regulation of RNA-protein interactions (18) . From our proteomic studies, if decreased RS/DJ-1 is indeed involved in enhanced drug-induced apoptosis, then introduction of RS/DJ-1 should reverse this apoptotic process. To directly address this issue, a RS/DJ-1 expression plasmid was transiently transfected into H157 cells, and the effect of RS/DJ-1 on apoptosis was measured. Control cells were transfected with an empty vector. Cells were treated with paclitaxel (250 nM), U0126 (10 µM), or the combination of paclitaxel and U0126. Control cells treated with paclitaxel exhibited a dose-dependent increase in apoptosis, and U0126-treated cells exhibited a small increase in apoptosis, whereas the two drugs together enhanced apoptosis over each agent alone, as expected (Fig. 5aCitation , {blacksquare}). The transient introduction of RS/DJ-1 significantly reduced the level of apoptosis (Fig. 5a)Citation , indicating that RS/DJ-1 is a cell survival factor. This is consistent with the hypothesis that the reduction of RS/DJ-1 by a combination of paclitaxel and U0126 contributes to increased apoptosis and is consistent with the role of RS/DJ-1 as a possible oncogene (21) .



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  		Fig. 5. Role of RS/DJ-1 and RhoGDI{alpha} in tumor cell apoptosis. a, overexpression of RS/DJ-1 reversed paclitaxel and U0126-induced apoptosis. H157 cells were transiently transfected with pCMV empty vector or pCMV-driven RS/DJ-1. After 24 h, cells were treated with paclitaxel and 10 µM U0126 for 24 h, and apoptosis was assayed by ELISA that measures DNA-histone fragments. b, immunoblot of lysates from cells transfected with control siRNA or RhoGDI{alpha} siRNA and probed with RhoGDI{alpha}-specific antibody. The blot was stripped and reprobed with actin antibody (bottom panel) for equal loading. Interference with RhoGDI{alpha} transcript increased paclitaxel and U0126-induced apoptosis. H157 cells were transfected with control siRNA or RhoGDI{alpha} siRNA. After 72 h, cells were treated with paclitaxel and 10 µM U0126 for 24 h, and apoptosis was assayed by ELISA that measures DNA-histone fragments. c, control, irrelevant, or RhoGDI{alpha} siRNAs were introduced into H157 cells for 72 h, after which they were stained with PI, and cell cycle analysis was performed. Values correspond to the percentage of sub-G1 cells or cell death as measured by flow cytometry. Error bars represent SD.

 
To functionally characterize the role of altered RhoGDI{alpha} on apo-ptosis, we attempted to overexpress the gene, but we did not see a significant effect. Instead we used siRNA (22, 23, 24) to target and knock down RhoGDI{alpha}. RNAi, RNA interference, is a powerful research tool to selectively inhibit gene expression, leading to the selective silencing of the target mRNA and protein encoded. RhoGDI{alpha} protein levels were reduced with a siRNA specific to RhoGDI{alpha}, but not a control siRNA, with the most dramatic reduction occurring 96 h after transfection (Fig. 5b)Citation . The timeframe is necessary for the targeting and degradation of nascent RhoGDI{alpha} and turnover of the existing abundant RhoGDI{alpha} protein. Importantly, the reduction of RhoGDI{alpha} increased baseline apoptosis in control cells by >3-fold, to a level equivalent to the efficacy of paclitaxel (Fig. 5b)Citation . RhoGDI{alpha} siRNA also increased apoptosis in U0126-treated cells by 3-fold. The siRNA modestly increased apoptosis in cells treated with 10 nM paclitaxel. However, siRNA targeting RhoGDI{alpha} did not further enhance apo-ptosis in cells treated with the combination of paclitaxel and U0126, indicating that the decrease of RhoGDI{alpha} by the combination of paclitaxel and U0126 may have reached a threshold, and the reduction caused by siRNA no longer produces any additional effect. These experiments show a role for RhoGDI{alpha} in controlling apoptosis, although it does not completely duplicate the significant apoptosis induced by both paclitaxel and MEK inhibition. At least two explanations can be put forth: the first is that the transient expression system is not likely to cause efficient siRNA introduction into all cells. Improvements in this step may greatly enhance apoptosis. The second is that other concerted changes that occur upon combination drug treatment may be required to produce the greatly enhanced apoptotic effects. Nonetheless, these studies indicate that even the partial introduction of siRNA targeted to RhoGDI{alpha} still caused apoptosis at a level similar to that induced by a well-known antitumor drug, paclitaxel.

Because the most dramatic results with siRNA targeting RhoGDI{alpha} were observed in control-treated cells where the level of RhoGDI{alpha} is substantial, we subjected these cells to further analysis by flow cytometry. The siRNA-mediated reduction of RhoGDI{alpha} increased the percentage of cell death from 3% to 10% in untreated cells as compared with nonsilencing control siRNA cells as measured by flow cytometry (Fig. 5c)Citation .


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Research discoveries of cell survival proteins activated or apoptotic proteins down-regulated are important to improve tumor cell killing. Small molecule inhibitors that selectively target these proteins in combination with effective conventional cancer chemotherapeutic agents is the way forward to combat cancer (25 , 26) . The effectiveness of these small molecules as single agents has given additional weight to combination treatments. The combination of paclitaxel and MEK inhibitors involves a two-component system to enhance apoptosis. Paclitaxel causes G2-M block and activation of proapoptotic JNK, whereas MEK inhibitor leads to G1 arrest and blockade of ERK-regulated cellular proliferation and survival. In combination, the apoptotic response is much greater than additive, lending support to the argument that several proteins are differentially regulated beyond what is already known. Therefore, integrating genomic/proteomic approaches is vital to understand the genes/proteins that control apoptosis and cell fate during combination chemotherapy. Ultimately, the identification of cell survival and apoptotic targets in cancer will lead to new and improved therapeutic strategies.

The role of paclitaxel as a single agent in inducing apoptosis is important. Indeed, eight proteins were up-regulated with all treatments containing paclitaxel, and six proteins were down-regulated >2.5-fold. In total, seven unique protein spots were altered exclusively upon paclitaxel treatment. For this study, we have focused on proteins altered with only the combination of paclitaxel and U0126 treatment. We could positively identify RS/DJ-1 and RhoGDI{alpha} as altered and unique to only the combination treatment. Our functional studies with these two protein targets provide insight into the mechanisms of apoptosis. Although we are aware that proteins displaying spot volume difference that were <2.5 changes are also of interest, an arbitrary cutoff at 2.5x allowed us to focus on the most dramatic differences in protein spot expression. Lowering the expression threshold and decreasing the fold change filtering criteria will allow us to identify and characterize many more proteins involved in cancer cell apoptosis.

Proteomic research is its share of limitations. The difficulty with proteomic research is the technical challenges are still tremendous. These difficulties arise from the sheer number of proteins and each possible modification. Developments in mass spectrometry have fueled recent advancements; however, future progress will require tremendous discoveries in chemistry, biology, and bioinformatics. Nonetheless, integrating proteomic approaches to ask and solve complex biological questions can be accomplished despite these challenges. Technology advances have allowed for genomic research to proceed at a rapid and straightforward pace. The technological advances of proteomics are still in their infancy as compared with genomic research. Recent advances in mass spectrometry and protein sciences have set the stage for current proteomics research.

Two-dimensional gel electrophoresis technology has become a useful tool for the analysis of global and comparative protein expression analysis. The power of two-dimensional gel electrophoresis is the resolution of thousands of proteins on a single gel. The difficulties with two-dimensional gel electrophoresis have been reproducible protein separation and quantification. Our proteomic analysis uses the latest two-dimensional gel electrophoresis improvements and overcomes some of these prior limitations. Unfortunately, the state of two-dimensional gel electrophoresis still yields the most abundant proteins and excludes many low copy number proteins. Additional difficulties with analysis of hydrophobic proteins and basic proteins remain a limitation in two-dimensional gel electrophoresis. Critical improvements in proteomics research will require adding complementary tools. Alternative approaches include liquid chromatography and capillary electrophoresis based separations coupled with mass spectrometry, such as Multidimensional Protein identification technology or MudPIT (27 , 28) . The limitation with these approaches is overcoming sample complexity. Seriously needed improvements in database and bioinformatics tools will allow for identification and quantification of single peptides/proteins from these complex peptide mixtures.

The comprehensive analysis of protein complexes and signal transduction pathways can be achieved using new proteomic tools. In isotope-coded affinity tagging, proteins are labeled with heavy (d8 or d7) deuterium atoms from one total protein sample, whereas the other sample is labeled with a light (d0) atoms (29) . In connection with our studies, carcinoma cells or tumors after treatment could be lysed, labeled, combined, and proteolytically cleaved to yield a complex mixture of peptides differentially labeled with either d8 or d0 atoms. The subsequent labeled peptides are then affinity purified, fractionated, and analyzed by mass spectrometry. The d8 and d0 isotope-coded affinity tagging reagents have a mass difference of exactly 8 Da; therefore, relative abundance of each peptide in each sample can be determined by differences in mass spectra peak height.

In this study, we present the first evidence that RS/DJ-1 is overexpressed in primary NSCLC and identify RS/DJ-1 as a potential anticancer target. RS/DJ-1 is a recently described protein whose function is not well understood, but it is known to inhibit RNA-protein complexes in a cell-free system and to exhibit oncogenic potential (18 , 21) . Our results with RS/DJ-1 in apoptosis are consistent with the recent report on the role of DJ-1 mutations in early onset Parkinson’s disease (30) , namely, that a reduced level of DJ-1 is consistent with neuronal apoptosis and neurodegneration. As this manuscript was being prepared, the crystal structure of DJ-1 has been described and implicates DJ-1 in transcriptional regulation after oxidative stress (31) . Our study shows that the reduction of RS/DJ-1 is associated with greater apoptotic cell death, whereas the introduction of RS/DJ-1 into cells enhanced cell survival. This role of RS/DJ-1 is supported by earlier evidence that it weakly transforms NIH3T3 cells but exhibits significantly more transforming activity when coexpressed with H-Ras or c-myc. Using a similar transformation assay, other proteins have been identified as potential oncogenes, but this report highlights an important role for RS/DJ-1 in chemoresistance. Another recent study has identified RS/DJ-1 as a potential tumor antigen that is found in the circulation of tumor-bearing patients (32) . This report raises the intriguing possibility that RS/DJ-1 may be secreted or released into the circulation, further emphasizing its potential as a tumor marker. RS/DJ-1 was significantly up-regulated in six of seven primary NSCLC tumor tissues as compared with matched control for each tissue. Overexpressed proteins provide better potential drug targets for the development of small molecule therapeutics.

RhoGDI is an interesting drug target. RhoGDI{alpha} (RhoGDI-1), RhoGDIß (GDI/D4), and RhoGDI{gamma} are all members of the RhoGDI family. Our siRNA experiments directed at RhoGDI{alpha} show that inhibition of this protein enhances apoptosis in control-, paclitaxel-, and U0126-treated cells. Implicating RhoGDI{alpha} in cell death suggests the importance of the traditional role RhoGDI{alpha} plays in controlling cellular responses through the small GTPases Rac, Rho, and Cdc42. As its name implies, RhoGDI inhibits the dissociation of GDP from the GDP-bound form and sequesters Rac, Rho, and Cdc42 in the inactive form. Our results add a novel role for RhoGDI as an important event in the apoptotic response in cancer cells. Review of the literature indicates that RhoGDI{alpha} has been identified as down-regulated during all-trans retinoic acid and the cyclin-dependent kinase inhibitor bohemine treatment (33 , 34) . In this study, we identify and characterize RhoGDI{alpha} as a cell death mediator and as a potential anticancer target. Recently, RhoGDIß has been shown to be overexpressed in pancreatic cancers (35) .

The widespread expression of RS/DJ-1 and RhoGDI{alpha} suggests that these proteins may operate in several cell types and cancers. RS/DJ-1 is ubiquitously expressed in over 22 human tissues, including stomach, brain, muscle, skin, lymph, kidney, breast, prostate, and lung. Our data, based on the comparative analysis of seven NSCLC tumors and their adjacent normal controls, indicate that RS/DJ-1 is overexpressed in NSCLC. Its expression level is increased in tumor-bearing patients, consistent with a role in the survival of tumors. Thus, extending the expression studies to a variety of cancers could be beneficial to understand the extent of overexpression. RhoGDI{gamma} is preferentially expressed in brain and pancreas (36 , 37) , and it is also up-regulated and overexpressed in malignant tissue, such as colon polyps and ovarian carcinoma (38) . Thus, using siRNA to specifically target RhoGDI{alpha} over other family members could be a future approach to avoid potential neurotoxicities. In summary, this work provides proteomic and functional evidence for the importance of RS/DJ-1 and RhoGDI{alpha} in cell survival and apoptosis. Modulating these proteins with siRNA or small molecules in combination with cancer chemotherapeutic agents may be a valuable approach to combat cancer.


   ACKNOWLEDGMENTS
 
We thank Dr. Lee Graves and Tom Hilder for helpful comments and discussions. We thank Evangeline Reynolds at the Lineberger Comprehensive Cancer Center Tumor Procurement Facility for the primary lung carcinoma tissues.


   FOOTNOTES
 
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.

1 Supported by NIH Grant CA-58233 and by a seed grant from the Lineberger Comprehensive Cancer Center. Back

2 To whom requests for reprints should be addressed, at Lineberger Comprehensive Cancer Center, Campus Box Number 7295, University of North Carolina, Chapel Hill, NC 27599. Phone: (919) 966-5538, Fax: (919) 966-8212; E-mail: panyun{at}med.unc.edu Back

3 The abbreviations used are: MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; JNK, c-Jun NH2-terminal kinase; 6FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine; siRNA, short interfering RNA; NSCLC, non-small cell lung carcinoma; MALDI, matrix-assisted laser-desorption/ionization; TOF, time-of-flight; RT-PCR, reverse transcription-PCR; PI, propidium iodide. Back

Received 3/25/03. Revised 7/17/03. Accepted 7/22/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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