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Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas (S.N.K., A.B., M.U., C.S.L., M.R., T.C.C.); and Geriatric Research, Education, and Clinical Center, Central Arkansas Veterans Healthcare System, North Little Rock, Arkansas (R.A.D.)
Received July 9, 2007; accepted October 3, 2007
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Abstract |
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). Vinblastine binds to tubulin subunits and inhibits tubulin polymerization, thus disrupting the dynamic instability of spindle microtubules (Jordan and Wilson, 2004). This leads to mitotic arrest and subsequent cell death by apoptosis, either soon after metaphase arrest, after a delay, or after resumption of an impaired cell cycle entailing DNA endoreduplication (Khan and Wahl, 1998; Casenghi et al., 1999). The mechanisms that trigger apoptosis after metaphase arrest are under active investigation. Antimitotic drugs such as vinblastine activate several signal transduction pathways that may promote apoptosis. Two prominent and perhaps universal responses are those that lead to the activation of c-Jun NH2-terminal kinase (JNK) (Fan and Chambers, 2001) and to phosphorylation of Bcl-2 and Bcl-xL (Ruvolo et al., 2001). Although some evidence has been presented to suggest that JNK catalyzes Bcl-2/Bcl-xL phosphorylation occurring in response to antimitotic drugs (Yamamoto et al., 1999), more recent work has indicated that a distinct kinase is responsible (Du et al., 2005).
We have documented previously that vinblastine induces JNK activation, c-Jun expression and phosphorylation, and AP-1 activation in KB-3 carcinoma cells (Berry et al., 2001; Fan et al., 2001; Brantley-Finley et al., 2003). The increase in c-Jun expression is blocked by the JNK inhibitor SP600125, suggesting that c-Jun induction occurs via a JNK-dependent pathway (Brantley-Finley et al., 2003). Various approaches have been used to decipher the role of this pathway in the cellular response to this drug, but these have not produced a consistent answer. For example, whereas a dominant-negative to c-Jun rendered KB-3 cells more vinblastine-resistant (Fan et al., 2001), suggesting a proapoptotic role, c-Jun knockout fibroblasts, predicted to be more resistant, were equally as sensitive as wild-type cells (Obey et al., 2005). In addition, stable or inducible overexpression of c-Jun renders cells markedly resistant to vinblastine (Obey et al., 2005; Duan et al., 2007). These results may reflect cell type-specific differences in c-Jun function and probably reflect the fact that c-Jun plays a role in both cell proliferation and cell death and can functionally participate in many different protein complexes.
The mechanism(s) underlying the robust induction of c-Jun protein observed in vinblastine-treated cells has not been established. Several possibilities exist. One is through transcriptional regulation. The human c-Jun promoter has several regulatory elements responsive to different signal transduction pathways. An AP-1 site at -71 to -64 is particularly responsive to c-Jun/ATF2 heterodimers and confers the ability of the c-Jun promoter to be autoregulated by its product (Angel et al., 1988). Another is through enhanced protein stability. For example, c-Jun is targeted for ubiquitin-mediated degradation by inactive JNK, and JNK-mediated phosphorylation protects against degradation (Fuchs et al., 1998).
In this article, we show that the increase in c-Jun expression in response to vinblastine is through a transcriptional autoamplification mechanism involving the proximal AP-1 site in the c-Jun promoter. This suggested the use of small interfering RNA (siRNA) to block the amplification cycle, and this approach effectively eliminated c-Jun mRNA and protein induction in response to vinblastine. Apoptosis was significantly inhibited in c-Jun siRNA-treated cells compared with control cells, indicating that c-Jun induction plays an important proapoptotic role, and we present evidence implicating the cyclin-dependent kinase inhibitor, p21, as a key target.
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Materials and Methods |
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Cell Culture. The KB-3 human cervical carcinoma cell line was maintained in monolayer culture at 37°C and 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 4.5 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. HCT116 colon carcinoma cells, both wild-type and p53-null, were grown in McCoy's 5A medium supplemented with 10% fetal bovine serum.
Cell Extraction and Immunoblotting. Whole-cell extracts were prepared, and immunoblotting was performed as described previously (Berry et al., 2001) using 15 µg of protein per lane for samples derived from experiments involving siRNA and 50 µg of protein per lane otherwise. Quantitation of relative protein expression from immunoblot films was determined by scanning densitometry using an HP ScanJet 6200C scanner (Hewlett Packard, Palo Alto, CA) and ImageQuaNT software (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK).
AP-1 Luciferase Assay. Cells (1.5 x 105 cells/well) were plated in 12-well plates 1 day before transfection and then cotransfected with 1 µg of 7x pAP-1-Luc (firefly luciferase under control of seven copies of an AP-1 consensus element) (Stratagene, La Jolla, CA) and 0.1 µg of pRL-TK (Renilla reniformis luciferase under control of thymidine kinase promoter) (Promega) using Lipofectamine Plus reagent (Invitrogen, Carlsbad, CA). After 24 h, cells were treated with vehicle (0.1% DMSO) or 30 nM vinblastine for various times and harvested for the determination of firefly and R. reniformis luciferase activities using the Dual-Luciferase Assay System (Promega) and a TD-20/20 Luminometer (Turner Biosystems, Sunnyvale, CA). Luciferase reporter constructs containing the human c-Jun promoter region -79 to +170, either wild-type or with the AP-1/CRE site at -71 to -64 mutated were used in similar experiments. The results are expressed as average relative firefly luciferase activity normalized to R. reniformis luciferase activity.
Real-Time Polymerase Chain Reaction. Quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) was performed according to the manufacturers' protocols using reagents from Bio-Rad Laboratories (Hercules, CA) and the Prism 7700 Sequence Detection System and software of Applied Biosystems (Foster City, CA). Total RNA was isolated using RNeasy extraction kit (Qiagen, Valencia, CA). Total RNA (1 µg) was used to synthesize cDNA using the iScript cDNA kit to quantify transcripts for c-Jun, and the control housekeeping gene GAPDH. Real-time RT-PCR reactions were performed using the iTaq SYBR Green Supermix with ROX and specific primers designed using the Taqman Probe and Primer Design function (ignoring the probe) of the Primer Express version 1.5 software. The c-Jun (5'-GAGAGGAAGCGCATGAGGAA-3' and 5'-CCAGCCGGGCGATTC-3'; accession number NM_002228 [GenBank] , National Center for Biotechnology Information database) and GAPDH primers (5'-GAAGGTGAAGGTCGGAGTC-3' and 5'-GAAGATGGTGATGGGATTTC-3'; accession number NM_002046 [GenBank] , National Center for Biotechnology Information database) were synthesized by IDT DNA (Coralville, IA). Standard curves for each assay were generated using 4-fold serial dilutions of a cDNA pool containing all samples. Standard curves ranged from 20.0 to 0.08 ng (RNA equivalents of cDNA). Optimal primer concentrations were determined to be 160 nM for c-Jun and 300 nM for GAPDH to reach at least 91% amplification efficiency. Relative gene expression in 1.25 ng of RNA equivalents of cDNA for each individual sample was calculated using standard curves, plotted as log of the nanograms of input RNA versus cycle number. Results are presented as the ratio of c-Jun to GAPDH expression relative to a standard curve for each assay.
Cell Death ELISA. Untreated and vinblastine-treated KB-3 cells were diluted to a concentration of 5 x 104 cells/ml for measurement of apoptosis using the Cell Death Detection ELISA kit (Roche, Penzberg, Germany).
Optimization of siRNA Transfection. siRNA transfection conditions were optimized using Silencer GAPDH siRNA control (Ambion). Fluorescein labeling of validated GAPDH siRNA using 6-carboxy-fluorescein was performed using Label IT Nucleic Acid Labeling kit following the manufacturer's protocol (Mirus Corporation, Madison, WI). Transfection of fluorescent-labeled siRNA was performed using KB-3 cells grown on 8-chamber culture slides (BD Falcon; BD Biosciences, San Jose, CA). Cells at 40% confluence were transfected with 50 nM fluorescein-labeled GAPDH siRNA using Oligofectamine (2 µl per 250 µl of Opti-MEM low-serum media) (Invitrogen). Twenty-four hours after transfection, cells were fixed using 4% paraformaldehyde, and slides were analyzed as described previously (Bene et al., 2004).
Transfection of c-Jun siRNA. KB-3 cells were plated in 24-well plates to achieve 30 to 40% confluence by the next day. Cells were washed twice with OPTIMEM low-serum media and transfected with 50 nM concentration of a pool of four siRNAs targeting different regions against c-Jun (SMART pool; Dharmacon, Lafayette, CO) or a pool of four nontargeting control siRNAs recommended by the manufacturer (siCONTROL; Dharmacon). After 24 h, cells were treated with vehicle (0.1% DMSO) or 30 nM vinblastine for 24 h. For longevity of siRNA action, KB-3 cells were transfected with the pool of c-Jun siRNAs for 24, 36, 48, or 72 h before application of 30 nM vinblastine for another 24 h.
Caspase-3 Assay. After treatment, cells were pelleted, washed twice in phosphate-buffered saline and lysed in 20 mM HEPES, pH 7.5, 10% sucrose, 0.1% CHAPS, 2 mM dithiothreitol, 0.1% Nonidet P-40, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A at 4°C. After centrifugation (16,000g, 10 min), caspase-3 activity in the supernatant was assayed with DEVD-amino-4-methyl coumarin (AMC) as substrate. Extracts (50 µg of protein) were incubated in 0.25 ml of 100 mM HEPES, pH 7.4, 10% sucrose, 0.1% CHAPS, 10 mM dithiothreitol, and 50 µM DEVD-AMC for 60 min at 30°C. The amount of liberated fluorescent AMC was determined using a Spectrofluorometer (PerkinElmer Life and Analytical Sciences, Waltham, MA) with an excitation wavelength of 380 nm and an emission wavelength of 460 nm (Stennicke and Salvesen, 2000).
Chromatin Immunoprecipitation. Chromatin immunoprecipitation was performed as described previously (de Belle et al., 2000) with slight modifications. All solutions contained the following protease and phosphatase inhibitors: EDTA-free protease inhibitor cocktail tablet (Roche), 20 mM β-glycerophosphate, 1 mM Na3VO4, 1 µM okadaic acid, and 1 mM phenylmethylsulfonyl fluoride. After cross-linking of cells with formaldehyde, nuclear extracts were prepared and sonicated to shear chromatin to an average size of 
500 base pairs. Samples were precleared with Protein A/G Sepharose beads, and c-Jun antibody (1-100 dilution) (Santa Cruz Biotechnology) was added to 1.5 mg of protein sample. A part of each captured immunocomplex was used for immunoblot analysis to confirm the presence of c-Jun in the chromatin pull-down. For the remaining sample, cross-links in the immunoprecipitated chromatin were reversed by heating with proteinase K at 65°C overnight, and DNA was purified by the MinElute Reaction Cleanup column (Qiagen) and resuspended in water. The chromatin immunoprecipitation (ChIP)-captured DNA sample and sonicated nuclear extract consisting of genomic DNA were subject to RT-PCR with p21 promoter-specific primers (IDT DNA), which amplify 446 base pairs of the 2101 to 2547 region of the human p21 promoter (accession number U24170
[GenBank]
) (p21 promoter sense: 5'-CTC ACA TCC TCC TTC TTC AG-3'; p21 promoter antisense: 5'-CAC ACA CAG AAT CTG ACT CCC-3'). The PCR profile was 3 min at 95°C (denaturation); 30 cycles of 30 s at 95°C; 30 s at 54°C (annealing temperature); 45 s at 72°C (primer extension); and 5 min at 72°C (final extension). PCR products were analyzed by gel electrophoresis using 1.2% agarose.
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Results |
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; Stone and Chambers, 2000; Brantley-Finley et al., 2003). Consistent with these results, vinblastine increased AP-1-dependent transcription with a similar time course, as determined by the use of a luciferase reporter driven by several copies of an AP-1 consensus element (Fig. 1B). To determine whether the increase in c-Jun protein expression was at the mRNA level, c-Jun mRNA was measured by real-time RT-PCR. c-Jun mRNA, determined as a ratio with respect to GAPDH mRNA, increased 10-fold with a maximum after 16 h of vinblastine treatment (Fig. 1C).
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; van Dam et al., 1993). We therefore transfected cells with a luciferase reporter controlled by a -79 to +170 sequence of the c-Jun promoter. Vinblastine strongly stimulated the reporter, whereas a reporter under the control of a mutated promoter lacking a functional AP-1 site was almost completely nonresponsive to vinblastine (Fig. 1D). These results suggest that vinblastine induces c-Jun at the transcriptional level through the proximal AP-1 site in the c-Jun promoter. Vinblastine-Induced c-Jun Expression Was Blocked by Inhibitors of Transcription or Translation. To test the possibility that vinblastine-induced c-Jun expression may be dependent on both transcription and translation, KB-3 cells were pretreated with cycloheximide or actinomycin D for 30 min and then treated with 30 nM vinblastine for 16 h. Whole-cell extracts were prepared and subjected to immunoblotting for c-Jun and phospho-c-Jun. As shown in Fig. 2A, the inhibitors completely blocked vinblastine-induced expression of c-Jun and phospho-c-Jun. We next determined whether inhibition of c-Jun synthesis influenced vinblastine-induced apoptosis. KB-3 cells were pretreated with cycloheximide or vehicle for 30 min and then treated with 30 nM vinblastine or vehicle for 48 h. Apoptosis was quantitatively assessed as described under Materials and Methods (Fig. 2B). Apoptosis was induced by vinblastine alone as expected. Cycloheximide alone also induced apoptosis. In contrast, the combination of both vinblastine and cycloheximide led to a significantly decreased level of apoptosis relative to vinblastine alone. These results suggest that protein translation is required for vinblastine-induced apoptosis.
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Vinblastine-Induced Apoptosis Was Inhibited in Cells Transfected with c-Jun siRNA. Having established a highly effective method to block vinblastine-induced c-Jun expression, we next evaluated the effect of c-Jun siRNA on vinblastine-induced apoptosis (Fig. 3D). In the absence of vinblastine, a very low basal level of apoptosis was observed that was similar for untransfected cells or cells transfected with control or c-Jun siRNA. When cells were treated with vinblastine, apoptosis was highly significantly reduced in cells pretransfected with c-Jun siRNA compared with untransfected cells or cells pretransfected with control siRNA (Fig. 3D). To further document the effect of siRNA on vinblastine-induced apoptosis, caspase-3 assays were conducted. As shown in Fig. 3E, caspase-3 activity was detected in KB-3 cells at 24 h and increased over the next 24 h, as observed previously (Fan et al., 2001). At the 24- and 36-h time points, caspase-3 activity was increased slightly but not significantly by control siRNA (p value < 0.09 at 24 and 36 h). In contrast, significantly lower caspase-3 activity was observed in KB-3 cells transfected with c-Jun siRNA (p value < 0.02 at 24 h, <0.0004 at 36 h). By 48 h after treatment, caspase-3 activity was comparable in control and c-Jun siRNA-transfected KB-3 cells and not significantly different. These results indicate a marked decrease in apoptosis and a delay in caspase-3 activation in response to vinblastine when c-Jun induction is blocked, implicating a proapoptotic role for c-Jun in this context. The reduced apoptosis at 48 h may be a direct consequence of the earlier delay in caspase-3 activation or, because caspase-3 activity returned to normal, may reflect the coexistence of caspase-independent apoptotic pathways.
p21 as a Key Target of c-Jun. To search for target genes that might mediate the proapoptotic effects of c-Jun, we used ChIP and hybridization to AP-1 arrays to identify promoters preferentially bound by c-Jun in vinblastine-treated KB-3 cells. c-Jun was readily immunoprecipitated from KB-3 cell chromatin, with much greater relative abundance after vinblastine treatment as expected and with no c-Jun signal when antibody was omitted (Fig. 4A). ChIP-enriched DNA was subjected to hybridization to AP-1 arrays as described previously (Hayakawa et al., 2004). The results of this study will be reported separately. One putative target gene uncovered was the CDK inhibitor p21WAF1/CIP1. ChIP-enriched DNA was used for PCR amplification using primers corresponding to the p21 promoter, as described under Materials and Methods. The p21 promoter sequence was highly enriched in the immunoprecipitate from vinblastine-treated cells and undetectable in the immunoprecipitate from control cells or when antibody was omitted (Fig. 4B, lanes 3, 4, and 5). The p21 promoter sequence was detectable with equal abundance when total chromatin from untreated cells or vinblastine-treated cells was used in parallel experiments (Fig. 4B, lanes 1 and 2). Previous studies have indicated that c-Jun can regulate the p21 promoter, and several reports have indicated that c-Jun can directly repress p21 (Tsao et al., 1996; Wang et al., 2000). Consistent with this possibility and with the demonstration of an association of c-Jun with the p21 promoter (Fig. 4B), we observed a time-dependent decrease in p21 expression in vinblastine-treated KB-3 cells (Fig. 4C). Indeed, the pattern of p21 expression was reciprocal to that of c-Jun. Figure 4C also shows p53 expression, which, as reported previously (Fan et al., 2001), is found in relatively low levels in KB-3 cells and undergoes a transient reduction in expression after vinblastine treatment.
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). KB-3 cells are a human cervical carcinoma HeLa subline, and as such, they are considered compromised for p53 function by virtue of expression of the HPV E6 oncoprotein. Although the data of Fig. 4C show a reciprocal relationship between c-Jun and p21 expression after vinblastine treatment, they do not address whether p53 acts as an intermediary regulator. To answer this question, we used HCT116 cells, both wild-type and p53-null. The cell lines were treated with vinblastine for different periods up to 48 h, and cell extracts were subjected to immunoblotting. The 24- and 48-h time points are presented in Fig. 5. In HCT116 wild-type cells, vinblastine robustly induced c-Jun expression with a maximum at 24 h and a decrease in expression at 48 h. However, p53 and p21 levels remained unaltered. In p53-null cells, vinblastine induced c-Jun expression to a similar extent and with a similar time course as observed in wild-type cells (Fig. 5). However, in p53-null cells, p21 levels declined markedly with vinblastine treatment. Taken together, these results suggest that vinblastine-induced c-Jun may down-regulate p21 only in cells that lack wild-type p53 function.
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Discussion |
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B, Raf-1, Bcl-2 proteins, and others (Bhalla, 2003; Mollinedo and Gajate, 2003). Despite this range of responses, one feature common among all cell lines examined is activation of the JNK pathway, first documented in KB-3 cells (Osborn and Chambers, 1996). Although JNK can phosphorylate a diverse spectrum of substrates, JNK activation usually entails phosphorylation of c-Jun and activation of AP-1-dependent transcription (Davis, 2000). We have shown previously (Berry et al., 2001) and confirmed in this study that treatment of KB-3 cells with vinblastine leads to c-Jun induction and AP-1 activation. The aims of this study were to define the mechanism of c-Jun induction and to determine its role in apoptosis. The results support a model for regulation at the transcriptional level and provide evidence for a proapoptotic role involving p21 regulation.
The robust increase in c-Jun expression observed in vinblastine-treated cells is JNK-dependent, because we have shown previously that specific inhibition of JNK by SP600125 completely blocks induction (Brantley-Finley et al., 2003). JNK can regulate c-Jun levels both by increasing c-Jun transcription (Karin, 1995) and/or by increasing c-Jun stability (Fuchs et al., 1998). Although the present data do not exclude the latter as a contributory factor, the evidence strongly supports a transcriptional autoamplification mechanism: (1) c-Jun expression is blocked by inhibitors of transcription or translation; (2) c-Jun mRNA levels increase before and parallel with the increase in c-Jun protein; (3) vinblastine activates the c-Jun promoter via the proximal AP-1 site; and (4) siRNA to c-Jun blocks c-Jun induction at both the mRNA and protein levels. Given these data, it seems likely that the increase in c-Jun expression proceeds via JNK-mediated phosphorylation of pre-existing c-Jun and ATF2, which combine to activate the c-Jun promoter at the proximal AP-1 site, leading to an increase in c-Jun mRNA and protein, which is in turn phosphorylated by JNK, resulting in further amplification of this autoregulatory loop. Although other stressful stimuli that result in c-Jun induction, such as UV irradiation, have been shown previously to operate via such a mechanism (Angel et al., 1988; Karin, 1995), these results are the first to provide definitive evidence that microtubule inhibitors also increase c-Jun levels through an analogous autoregulatory mechanism.
Vinblastine and other antimitotic drugs promote a robust increase in c-Jun expression not only in KB-3 cells but in all cell lines examined to date (C. Lyle and T. Chambers, unpublished observations), suggesting that it is a key response to microtubule inhibition. However, in the absence of an effective and specific way to block c-Jun function or expression, the role of this pathway has been difficult to decipher. The most compelling results were derived from stable expression of TAM-67, a dominant-negative c-Jun mutant, which rendered KB-3 cells more resistant to vinblastine (Fan et al., 2001). Given the present results that vinblastine induces c-Jun mRNA expression, an opportunity was presented to test siRNA as an approach to block expression and probe function. In general, siRNAs have been used to silence constitutively expressed mRNAs, and there are few reports describing the knockdown of an mRNA induced by a stress stimulus (Devi, 2006). In our system, c-Jun siRNA was extremely effective in reducing the increase in c-Jun mRNA and protein in vinblastine-treated cells. Under these conditions, vinblastine-induced apoptosis was significantly decreased to approximately 60% of control levels (Fig. 3D). These results indicate that c-Jun plays a major proapoptotic role. However, the fact that there remains significant cell death when c-Jun induction is repressed suggests the existence of alternate pathways. Indeed, a major effect of c-Jun depletion seemed to be in delay, not overall inhibition, of caspase-3 activation (Fig. 3E).
To gain insight into the mechanism of c-Jun in vinblastine-induced apoptosis, we sought to identify candidate target genes. Using chromatin immunoprecipitation and promoter array hybridization, as described previously (Hayakawa et al., 2004), one of the promoters identified by this method was that of p21 (N. Upreti and T. Chambers, unpublished observations). Chromatin immunoprecipitation confirmed an association of c-Jun with the p21 promoter after vinblastine treatment (Fig. 4B). c-Jun has been shown capable in different studies of up-regulation or repression of p21 transcription. In one study, c-Jun transactivated the p21 promoter in collaboration with Sp1 but was unable to transactivate the p21 promoter in the absence of Sp1 (Kardassis et al., 1999). In several other studies, c-Jun was shown to repress p21 transcription (Tsao et al., 1996; Wang et al., 2000). We observed down-regulation of p21 concomitant with c-Jun up-regulation in KB-3 cells, suggesting that c-Jun occupancy causes repression of the p21 promoter.
The down-regulation of p21 by c-Jun may be a mechanism to prevent cell cycle arrest after aberrant mitotic exit, promoting cycling of impaired cells and thus facilitating an apoptotic outcome. In this regard, the function of c-Jun in the response to microtubule inhibition may be analogous to that in the UV response, in which a major role of c-Jun is to promote cell cycle re-entry of UV-damaged cells (Shaulian et al., 2000). However, in the UV response, c-Jun acts as a negative regulator of p53 and indirectly causes p21 down-regulation. This prompted us to determine whether the down-regulation of p21 in response to vinblastine occurred in a p53-dependent or -independent manner. We found that in cells with wild-type p53 function, vinblastine failed to down-regulate p21, whereas in p53-null cells, p21 down-regulation was observed, similar to the results with KB-3 cells, which have compromised p53 functionality. Although a broader spectrum of cells will need to be examined before a general consensus can be formed, it is interesting to note that the binding sites for c-Jun in the p21 promoter overlap with those of p53. Thus, the distal promoter region, at -1394 to -2301, contains several binding sites for p53, and within this region, there is also a binding site for c-Jun (Chung et al., 2002; Gartel and Radhakrishnan, 2005). Thus, p53 and c-Jun may compete for binding, and this may explain why c-Jun is an effective regulator only in the absence of wild-type p53. A testable hypothesis is that c-Jun may play a greater role in p21 regulation in cells with compromised p53 function, which includes the majority of cancer cells. It is interesting that previous studies have shown that recruitment of myc to the p21 promoter blocks p21 induction by p53 (Seoane et al., 2002). Thus, competitive interaction of transcription factors seems to be a key mechanism for regulation of p21 transcription.
In summary, we have demonstrated that siRNA is an effective means to knock down c-Jun mRNA induced in response to vinblastine treatment, and we presented evidence for a proapoptotic role that may be mediated, at least in part, through p21 down-regulation. The demonstration of highly effective silencing of a gene induced by a stressful stimulus represents a powerful application of siRNA. The approach described here may be of considerable usefulness in further exploration of the role of c-Jun in the response to microtubule inhibition and other systems in which c-Jun induction plays a key role.
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Acknowledgements |
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Footnotes |
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S.N.K. and A.B. contributed equally to this work.
ABBREVIATIONS: JNK, c-Jun NH2-terminal protein kinase; AP-1, activating protein 1; DMSO, dimethyl sulfoxide; RT, reverse transcription; siRNA, small interfering RNA; PCR, polymerase chain reaction; VBL, vinblastine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]1-propane-sulfonate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ChIP, chromatin immunoprecipitation; ELISA, enzyme-linked immunosorbent assay; AMC, amino-4-methyl coumarin; SP600125, anthra(1,9-cd)pyrazol-6(2H)-one 1,9-pyrazoloanthrone.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Timothy Chambers, Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Mail Slot 516, 4301 W. Markham Street, Little Rock, AR 72205-7199. E-mail: chamberstimothyc{at}uams.edu
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