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[Cancer Research 62, 3257-3263, June 1, 2002]
© 2002 American Association for Cancer Research

Molecular Biology and Genetics

Targets of c-Jun NH2-terminal Kinase 2-mediated Tumor Growth Regulation Revealed by Serial Analysis of Gene Expression

Olga Potapova1, Sergey V. Anisimov, Myriam Gorospe, Ryan H. Dougherty, William A. Gaarde, Kenneth R. Boheler and Nikki J. Holbrook2

Cell Stress and Aging Section, Laboratory of Cellular and Molecular Biology [O. P., M. G., R. H. D., N. J. H.] and Molecular Cardiology Unit, Laboratory of Cardiovascular Science [S. V. A., K. R. B.], Gerontology Research Center, National Institute on Aging-IRP, Baltimore, Maryland 21224, and Isis Pharmaceuticals, Inc., Carlsbad, California 92008 [W. A. G.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although the c-Jun NH2-terminal kinase (JNK) pathway has been implicated in mediating cell growth and transformation, its downstream effectors remain to be identified. Using JNK2 antisense oligonucleotides (JNK2AS), we uncovered previously a role for JNK2 in regulating cell cycle progression and survival of human PC3 prostate carcinoma cells. Here, to identify genes involved in implementing JNK2-mediated effects, we have analyzed global gene expression changes in JNK2-deprived PC3 cells using Serial Analysis of Gene Expression. More than 40,000 tags each were generated from control and PC3-JNK2AS libraries, corresponding to 15,999 and 20,698 unique transcripts, respectively. Transcripts corresponding to transcription factors, stress-induced genes, and apoptosis-related genes were up-regulated in the PC3-JNK2AS library, revealing a significant stress response after the inhibition of JNK2 expression. Genes involved in DNA repair, mRNA turnover, and drug resistance were found to be down-regulated by inhibition of JNK2 expression, further highlighting the importance of JNK2 signaling in regulating cell homeostasis and tumor cell growth.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The JNK3 signaling pathway has been studied extensively within the context of cellular stress, where activation and/or stabilization of target transcription factors involved in mediating stress-induced gene expression (including c-Jun, JunB, ATF2, Elk-1, c-myc, and p53) are regulated via JNK phosphorylation (reviewed in Refs. 1, 2, 3, 4 ). Much less is known about the role of JNK in regulating basal cell growth, but recent studies have implicated nonactivated JNK in regulating the ubiquitination and degradation of several of its substrates, suggesting important regulatory functions of the proteins in the absence of their phosphorylation (5, 6, 7) . Among three JNK genes identified, JNK1 and JNK2 are ubiquitously expressed. The roles of the resulting proteins during mouse development have been investigated through gene-targeting experiments (8, 9, 10, 11) . Although it has been suggested that JNK1 and JNK2 might have selective or preferential roles in mediating stress-induced apoptosis and regulating gene expression (12, 13, 14) , the functional significance of the different JNK isoforms in human cells is far from clear.

We have used isoform-specific JNKAS to investigate the roles of JNK1 and JNK2 in influencing human tumor cell growth (15, 16, 17, 18) . Although JNKAS treatment had little or no effect on either untransformed cells or tumor cells possessing functional p53 protein, it caused marked growth inhibition of several p53-deficient human tumor cell type lines (17 , 18) . Such JNK-mediated growth regulation was associated with a block in DNA synthesis in certain cell types (17) , whereas it triggered S phase accumulation and apoptosis in other cells (18) . Importantly, administration of JNKAS to athymic mice bearing human prostate carcinoma PC3 xenografts was also shown to inhibit tumor growth significantly (19) , providing in vivo evidence that interruption of the JNK signaling inhibits tumor progression.

Considering the vast number of studies dedicated to the elucidation of JNK function, relatively little is known about JNK’s influence on gene expression under normal growth conditions. To identify novel targets of JNK2 signaling in human prostate carcinoma PC3 cells, we used antisense technology to acutely inhibit JNK2 expression in otherwise unstressed cells, then investigated the resulting gene expression patterns using SAGE (20) . This high-throughput method of gene expression analysis has been used previously to identify and clone genes differentially expressed in cancer versus normal cells and to clarify functions of key growth regulatory molecules, such as p53 (21, 22, 23) .


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture, Treatment with Oligonucleotides, and Cell Growth Assay.
Human prostate carcinoma PC3 cells (American Type Culture Collection) were cultured in RPMI medium (Life Technologies, Inc.). Phosphorothioate oligonucleotides targeting JNK expression were delivered to cells by transfection as described previously (24 , 25) . Briefly, oligonucleotides diluted to a final concentration of 0.4 µM in 10 µg/ml lipofectin (Life Technologies, Inc.) were applied to cells for 16 h. Cell viability, measured in triplicate, was determined using the MTS reagent (Promega) in 96-well tissue culture plates. Cell viability is expressed as percentage of viable cell mass after treatment with oligonucleotides relative to that of untreated cells.

Northern Blot Analysis.
Total RNA was isolated using RNA Stat-60 (Tel-Test B) and processed using standard methodologies. cDNAs were isolated from plasmids or obtained by PCR and labeled with [{alpha}-32P]-dATP by the random primer method. Oligonucleotides complementary to human p21Cip1/Waf1 mRNA (3'-CTCCGTGACGAAGTCAAAGTTCCACCGTTCTCGGGCCTCCTGGAGACAGCC-5') and 18S rRNA (3'-ACGGTATCTGATCGTCTTCGAACC-5') were labeled with [{alpha}-32P]-dATP and Terminal Transferase (Boehringer Mannheim). Hybridization was performed by the method of Church and Gilbert (26) , and signals were visualized using a STORM system (Molecular Dynamics).

Flow Cytometric Analysis and Biochemical Assessment of Apoptosis.
Cells were prepared using the Cellular DNA Flow Cytometric Analysis kit (Boehringer Mannheim) and analyzed using a FACScan flow cytometer (Becton Dickinson) and the Multicycle (Phoenix Flow) software program. Oligonucleosomal fragments released from nuclei because of degradation of genomic DNA and, therefore, present in cytoplasmic extracts were assayed using a Cell Death Detection ELISA Plus kit (Boehringer Mannheim). All experiments were carried out in triplicate.

Generation and Analysis of SAGE Libraries.
SAGE libraries were derived from mock- and JNK2AS-treated PC3 cells 24 h post-treatment. SAGE was performed essentially as described (20) with minor modifications (27) . Briefly, poly (A)+ mRNA was prepared using oligo(dT)25 magnetic beads (Dynal), and cDNA was synthesized with a 5' biotinylated oligo(dT)18 using a cDNA Synthesis System (Life Technologies, Inc.). Tags were generated using NlaIII and BsmFI restriction enzymes (New England Biolabs). Positive clones were selected on Zeocin-containing plates (Invitrogen), tested for the quality of inserts, and prepared for sequencing using the QIAprep 96 Turbo Miniprep system (Qiagen). Tag sequences were generated using SAGE analysis software 3.04-beta.4 To increase data reliability, clones with fewer than or equal to four tags were excluded from further consideration as they could contain random sequences recognized as tags. Abundances of remaining tags were normalized to the total number of reliable tags in each library. Statistical analysis was performed using the formula [N1 - k (N11/2)] - [N2 + k (N21/2)] > 0, where N1 is the higher tag frequency and k = 2.58 for P < 0.01 (23) . Genes corresponding to each tag were identified using the GenBank5 and UniGene6 databases.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition of JNK Expression by JNKAS Results in Growth Suppression of PC3 Cells.
To investigate the involvement of the JNK pathway in regulating basal growth of PC3 cells, we used specific JNK1 and JNK2AS (15 , 24) , delivered at nearly 100% transfection efficiency (Fig. 1A)Citation . A substantial isoform-specific reduction in the levels of mRNAs encoding JNK1 and JNK2 was accomplished after 16–18 h of treatment with 0.4 µM of the corresponding JNKAS oligonucleotides, whereas control treatments (mock transfection or transfection with each of four different control oligonucleotides) lacked any effect on JNK mRNA levels (Fig. 1B)Citation . Maximum mRNA suppression was seen 24 h after transfection, with JNK mRNA levels returning to normal ~72 h after transfection (data not shown). Inhibition of JNK expression was confirmed by Western analysis (Fig. 1C)Citation . Treatment with either JNK1AS or JNK2AS markedly attenuated both JNK46kDa and JNK54kDa proteins, although it did not completely eliminate either of them.



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  		Fig. 1. Effects of JNKAS on JNK expression and growth of PC3 cells. Cells treated with 0.4 µM the oligonucleotides indicated were analyzed 24 h later. A, PC3 cells were transfected with FITC-labeled oligonucleotide and subjected to confocal fluorescence microscopy (x800 magnification; left panel). Phase-contrast image of the same field (right panel) indicates nearly 100% efficiency of oligonucleotide uptake. B, inhibition of JNK1 and JNK2 mRNAs expression by JNKAS but not by JNKS or JNKScr. C, reduction in JNK protein levels as assessed by Western blot analysis using SC-571 JNK antibodies (Santa Cruz Biotechnology). JNK protein levels in the JNKAS treatment groups range between 25 and 40% of the JNK levels measured in JNKScr-treated cells. D, PC3 cell viability after JNKAS treatment. The degree of growth inhibition seen in JNK1AS- and JNK2AS-treated cells is significantly different (Student’s test P < 0.001).

 
To examine the influence of JNK inhibition on growth of PC3 cells, an MTS-based assay was used. Viable cell masses of mock- and JNKScr-treated cultures did not differ significantly from those of untreated cells. However, treatment with either JNK1AS or JNK2AS led to a significant reduction in viable cell mass within 24 h after transfection (Fig. 1D)Citation . Growth suppression was more pronounced with JNK2AS than with JNK1AS treatments (44 and 23% decrease, respectively). Assessment of DNA synthesis rates using BrdU showed no inhibition of DNA synthesis in JNKAS-treated cultures (data not shown).

Growth Inhibition of JNK2-deprived PC3 Cells Results from Cell Cycle Arrest and Apoptosis.
Morphological examination of JNK2AS-treated PC3 cultures clearly revealed features characteristic of cells undergoing apoptosis that were not apparent in either mock- or JNKScr-treated cells. JNKAS-treated cells exhibited membrane blebbing, rounding up of cells, and cell detachment, as seen by phase-contrast microscopy (Fig. 2A)Citation . Fragmented and condensed nuclei were visualized using 4'-6-diamidino-2-phenylindole (data not shown), and oligonucleosomal DNA fragments were apparent using an ELISA-based assay (Fig. 2B)Citation . Finally, changes in cell size and DNA content consistent with growth inhibition and apoptosis were evident by fluorescence-activated cell sorting (Fig. 2, C and D)Citation . With respect to this last parameter, the cell cycle distribution of mock- and JNKScr-treated PC3 cells was very similar to that of untreated cells: G1 = 28 ± 3%; S = 31 ± 5%; G2-M = 37 ± 5%. In contrast, JNK1AS- and JNK2AS-treated cultures showed a marked accumulation of cells in S phase (56 and 72%, respectively; Fig. 2CCitation ). Assessment of cells with reduced DNA content (sub-G1 cells) by flow cytometry revealed no cells with less than 2N DNA content in either mock- or JNKScr-treated cells, whereas >10% of JNK2AS-treated populations had less than 2N DNA content, indicative of apoptosis (Fig. 2D)Citation .



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  		Fig. 2. Cell cycle arrest and apoptosis in JNK-deprived PC3 cells. Cells were treated with 0.4 µM corresponding oligonucleotides and analyzed 24 h post-treatment. A, microscopic appearance of JNKAS-treated PC3 cells (x400 magnification). Untreated (data not shown), mock-, and JNKScr-treated control cultures were indistinguishable, whereas JNKAS-treated (particularly JNK2AS-treated) populations exhibited different morphologies. B, apoptosis assessment by quantitation of oligonucleosomal fragments ("Materials and Methods"). C, distribution of cells in each cell cycle compartment. D, representative flow cytometry profiles.

 
Changes in the Global Gene Expression Profile of PC3 Cells after JNK2AS Treatment.
The altered cell cycle distribution and presence of apoptotic cells in JNKAS-treated cultures suggested that expression of cell cycle/apoptosis regulatory proteins might be affected by the JNK proteins. Because of the more pronounced effects seen with JNK2AS treatment, additional studies were restricted to JNK2AS. To identify JNK2-regulated genes in PC3 cells, SAGE libraries were prepared from JNK2AS- and mock-treated PC3 cells, and differences in gene expression patterns compared. SAGE is based on the generation and analysis of libraries of short tags that are unique to individual transcripts. A total of 42,567 and 40,797 tags was obtained from the PC3-JNK2AS and PC3-Mock libraries, respectively. After exclusion of ~3–4% of artifactual tags, analysis of the remaining tags revealed 20,698 and 15,999 unique transcripts in the PC3-JNK2AS and PC3-Mock libraries, respectively. Individual tag numbers varied from 1 to 350 in the PC3-JNK2AS library and from 1 to 239 in the PC3-Mock library (Fig. 3, A and B)Citation . Most transcripts existed at equal levels in both libraries, and >70% of transcripts were detected only once (Fig. 3)Citation .



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  		Fig. 3. Gene expression profiles in SAGE libraries. Tags (39,133 and 40,998, respectively) were obtained from PC3-Mock and PC3-JNK2AS libraries. Tag frequencies in each library were continuous, suggesting normal distribution of the data. Designated with letters are the sums of transcript abundances in the range of 100–150 (a), 150–200 (b), 200–250 (c), and 250–300 (d). A, abundance of 15,999 unique transcripts identified in the PC3-Mock library. B, abundance of 20,698 unique transcripts identified in the PC3-JNK2AS library. In C, relative normalized frequencies of transcript in the SAGE libraries were determined by the dividing total number of tags observed in each library. Ratio plot contains 80,131 tags, of which 30,845 are unique transcripts. Asymmetrical appearance of the plot results from the uneven sizes of tag libraries.

 
Among 61 transcripts that were >=2-fold more abundant in the PC3-JNK2AS library (Table 1)Citation , 18 (30%) matched to ESTs, including 5 ESTs with no similarity to known genes and one tag (CGTTGCTGGG) that did not correspond to a known sequence. Three transcription factors were markedly up-regulated in the JNK2AS group: ETR101 (16.3-fold, 17/1 tags), FRA-1 (11.9-fold, 25/2 tags), and Jun-B (8.6-fold, 9/0 tags). Expression of two apoptosis-associated genes described previously as Gadd34 and Gadd45 was also markedly higher in the JNK2AS-treated cells (8.6- and 6.7-fold, respectively). Two additional groups of genes stood out among transcripts up-regulated in the PC3-JNK2AS library: (a) those encoding proteins involved in the electron transport chain (mitochondrial cytochrome b and NADH dehydrogenase); and (b) IFN-inducible proteins (IFI78, IFI17, ISGF-3, OAS3, IFI16b, ISG15, IFI-6-16, IFI27, and 1-8U).


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  		Table 1 Examples of transcripts up-regulated in JNK2AS-treated PC3 cells

 
Among 63 transcripts displaying >2-fold lower abundance in JNK2AS-treated cells, 18 (29%) matched to ESTs, including 4 ESTs with no similarity to known genes (Table 2)Citation . Transcripts whose expression was inhibited by JNK2AS treatment included genes involved in membrane structure/transport (exchange factor p532, maxiKCa subunit, clathrin, and caveolin-1) and growth regulation [integrin ß-4, cyclin D1, uracil DNA glycosylase, Rac1, HL14, and HMG-I(Y)]. Interestingly, expression of several genes encoding snRNA-associated proteins involved in RNA processing (Lsm3; Lsm5 and CROP/Luc7A) was inhibited in the absence of JNK2. The complete gene expression profiles are available from the authors.


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  		Table 2 Examples of transcripts downregulated in JNK2AS-treated PC3 cells

 
Validation of SAGE Results by Northern Blotting.
To verify the changes in the expression levels of potential JNK-regulated genes identified by SAGE, we carried out Northern blot analysis (Fig. 4)Citation . We first confirmed the inhibition of JNK2 mRNA expression in JNK2AS-treated cells (Fig. 4A)Citation . Next, we examined the expression of SAGE-identified transcription factors and, as predicted, detected significant increases in the levels of mRNAs encoding ETR101, JunB, and FRA-1 (Fig. 4BCitation , data not shown). Because JunB, a component of AP-1 transcription factor complexes, is a substrate of JNK (5) , we assessed the expression levels of c-Jun, another AP-1 component and major JNK substrate (28) . As shown, c-Jun mRNA levels were also up-regulated in JNK2AS-treated cells (Fig. 4B)Citation . Expression of c-fos was likewise elevated in JNK2AS-treated cells (data not shown), indicating that inhibition of JNK2 expression had a major effect on the expression of transcription factors that are constituents of AP-1.



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  		Fig. 4. Validation of SAGE results by Northern blotting. A representative Northern blot result from at least two independent experiments is shown. Numbers on the right indicate the expression levels of corresponding mRNAs measured in JNK2AS-treated cultures, indicated as a percentage of the expression levels measured in JNKScr-treated cultures. A, suppression of JNK2 mRNA expression in JNK2AS-treated cultures. B, genes up-regulated after inhibition of JNK2 expression. C, stress-activated/apoptosis-associated genes up-regulated in JNK2AS-treated cells. D, genes down-regulated in JNK2AS-PC3 cells (trans/bal, transcobalamin). D, genes nonspecifically up-regulated by oligonucleotide treatment.

 
Northern analysis of the apoptosis-associated genes Gadd34 and Gadd45 verified their higher expression in JNK2AS-treated cells (Fig. 4C)Citation , and the mRNA levels of Gadd153, another gene whose expression is often up-regulated by conditions that induce Gadd34 and Gadd45, were also elevated. The induction of the Gadd genes suggested that JNK2AS-treated cells were under stress and prompted us to examine the expression of two other stress-regulated genes: (a) Grp78, a molecular chaperone involved in protein folding; and (b) the cyclin-dependent kinase inhibitor p21Cip1/Waf1, which we had found previously to be up-regulated in other JNK2AS-treated cells (17 , 18) . Both genes were markedly up-regulated in JNK2AS-treated cells (Fig. 4C)Citation , suggesting that depletion of JNK2 in PC3 cells facilitates the induction of several independent stress-signaling pathways.

Activation of the JNK pathway is important for the phosphorylation and activation of protein components of AP-1 transcription factor complexes (1, 2, 3, 4 , 5, 6, 7 , 28) , suggesting that genes regulated by AP-1-dependent transcription could be negatively affected by JNKAS treatment. Indeed, we detected reduced expression of several AP-1-regulated genes, including lamin A/C (29) , and integrin ß-4 (Ref. 30 ; Fig. 4DCitation ). Among other genes whose lower expression after JNK2AS treatment was verified by Northern blotting were HMG-I(Y), a protein highly expressed in prostate cancer (31) and recently identified as a myc-regulated oncogene (32) , Lsm5 (33) , transcobalamin, and DSS1 (Ref. 34 ; Fig. 4Citation D, and data not shown). Importantly, although most JNK2AS-altered genes appeared to be specific for treatment with this oligonucleotide, some gene expression changes were also seen in the JNK1AS- and JNKScr-treated groups, indicating that a more global response to the uptake of oligonucleotides (Fig. 4E)Citation was elicited. Within this group were two IFN-inducible genes, ISGF-3 and ISG15, suggesting that application of phosphorothioate oligonucleotides could induce IFN-dependent responses.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The activation of the JNK pathway after stress has been extensively studied and activated JNK has been implicated in regulating important cellular processes including proliferation, adhesion, motility, survival, and apoptosis (1, 2, 3, 4) . However, under normal growth conditions, JNK has also been shown to play key regulatory functions, such as controlling the stability of a number of proteins (5, 6, 7) and maintaining the growth of certain tumor cells (17 , 18) . However, the precise effectors of JNK-mediated effects on cell growth and carcinogenesis remain poorly understood. Our earlier work revealed that basal growth of tumor cells requires JNK2 functions that are independent of its activation state, as reduction of the JNK2 protein resulted in growth arrest and apoptosis (17 , 18) . The cell’s p53 status was found to profoundly influence the response of tumor cells to JNK2 depletion as, in general, cells deficient in p53 function, but not cells with normal p53 status, underwent apoptosis (18) . Our findings presented here using the p53-null PC3 cells are consistent with our previous observations and suggest that inhibition of JNK2 activity and/or expression could be beneficial for the treatment of certain cancers harboring p53 mutations.

We used SAGE to identify genes differentially expressed in JNK2AS- and Mock-treated PC3 cells. The majority of the transcripts were detected only once, and only ~0.3% (61 up- and 63 down-regulated) were differentially expressed at >=2-fold level (P < 0.01). Among these transcripts, ~30% of the tags matched to ESTs, and ~15% of tags matched to multiple genes, thus hindering immediate identification of their products.

It was expected that the JNKAS-triggered reduction in JNK expression would lead to diminished AP-1-mediated transactivation because of the decreased availability of phosphorylated/activated c-Jun. Indeed, decreased expression of several such genes identified by SAGE was confirmed by Northern blot analysis (Fig. 4D)Citation . These include integrin ß-4, whose protein product has been shown to promote carcinoma invasion (30 , 35) , lamin A/C (36, 37, 38) , and HMG-I(Y), a novel oncogene involved in chromatin structure and transcriptional regulation, with a potential role in resistance to cisplatin (32 , 39 , 40) . Reduced expression of two other genes, CROP/hLuc7A (41) and HMG17, whose products might also be involved in cisplatin resistance, further suggest a link between JNK pathway and tumor cell sensitivity to cisplatin, as suggested by our earlier work (42) . Together, this pattern of gene expression supports an important role for JNK in the restructuring of chromatin, in influencing cell-cell and cell-matrix interactions and in sensitizing human tumor cells to cisplatin.7 Among other interesting genes that are also differentially expressed as a function of JNK2 levels are ETR101, a putative transcription factor (43) , and caveolin-1, which functions in tyrosine kinase signaling and has been implicated in drug resistance and growth regulation (44, 45, 46, 47) . Several EST-matched tags (e.g., RAD51-interacting protein homologue and heat shock protein 90{alpha} homologue) are also worthy of further analysis.

Unexpectedly, mRNAs encoding several AP-1 up-regulated genes, including c-jun, Gadd153, and p21Cip1/Waf1 (48) , were markedly up-regulated in JNK2AS-treated cells (Fig. 4)Citation . Several other genes whose expression was significantly higher in JNK2AS-PC3 cells were also of special interest because of their involvement in cellular growth, cell cycle regulation, and apoptosis. We confirmed that inhibition of JNK2 expression led to the up-regulation of stress-response genes, including Gadd34, Gadd45, Gadd153, p21Cip/Waf1, Grp78, c-fos, c-Jun, and JunB (Fig. 4C)Citation , as well as genes involved in mitochondrial respiration. The heightened expression of these genes indicates the activation of diverse stress response pathways in JNK2AS-treated cells and supports the importance of the JNK pathway for homeostatic functions. The exact mechanism of induction of these genes awaits further investigation. One possible mechanism involves a feedback loop whereby the turnover of c-Jun and JunB (and that of other proteins whose degradation also depends on the presence of unstressed JNK; Refs. 5, 6, 7 ) would decrease because of the great reduction in JNK expression, thus resulting in the induced transcription of their own genes. Other possible mechanisms to explain the differential gene expression reported here may be based on changes in the activity of certain transcription factors or post-transcriptional regulatory proteins, whose activity is perturbed by inhibition of JNK2 expression. In this regard, JNK2-regulated events may directly influence mRNA turnover; among the transcripts down-regulated (and confirmed; data not shown) in the JNK2AS populations were the U6 snRNA-associated Sm-like proteins Lsm3 and Lsm5, whose yeast homologues are essential for mRNA decay (33) . This finding suggests that inhibition of their human counterparts in JNK2-deprived cells may result in the stabilization of target mRNAs and is in keeping with earlier reports, implicating JNK in regulating the turnover of mRNAs containing Adenine and Uracil (AU)-rich elements (49 , 50) .

That some JNKAS differentially expressed genes were not identified as such in the SAGE libraries (p21Cip1/Waf1 and Gadd153, among others) can be explained, at least in part, by limitations in the SAGE methodology, e.g., one tag corresponding to p21Cip1/Waf1 was omitted from the analysis during the "clean-up" procedure, thus preventing differences in expression of p21Cip1/Waf1 from being recognized as statistically significant. Additional SAGE omissions can occur from the lack of NlaIII recognition sites in the transcript or the absence of a poly(A) tail. Therefore, it is imperative that SAGE results be confirmed by other means.

Other important limitations of this study were imposed by two processes occurring concomitantly with the reduction in JNK levels: (a) transfections were carried out in serum-free medium, and gene expression assessments in the first several hours (0–12 h) after the addition of complete medium would thus be heavily confounded by "serum effects;" and (b) late time points may be strongly influenced by the apoptosis that ensues in JNK2AS-treated cells ~48 h after transfection. Taken into consideration these early and late complicating matters (serum effects and apoptosis, respectively), we deemed the time point chosen (24 h after removal of oligomers) to be the most adequate for the evaluation of bonafide JNK2-regulated gene expression. Genes implicated in apoptosis (such as Gadd45 and Gadd153) and cell cycle arrest (such as c-jun and p21) were found to be induced at this time, suggesting that both mechanisms of growth inhibition play a role in PC3 cell growth reduction by JNK2AS. Indeed, we reported previously that treatment with JNKAS can result in both cell cycle arrest (17) and apoptosis (18) of human tumor cells.

Despite the effort that has been devoted to understanding the physiological importance of JNK signaling, and the vast increase in our knowledge of its regulation, relatively little is known about the downstream effectors of JNK that mediate its diverse effects. Acute inhibition of JNK2 expression with JNK2AS oligonucleotides allowed us to suppress all JNK2 functions, including those distinct from its kinase activity. This intervention enabled us to identify a number of candidate genes that could be involved in implementing JNK’s effects on tumor cell growth. It is important to note that although some of the genes displaying altered expression represent direct targets of JNK2 signaling and are likely to be causal in inducing growth inhibition and apoptosis, others reflect secondary effects of JNK2AS-induced cell death. Obviously, additional experiments will be required to discern between these possibilities and determine the specific roles of the putative targets identified here. The current studies have provided a framework for future studies and offer novel insight into the role of JNK in regulating cell growth and homeostasis.


   ACKNOWLEDGMENTS
 
We thank the members of the National Institute on Aging-IRP Flow Cytometry Unit F. J. Chrest, C. Morris, and Dr. R. P. Wersto for valuable help with flow cytometry data acquisition and analysis and Natalia Podlutskaya for excellent technical assistance.


   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 Present address: SUGEN, Inc., 230 East Grand Avenue, South San Francisco, CA 94080. Back

2 To whom requests for reprints should be addressed, at Yale School of Medicine, 300 George Street, 8th floor, New Haven, CT 06510. Phone: (203) 737-5847; Fax: (203) 737-1801; E-mail: nikki.holbrook{at}yale.edu. Back

3 The abbreviations used are: JNK, c-Jun NH2-terminal kinase; SAGE, serial analysis of gene expression; JNKAS, c-Jun NH2-terminal kinase antisense oligonucleotide; JNKScr, c-Jun NH2-terminal kinase scrambled oligonucleotide; EST, expressed sequence tag; Gadd, growth arrest and DNA damage inducible; AP-1, activator protein 1. Back

4 Internet address: http://www.sagenet.org. Back

5 Internet address: http://ftp.ncbi.nlm.nih.gov/genBank. Back

6 Internet address: http://www.ncbi.nlm.nih.gov/SAGE/SAGEtag.cgi. Back

7 Potapova and Holbrook, unpublished observations. Back

Received 10/ 3/01. Accepted 3/22/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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