c-jun n-terminal kinase is essential for growth of human ... filec-jun n-terminal kinase is...

O. Potapova et al. Suppression of JNK expression inhibits tumor cell growth

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Revised 05-09-2000

c-Jun N-Terminal Kinase is Essential for Growth of

Human T98G Glioblastoma Cells

Olga Potapova1, Myriam Gorospe1, Frédéric Bost2, Nicholas M. Dean3, William A.

Gaarde3, Dan Mercola2, and Nikki J. Holbrook1,*

1Cell Stress and Aging Section, Laboratory of Biological Chemistry, Gerontology Research

Center, National Institute on Aging, 5600 Nathan Shock Drive, Baltimore, MD 21224;

2Sidney Kimmel Cancer Center, 10835 Altman Row, San Diego, CA 91212;

3Isis Pharmaceuticals Inc., 2280 Faraday Avenue, Carlsbad, CA 92008.

Running Title: Suppression of JNK expression inhibits tumor cell growth

*Corresponding author: Nikki J. Holbrook, phone (410)558-8446; FAX (410)558-8386

E-mail: [email protected]

Mailing address: 5600 Nathan Shock Dr., Box 12, Baltimore, MD 21224

File M9:04591 second revision.doc

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SUMMARY

The c-Jun N-terminal Kinase/Stress-Activated Protein Kinase (JNK/SAPK) pathway is

activated by numerous cellular stresses. Although it has been implicated in mediating

apoptosis and growth factor signaling, its role in regulating cell growth is not yet clear. Here,

the influence of JNK on basal (unstimulated) growth of human tumor glioblastoma T98G cells

was investigated using highly specific JNK antisense oligonucleotides to inhibit JNK

expression. Transient depletion of either JNK1 or JNK2 suppressed cell growth associated

with an inhibition of DNA synthesis and cell cycle arrest in S phase. The growth inhibitory

potency of JNK2 antisense (JNK2IC50 = 0.14 µµµµM) was greater than that of JNK1 antisense

(JNK1IC50 = 0.37 µµµµM), suggesting that JNK2 plays a dominant role in regulating growth of

T98G cells. Indeed, JNK2 antisense-treated populations exhibited greater inhibition of DNA

synthesis and accumulation of S-phase cells than did the JNK1 antisense-treated cultures, with

a significant proportion of these cells detaching from the tissue culture plate. JNK2 (but not

JNK1) antisense-treated cultures exhibited marked elevation in the expression of the cyclin-

dependent kinase inhibitor p21Cip1/Waf1 accompanied by inhibition of cdk2/cdc2 kinase

activities. Taken together, these results indicate that JNK is required for growth of T98G cells

in non-stress conditions and p21Cip1/Waf1 may contribute to the sustained growth arrest of

JNK2-depleted T98G cultures.

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INTRODUCTION

The c-Jun N-terminal Kinase/Stress-Activated Protein Kinase (JNK/SAPK)1 signal

transduction pathway consists of a cascade of protein phosphorylation reactions leading to the

activation of c-Jun N-terminal kinase (JNK). Numerous pro-inflammatory factors and other stressful

stimuli activate the JNK pathway by dual-specificity kinases (JNKKs), which are in turn activated

via phosphorylation by upstream kinases (1-3). The function of the JNK pathway has mostly been

studied within the context of cellular stress, where its activation causes both stabilization and

elevated activity of targeted transcription factors via their phosphorylation by JNK (1-4). Major

substrates of JNK include the transcription factors c-Jun (5-7), ATF-2 (8-10), Elk-1 (11-12), and p53

(13-14). Much less is known about the role of JNK in normally growing cells, but recent studies

suggest that non-activated JNK is important for regulating degradation of its substrates (4).

Three different JNK genes, designated JNK1, JNK2, and JNK3, have been described. JNK1

and JNK2 are ubiquitously expressed, while JNK3 is largely restricted to brain, heart and testis.

Each of the JNK genes produces several isoforms derived from alternative splicing, but the

functional significance of the different isoforms is unclear. JNK1 and JNK2 display distinct

substrate affinities (10, 15) and may have selective or preferential roles in different biological

processes. For example, JNK1 plays a role in the production of neurite-like structures in PC12 cells

(16) while JNK2 has been implicated in regulating expression of E-selectin (17) and EGF-stimulated

growth of A549 cells (18-19). The role of JNK in cell survival and death is complex (1-2, 20). For

instance, a rapid increase in JNK activity is required for apoptosis in cells of neuronal origin

following withdrawal of nerve growth factor (21), but in other instances, such as following TNF-α

treatment, JNK activation appears to inhibit apoptosis (22-23).


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Several lines of evidence suggest that the JNK pathway may play a role in cellular

transformation and tumor cell growth. JNK activation is required for cellular transformation by

certain oncogenes as well as by constitutively activated c-Ras, Rac, and PAK-65 (24-29), and

inhibition of the JNK pathway can reverse a transformed phenotype (30-31). In addition, growth

factors such as EGF and PDGF can cause activation of the JNK pathway (18, 32-34) and systemic

treatment of PC3 tumor-bearing mice with JNK antisense oligonucleotides inhibits tumor growth and

promotes regression in a high proportion of cases (35).

We have recently reported that treatment with JNK2-specific antisense oligonucleotides

(JNK2AS) led to apoptosis in several cell types in a p53-dependent fashion, and provided evidence

that p21Cip1/Waf1, a downstream target of p53, was an important factor in the survival of cells with

wild type p53 status (36). In a panel of human tumor cell lines containing null/mutant p53, the

human T98G glioblastoma cell line stood out as an exception, in that we failed to see any apoptosis

following JNKAS treatment. The present study was initiated to further investigate the role of JNK in

regulating basal growth (normal culture conditions) of human glioblastoma T98G cells. We

demonstrate that targeted inhibition of JNK expression, accomplished through use of specific

antisense oligonucleotides, does not result in significant apoptosis of T98G cells, but rather results in

marked growth suppression that is associated with inhibition of DNA synthesis, cell cycle arrest in S

phase and p53-independent induction of the cyclin-dependent kinase inhibitor p21Cip1/Waf1. These

findings indicate that JNK is essential for normal growth of T98G cells and suggest that the JNK

pathway regulates molecules vital for replication of these tumor cells.


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EXPERIMENTAL PROCEDURES

Cell Culture and Metabolic Labeling - Human glioblastoma T98G cells were cultured in

Dulbecco’s Modified Essential Medium (Gibco BRL), supplemented with 10% fetal bovine serum

(Irvine Scientific). For metabolic protein labeling, cells were incubated in methionine-free, cysteine-

free, and serum-free medium for 4 h in the presence of 200 µCi of TranslabelTM (ICN Chemical)

containing carrier-free [35S]-methionine/cysteine. Immediately after incubation, cells were washed

with cold PBS and lysed in 0.5 ml RIPA buffer containing protease and phosphatase inhibitors (0.15

M NaCl, 50 mM Tris-HCl, pH 7.2, 1% deoxycholic acid, 1% Triton X-100, 1 mM EDTA, 1 mM

phenylmethylsulfonyl fluoride (PMSF), 1 mM orthovanadate, 10 mM NaF and 10 mM sodium

pyrophosphate).

Treatment of Cells With Oligonucleotides - All phosphorothioate oligonucleotides used in

this study were prepared and characterized by Isis Pharmaceuticals, Inc. as previously described (18,

37). Two oligonucleotides: ISIS12539 (5'-CTCTCTGTAGGCCCGCTTGG-3') and ISIS12560 (5'-

GTCCGGGCCAGGCCAAAGTC-3'), termed JNK1AS and JNK2AS, respectively, specifically

eliminate the expression of respective JNK proteins (17-19). To control for nonspecific events

“sense” sequence oligonucleotides (JNK1S, ISIS14320; and JNK2S, ISIS14318), and “scrambled"

sequence oligonucleotides (JNK1Scr, ISIS14321; and JNK2Scr, ISIS14319) with the same base

composition as the antisense oligonucleotides, but in arbitrary order, were employed.

Oligonucleotides were delivered to cells by lipofection as previously described (18). Briefly,

solutions for lipofection were made by mixing 10 µg/ml LipofectinTM (Gibco BRL) reagent in MEM

(Gibco BRL) with an equal volume of oligonucleotide solution, incubating this mixture at room

temperature for 15 min and diluting it with LipofectinTM solution to a final oligonucleotide


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concentration of 0.4 µM. Cells were incubated with the LipofectinTM-oligonucleotide solution at

37oC in 10% CO2 for 12-16 h, after which they were washed once with serum-free medium and

cultured in complete medium.

Cell Growth and DNA Synthesis Assays - To determine the proliferation rates, cells were

seeded at 25 x 103 cells/cm2 in 6-well cluster plates and growth was assessed by daily cell counting

(Coulter counter) in triplicate. For determination of cell viability and assessing DNA synthesis, cells

were seeded at 1-5 x 103 cells/well in 96-well tissue culture plates. The viable cell mass was

determined by the addition of 3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl)-2-(4-

sulfophenyl)-2H-tetrazolium inner salt (MTS) as described in the manufacturer's protocol (Promega).

Cell viability is expressed as the ratio of viable cell mass following a given treatment to that of

untreated cells x 100 (Viable Cell Number, %). Rates of DNA synthesis were measured by

incorporation of [3H]-Thymidine into polymeric DNA. Cells were treated with oligonucleotides and

pulsed with 0.5 µCi [3H]-Thymidine/well for 3 h at the indicated times. Harvested lysates were

transferred to paper spots with a PhD-200A cell harvester (Cambridge Technologies), and the

amount of radioactive DNA quantitated by scintillation counting using Biosafe-II scintillation liquid.

Northern and Western Analyses - Total RNA was isolated from treated cells using RNA

Stat-60 (Tel-Test B) according to the manufacturer's instructions. RNA (30 µg/lane) was size-

separated in agarose-formaldehyde gels and transferred to GeneScreen Plus nylon membranes

(DuPont/NEN). JNK1 and JNK2 cDNAs, isolated from pBSJNK2-1xHA and 3xHA-JNK1SRα3,

respectively, were labeled with [α-32P]-dATP using a random-primer labeling kit (Boehringer

Mannheim). Hybridization and washes were performed by the method of Church and Gilbert (38),

and hybridization signals were visualized and quantified using a PhosphorImager (Molecular


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Dynamics). To monitor the quality of samples loading and transfer membranes were hybridized to a

24-base oligonucleotide 3’-ACGGTATCTGATCGTCTTCGAACC-5’complementary to 18S RNA.

For western analyses, 50 µg of cell whole-cell lysates (18) were size-fractionated in 12%

SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. Proteins were

detected using an enhanced chemiluminescence system (Amersham Chemicals) following incubation

of the PVDF membranes with specific antibodies (Santa Cruz Biotechnology).

Flow Cytometric Analyses - Cell cycle distribution was analyzed using PI-stained cells.

Briefly, 2-5 x 106 cells were fixed in 70% ethanol, incubated with 1 µg/ml RNase A, stained with 1

µg/ml propidium iodide (Boehringer Mannheim), and analyzed on FACScalibur flow cytometer

(Becton Dickinson). The percentages of cells in the various stages of the cell cycle were determined

using ModFitLT software program.

S-phase cells were detected using 5-Bromo-2’-deoxy-uridine (BrdU) Labeling and Detection

Kit I (Boehringer Mannheim) as described by manufacturer with some modifications. Briefly, the

cells were treated with oligonucleotides and 24 h later pulsed for 1 h with 10 µM of BrdU labeling

reagent to allow incorporation of BrdU into DNA in place of thymidine. The cells were fixed for 24

h with 70% ethanol diluted in glycine buffer (50 mM glycine, 0.01% Triton X-100, 0.15 M NaCl; pH

2.0) and incubated with anti-BrdU monoclonal antibodies. After incubation with anti-mouse-Ig-

fluorescein, bound anti-BrdU monoclonal antibodies were visualized by FACS analysis.

The number of apoptotic cells following treatment with JNKAS was determined using an

APO-BRDUTM kit (Pharmingen) following the manufacturer’s protocol. Briefly, 1-2 x 106 cells

were fixed in 1% methanol-free formaldehyde following incubation in 70% ethanol at –20oC. To


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detect genomic DNA degradation bromodeoxyuridine triphosphate (Br-dUTP) was incorporated into

DNA breakes by terminal deoxynucleotidyl transferase enzyme (TdT). Finally, cells were stained

with fluorescein-labeled anti-BrdU antibody to detect DNA breaks and with Propidium Iodide

(PI)/RNase A solution for counter staining of the total DNA. Dual parameter display was used to

assess the number of apoptotic cells following analysis on FACScalibur flow cytometer.

In vitro Kinase Activity Assays – To detect JNK activation following UV-C exposure,

JNKAS-treated and control cultures were irradiated with 40 J/m2 UV-C 24 h post-lipofection,

washed with cold PBS 30 min later, and suspended in whole-cell extract (WCE) buffer (18). An in

vitro JNK kinase assay was performed using a fusion protein containing glutathione linked to the 1-

222 fragment of human c-Jun (GST-cJun) as substrate, as described (6). Briefly, 50 µg of cell lysate

was incubated for 3 h at 4oC with 10 µg of GST-cJun bound to Glutathione Sepaharose-4B

(Pharmacia Biotech). After washing three times in WCE buffer and once in kinase reaction buffer

(20 mM HEPES pH 7.7, 20 mM MgCl2, 20 mM β-glycerophosphate, 20 mM p-nitrophenyl

phosphate, 0.1 mM sodium vanadate, 2 mM DDT), the beads were incubated with 30 µl of kinase

reaction buffer containing 20 µM ATP and 5 µCi [α-32P]-ATP for 30 min at 300C. The reaction was

stopped by addition of Laemmli sample buffer. Samples were boiled for 5 min and resolved by

electrophoresis through 12% polyacrylamide-SDS gels (SDS-PAGE).

To detect cdk2 and cdc2 kinase activities following JNKAS treatment, cells were harvested

in cdk2/cdc2 lysis buffer (50 mM Tris-HCl, pH 7.4, 250 mM NaCl, 5 mM NaF and 0.1% Triton X-

100, 1 mM dithiothreitol, 0.1 mM sodium orthovanadate) supplemented with protease inhibitors and

cellular debris were removed from soluble extracts by centrifugation at 16,000 x g for 10 min at 40C.

Cdk2 and cdc2 were immunoprecipitated from 100-µg aliquots after a 16 h incubation at 40C in the


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presence of either anti-cdk2 (Pharmingen), or anti-cyclin B1 antibodies (Santa Cruz), respectively.

Immunoprecipitates were washed with cdk2/cdc2 lysis buffer and with H1 kinase buffer (50 mM

Tris-HCl, pH 7.4, 10 mM MgCl2 and 1 mM dithiothreitol). Kinase activities associated with anti-

cdk2 and anti-cyclin B1 immunoprecipitates were assayed in 50 µl of H1 kinase buffer containing 10

µg of histone H1 (Ambion) supplemented with 2 mM EGTA and 10 µCi of [ γ- 32P]ATP. Reactions

were carried out for 30 min at 370C and stopped by addition of Laemmli sample buffer. Reaction

products were electrophoresed in 15% SDS-polyacrylamide gels. Results were visualized with a

PhosphorImager (Molecular Dynamics).


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RESULTS

Specific Inhibition of JNK1 and JNK2 Expression by JNK Antisense Oligonucleotides

(JNKAS) - To examine a role for the JNK pathway in mediating basal growth of T98G cells and to

test whether there is a preferential role for JNK1 or JNK2, we employed specific JNK1 and JNK2

antisense oligonucleotides (18-20). Preliminary experiments using a control fluorescein (FITC)-

labeled oligonucleotide (c-rafISIS13153) revealed that the efficiency of uptake of the phosphorothioate

oligonucleotide, delivered as described in the Experimental Procedures section, was nearly 100%

(Fig. 1). The specificity of the elimination of JNK1 and JNK2 mRNAs by JNKAS in T98G cells is

shown in Fig. 2. While neither mock treatment nor treatment with control “scrambled” (JNKScr)

oligonucleotides had any effect on JNK mRNA levels in T98G cells, treatment with JNK1AS and

JNK2AS resulted in >95% elimination of the respective mRNAs 24 h post-lipofection (Fig. 2A).

Although the JNK1 and JNK2 mRNA levels have largely recovered by 72 h, an effective "window of

observation” was provided by the antisense treatment as indicated in Fig. 2B (shaded area). The

efficiency of inhibition of JNK was confirmed by western analysis (Fig. 3A). Major JNK isoforms

migrate with apparent molecular weights of 46 kDa and 54 kDa. The slower migrating proteins

(p54JNK) are largely, but not exclusively, composed of the JNK2 isoforms, whereas the faster

migrating proteins (p46JNK) are largely, but not exclusively, composed of JNK1 isoforms (1).

Treatment with JNK1AS and JNK2AS markedly attenuated the 46-kDa and 54-kDa components,

respectively (Fig. 3A), consistent with the known distribution of JNK isoforms. Time-course studies

of S35-metabolically-labeled T98G cells indicated that the half-lives for degradation of JNK1 and

JNK2 proteins are 14 h and 3.4 h, respectively (Fig. 3B). This is consistent with the marked

reduction in steady-state levels of both JNK proteins (a reflection of both reduced synthesis and


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degradation of existing proteins) between 30 and 50 h post treatment (data not shown).

Determination of kinase activity in cells irradiated with UV-C (40 J/m2) 24 h post-lipofection

revealed a markedly lower level of JNK activation in JNKAS-treated cells relative to control cells

(Fig. 3C).

Growth Inhibition of T98G Cells Treated With JNKAS - To determine whether inhibition

of JNK has an effect on the growth of T98G cells, we assessed the viability of T98G cells over a 24

to 120 h time period following JNKAS treatment. Viable cell mass was measured both using an

MTS-based colorimetric assay and by direct cell counts. The viability of mock- and JNKScr-treated

cultures was very similar to that of untreated cells over the entire time course (Fig. 4, A and B). In

contrast, treatment with either JNK1AS or JNK2AS led to a marked reduction in viable cell mass.

Growth suppression following treatment with JNK2AS was more pronounced than seen with

JNK1AS treatment (~40% vs 65% that of controls at 24 h post-lipofection, respectively), especially

at later time points (~15% vs 75%, respectively, at 120 h post-lipofection), although statistically

significant differences in growth inhibition by both JNKAS were observed as early as 24 h post-

lipofection. As determined by direct counting from 24-72 h post-lipofection, the number of viable

cells in attached JNK1AS-treated cultures was significantly diminished relative to that of control

cultures (Fig. 4C). Consistent with the transient loss in JNK expression, JNK1AS-treated cells

resumed growth at later times with cell numbers recovering to 70% of that seen in control cultures by

day 5 (Fig. 4B). JNK2AS-treated cells exhibited a greater loss in cell mass than their JNK1AS-

treated counterparts at all times, and, unlike that seen with JNK1AS, their growth did not recover

with time. Five days post-lipofection the viable cell mass of JNK2AS-treated population was 15%

of that seen for control groups (Fig. 4B). In summary, although the magnitude the growth inhibition

achieved by JNKAS differed somewhat depending on the method used to assess growth (MTS assay


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vs cell counting), the general observation of a growth inhibition in response to JNKAS treatment was

apparent regardless of the method employed.

To examine the dose-dependency for growth inhibition by JNK1AS and JNK2AS treatment,

cells were incubated with various amounts of antisense oligonucleotides ranging from 0 to 0.4 µM.

The total amount of oligonucleotides added to cells was kept constant by addition of appropriate

concentrations of corresponding JNKScr oligonucleotide. Both JNK1AS and JNK2AS exerted their

growth inhibitory effects in a dose-dependent manner (Fig. 5). Cells treated with JNK2AS exhibited

a markedly steeper decline in viable cell mass (JNK2IC50 = 0.14 µM) compared to cells treated with

JNK1AS (JNK1IC50 = 0.37 µM). These effects were not apparent in cells treated with similar doses of

JNK1S and JNK2S oligonucleotides, or in cells receiving 0.4 µM JNKScr. Thus, the inhibitory

properties of JNKAS are manifested in a dose-dependent manner and do not reflect cytotoxicity of

oligonucleotide treatment per se, but rather the susceptibility of T98G cells to perturbations in JNK

expression.

The Growth Inhibition in JNK-depleted T98G Cultures is Associated With Cell Cycle

Arrest and Inhibition of DNA Synthesis - At least two possible cellular responses could account for

the reduction in viable cell mass seen following treatment with JNKAS: decreased DNA synthesis

and/or selective death of JNKAS-treated cells. First, we examined the treated cells for evidence of

apoptosis. JNKAS-treated cultures, particularly JNK2AS-treated cultures, exhibited some

morphological alterations frequently seen in cells undergoing apoptosis including fewer mitoses,

rounded-up cells (Fig. 6), and detachment of many cells (up to 30%) from the plate surface (Fig. 4C).

Detached cells were examined for viability. Up to 90% of detached cells in JNKAS-treated cultures

excluded trypan blue, and were therefore alive cells, although they did not reattach or grow when


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placed in fresh medium in new dishes (data not shown).

Four additional methods were used to assess apoptosis in JNKAS-treated cells including

detection of apoptotic bodies in DAPI-stained cells and various types of flow cytometric analysis:

i.e., cell-cycle distribution of PI-stained cells, detection of BrdU incorporation into cellular DNA

breaks (APO-BRDU), and detection of Annexin V-stained populations. Detection of DNA

fragmentation by DAPI staining revealed no evidence of apoptotic cells in any treatment groups

through 72 h post-lipofection (Figs. 7 and 8, data not shown). Interestingly, the structure of nuclei in

JNKAS-treated cells differed from that of either mock-treated cultures or cells treated with JNKScr

oligonucleotides (Fig. 7A). The cause of these alterations is no known, but may reflect the presence

of cells with doubled DNA content that are unable to finish progression through the S phase and

mitosis following JNKAS treatment. No cells with condensed or fragmented nuclei indicative of

apoptosis were evident in any treatment group. More sensitive methods for assessing apoptosis

based on detection of BrdU incorporation into DNA breaks (APO-BRDU) (Fig. 7B) and detection of

specific membrane alterations using Annexin V (data not shown) also showed no evidence of

apoptosis in any of the treatment groups up to 48 h. Although a small fraction of JNK2AS-treated

cells (2.9%) did appear to undergo apoptosis at 48-72 h (data not shown), this percentage of

apoptotic cells is similar to that seen when assaying the negative control provided with the kit

(3.3%). Thus, while JNK2-depletion might result in a low level of apoptosis in T98G cells, our

results indicate that apoptosis cannot account for the magnitude of the reduction in viable cell mass

seen in JNK2AS-treated cultures.

While no evidence for apoptosis of JNKAS-treated cultures was found, JNKAS treatment

resulted in profound alterations in the distribution of cells throughout the cell cycle, indicative of cell


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cycle arrest (Fig. 8). The percentage of cells in G1, S and G2/M phases for untreated and control

cultures subjected to mock-lipofection or treatment with control oligonucleotides (JNKScr) were

similar: G1 = 63 ± 7 %; S = 23 ± 6 %; G2/M = 15 ± 6 % (average data from three independent

experiments, Figs. 8A and 8C). In contrast, lipofection with either JNK1AS or JNK2AS resulted in

a two-fold reduction in number of cells in the G1 compartment and an increase in number of cells in

other cell cycle compartments. Interestingly, while JNK1AS-treated cells accumulated in both S and

G2/M phases (35 ± 5.5 %; 31 ± 4 %, respectively), JNK2AS-treated cultures displayed an

accumulation of cells exclusively in S phase (50 ± 2 %). Additional analysis of distribution of cells

through the cell cycle was done using a BrdU pulse incorporation assay, which allows detection of S-

phase cells by the presence of BrdU in their cellular DNA (Fig. 8B). For control populations (mock-

and JNKScr-treated cells) and for JNK1AS-treated cultures the percentage of S-phase cells

determined with this method (23% and 35%, respectively) were similar to those obtained with PI

staining (22% and 34.5%, respectively; data were analyzed using ModFitLT software). However, in

the case of JNK2AS-treated cells, BrdU incorporation analysis revealed much lower number of S-

phase cells than PI staining (28% vs 50%). This discrepancy is likely to reflect the fact that T98G are

not actively progressing though S phase and thus are not incorporating BrdU due to JNK2AS

inhibitory influence on DNA replication (see below).

To further explore the nature of the growth arrest by JNKAS, we directly examined DNA

synthesis in JNKAS-treated cultures. As assessed by uptake of [3H]-thymidine, DNA synthesis was

greatly inhibited in both JNK1AS- and JNK2AS-treated populations (Fig. 9). The effect was greater

for JNK2AS-treated cultures, which exhibited near-complete inhibition of DNA synthesis by 24 h

post-lipofection (Fig. 9A). Interestingly, while DNA synthesis in JNK1AS-treated cells started to


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recover by 72 h post-lipofection (consistent with the return of JNK1 expression and transient

reduction in viable cell mass; Figs. 1 and 4), no such recovery was observed in JNK2AS-treated

cultures (Fig. 9B). This is consistent with the lack of recovery in viable cell mass of JNK2AS-

treated cultures noted above (Fig. 4). Thus, while transient depletion of JNK1 led to a transient

inhibition of DNA synthesis and reduction in growth of T98G cells, similar elimination of JNK2

expression led to sustained inhibition of DNA synthesis and permanent growth arrest of T98G cells

Inhibition of Cell Growth by JNKAS is Associated with Induction of p21Cip1/Waf1

Expression and Inhibition of Cyclin-Dependent Kinase Activities - The cyclin-dependent kinase

inhibitor p21Cip1/Waf1 is believed to play an important role in mediating cell cycle arrest in response to

a variety of treatments. Therefore, we investigated its expression in T98G cells following treatment

with JNK1AS and JNK2AS. Marked elevation of both p21Cip1/Waf1 mRNA (Fig. 10A) and protein

(Fig. 10B) was detected in JNK2AS-treated cultures. Interestingly, this effect was not shared by

JNK1AS-treated cells, suggesting that induction of p21Cip1/Waf1 expression may contribute to the

specific features of growth arrest observed in JNK2-depleted cultures such as permanent inhibition

of DNA synthesis and profound sustained S-phase arrest. No changes in the expression of other cell

cycle regulatory proteins were observed following any treatment, except for a slight elevation of

p27Kip1 protein levels in JNK2AS-treated cultures (Fig. 10B). As expected, elevation of p21Cip1/Waf1

levels in JNK2-depleted cells was correlated with a marked inhibition of cyclin-dependent kinase

(cdk2 and cdc2) activities in JNK2AS-treated cells (Fig. 10C).


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DISCUSSION

The role of the JNK pathway in mediating cellular responses to extracellular stimuli has been

studied extensively over the past several years, and the pro-apoptotic function of activated JNK is

supported by numerous studies (1-3, 39). However, evidence supportive of a pro-survival role for

the JNK pathway has also been documented (21, 30, 40). One plausible explanation for these

seemingly disparate effects is that JNK serves different functions under normal growth conditions

and during stress. Support for this view has come from findings that, in contrast to activated

(phosphorylated) JNK, which activates and stabilizes its substrates during stress; nonactivated

(nonphosphorylated) JNK targets them to degradation (reviewed in ref. (4)). Regulation of normal

cell growth may also require basal JNK kinase activity. Indeed, N-terminal phosphorylation of c-Jun

has been implicated in the regulation of cell growth both in vitro and in vivo (41).

Using high-affinity and high-specificity phosphorothioate antisense oligonucleotides

targeting JNK1 and JNK2 (18, 19), we have demonstrated that inhibition of JNK1 and JNK2

expression in otherwise unstressed human T98G glioblastoma cells results in marked suppression of

growth. The inhibitory effect seen with JNK2AS was much greater than that observed with JNK1AS

(Figs. 4, 5), possibly reflecting specific functions of JNK2 not shared by JNK1. However, physical

properties of JNK proteins such as their half-lives could also influence the outcome. The half-life of

JNK1 is considerably longer than that of JNK2 (Fig. 3B), and significant elimination of JNK1

protein (< 20% of steady-state level) is not achieved until approximately 40 h post JNKAS treatment.

Thus, we cannot completely exclude the possibility that a more prolonged depletion of JNK1 in

T98G cells would lead to effects comparable to those observed here with depletion of JNK2. In any

event, examination of several other human tumor lines has revealed a similarly critical role of JNK2


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for cell growth. For instance, JNK2 is required for EGF-stimulated growth of A549 cells in soft agar

(18, 19) and JNK2AS, but not JNK1AS, treatment of mice bearing established xenografts of human

PC3 cells was found to inhibit tumor growth (35).

Our findings reported here with T98G cells differ from our recent observations in several

other tumor cell models in which we found that cells deficient of p53 function undergo apoptosis in

response to JNK2AS treatment (36). The survival of cells with wild type p53 status was associated

with elevated levels of p21Cip1/Waf1 expression, which was not seen in p53-deficient cells. The

HCT116 cells lacking p21Cip1/Waf1 (51) underwent extensive apoptosis in response to JNK2AS

treatment (36). These findings support the hypothesis that p21Cip1/Waf1 plays an important role in

p53-mediated protection of tumor cells from JNK2AS effects. Although exhibiting marked growth

arrest, T98G did not undergo apoptosis following JNK2AS treatment (Figs. 7 and 8) despite absence

of functional p53 in these cells. Moreover, only JNK2AS-treated T98G cells were found to express

elevated levels of p21Cip1/Waf1 (Fig. 10) suggesting that induction of p21Cip1/Waf1 may be executed by a

p53-independent mechanism resulting in cell cycle arrest and protection of T98G cells from

apoptosis. While the mechanism(s) contributing to the induction of p21Cip1/Waf1 and downstream

effects remains to be determined, it is clear that these effects are p53-independent.

Although we found that about 10% of JNK2AS-treated T98G cells detached from the tissue

culture plate (Fig. 4C), no apoptosis was detected in the detached populations. The vast majority of

these cells were found to be viable based on the criterion of trypan blue dye exclusion (data not

shown). The percentage of detached cells further increased with time (30% by 72 h post-

lipofection), suggesting a possible role for JNK2 in regulating cell adhesion. Indeed, using the same

JNKAS, others have reported JNK2AS-specific inhibition of TNF-α-mediated induction in the


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expression of E-selectin (17), a protein involved in cell adhesion. The role of the JNK pathway in

anoikis, apoptotic cell death resulting from detachment, has been postulated, but remains

controversial (42, 43). Although we cannot exclude the possibility that the majority of JNK2AS-

treated cells may eventually die by apoptosis subsequent to their detachment from the extracellular

matrix, this effect was not detected at the times examined in this study.

We have shown that JNK depletion in T98G cells results in a dramatic inhibition of DNA

synthesis (Fig. 9) associated with accumulation of cells in the S and G2 phases of cell cycle (Fig. 8).

In previous studies we observed that inhibition of c-Jun N-terminal phosphorylation in T98G cells

results in impaired DNA repair (40) suggesting that an intact JNK pathway is required for normal

DNA repair. Together with our current findings, these observations indicate a possible role for JNK

in DNA repair during progression of cells through late S phase. Other components of the DNA

replication and/or cell cycle machinery could also be downstream targets of JNK. The cdk inhibitor

p21Cip1/Waf1, which was specifically elevated only in JNK2AS-treated cells, is an attractive target of

JNK2 signaling. However, if so, it is likely to be an indirect target as we have noted that p21Cip1/Waf1

expression remains elevated up to 72 h post JNKAS treatment associated with sustained growth

arrest although JNK expression has nearly returned to pretreatment levels (data not shown, Fig.2).

It’s worth noting that a p21Cip1/Waf1 expression has been linked to S-phase arrest in several other

model systems including hyperoxia (44) and treatment of cells with the retinoid CD437 (45).

The phenotype of JNK2AS-treated cells shares certain characteristics with the "permanent"

cycle arrest observed following γ-irradiation of human fibroblasts (46-47). Like JNK2AS-treated

cells, these fibroblasts express elevated levels of p21Cip1/Waf1 and are unable to synthesize DNA or

divide for extended time, but remain viable by the criterion of trypan blue exclusion. Induction of


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p21Cip1/Waf1 plays an important role in growth arrest of p53-deficient cells such as T98G (48), as was

previously observed in human astrocytoma cells (49). Whether p21Cip1/Waf1 induction is required for

the growth inhibitory effects seen with JNK2AS remains to be proven. We have found that transient

over-expression of p21Cip1/Waf1 in T98G cells in the absence of JNKAS treatment results in G1-phase

arrest rather than S-phase arrest (data not shown). This observation strongly suggests that the growth

inhibitory effects of JNK2AS treatment are not limited to induction of p21Cip1/Waf1, but must act in

collaboration with other JNK2AS-regulated factors to cause S-phase arrest. Further studies are

underway to identify these components.

Unlike T98G cells and several other human cancer cell lines we have examined, JNKAS

treatment is not growth inhibitory for normal prostate epithelial cells (data not shown). Likewise,

although double knockouts of JNK1 and JNK2 are not viable, cells derived from either JNK1 or

JNK2 knock-out mice grow normally (50, 51). These observations suggest that JNK1 and JNK2

possess redundant functions (and can therefore compensate for each other) and that neither is crucial

for the growth of non-transformed cells. Thus, the requirement of JNK2 for growth may be

accentuated in tumor cells. The recent development of procedures to generate somatic gene-

knockouts in human tumor cells (52, 53) provides the tools to investigate the effects of abrogating

JNK expression in human cancer cells. While further investigations are necessary to better

understand the role of JNK in regulating tumor cell growth, and to identify the specific JNK targets

involved, our findings highlight the importance of JNK for maintaining tumor cell homeostasis in the

absence of overt stress and suggest that strategies aimed at eliminating JNK could have therapeutic

benefit.


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ACKNOWLEDGEMENTS

We thank M. Karin for gifts of plasmid reagents, R. Roberson for technical help, and J.

Chrest and C. Morrison for help with flow cytometry analysis.

This work was supported in part by grants from the U.S. Public Health Service, NCI

CA63783 and NCI CA76173 (D.A.M.), by the California Breast Cancer Research Program

#8CB0246 (D.A.M.), by la Ligue Nationale Contre le Cancer (F.B.), le Conseil Régional de Haute

Normandie (F.B.) by the American Cancer Society, Ray and Estelle Sephar Fellowship (F.B.) and by

the fellowship program of the Sidney Kimmel Cancer Center.


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1 Abbreviations used: JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; ATF, activation

transcription factor; TNF-α, tumor nectosis factor-α; EGF, epidermal growth factor; PAK, protein-activated

kinase; PDGF, platelet-derived growth factor; JNK1AS and JNK2AS, antisense oligonucleotides targeting

expression of JNK1 or JNK2, respectively; DAPI, 4'-6-diamidino-2-phenylindole; MTS, (3-(4,5'-

dimethylthiazol-2-yl)-5-(3-carboxymethoxylphenyl-2-(4-sulfophenyl)-2H-tetrazolium inner salt.; PI –

propidium iodine; UV-C, ultraviolet light C band.


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FIGURE LEGENDS

Fig. 1. Efficiency of phosphorothioate oligonucleotide uptake in T98G cells. T98G cells at 50%

confluence were incubated with 0.4 µM of 3’FITC-labeled control phosphorothioate oligonucleotide

in the presence of 10 µg/ml LipofectinTM reagent (Gibco BRL) as described in the Experimental

Procedures section. The cells were fixed 24 h following treatment (~80% confluent culture), and

subjected to confocal fluorescence microscopy. Nearly 100% of cells showed uptake of the

fluorescent oligonucleotide. Image was taken at 800x magnification.

Fig. 2. Transient elimination of JNK mRNA expression following JNKAS treatment. A,

northern blot analysis of T98G cells after lipofection with 0.4 µM of the indicated oligonucleotides

(0.2 µM of each oligonucleotide when used in combination). The cells were collected for analysis at

the designated times and RNA was prepared using Stat-60 reagent as described in the Experimental

Procedures section. Hybridization with a probe recognizing 18S RNA was used to assess the

differences in loading and transfer among RNA samples. B, kinetics of suppression and

reappearance of mRNA. The shaded area indicates the period of time when the significant

suppression (about 50%) of the corresponding mRNA was achieved as determined in Fig. 2A.

Fig. 3. Depletion of JNK1 and JNK2 proteins with JNKAS treatment. A, attenuation of JNK

protein levels 24 h post-lipofection. T98G cells were treated with 0.4 µM oligonucleotides (0.2 µM

each in combinations). One hundred-µg aliquots of whole-cell lysates were processed for western

blot analysis, and JNK levels were detected using SC-571 JNK antibodies (Santa Cruz

Biotechnology). B, half-life determination of JNK1 and JNK2. T98G cells were metabolically

labeled for 4 h with 35S-methionine as described in the Experimental Procedures section, and the

amount of labeled JNK proteins at designated times was determined by immunoprecipitation,


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separation by SDS-PAGE, and visualization by autoradiography (lower panel). JNK1-specific

antibodies SC-479 (Santa Cruz Biotechnology) were used to detect JNK1 levels; and SC-571

antibodies were used for detection of JNK2 levels in JNK1-depleted lysates. The log function of

normalized intensities of the resulting bands for each protein was plotted as a function of the time

after the end of the metabolic labeling period. Slopes (least squares analysis) were used to calculate

the half-lives. This experiment was repeated three times and data from a representative experiment

are shown. C, inhibition of JNK activation in JNK-depleted T98G cells. Cells treated with a

combination of 0.2 µM each JNK1AS and JNK2AS (labeled JNKAS) or 0.2 µM each JNK1Scr and

JNK2Scr (labeled JNKScr) were exposed to 40 J/m2 UV-C (lanes labeled +) or left untreated (-) 24 h

post-lipofection. JNK activity was measured 30 min later by in vitro kinase assay as described in the

Experimental Procedures section.

Fig. 4. Depletion of JNK results in growth inhibition of T98G cells. A and B, assessment of the

viable cell mass by MTS tetrazolium dye conversion at 24 h (A) and 120 h (B) following treatment

with oligonucleotides. T98G cells were seeded in 96-well cluster plates at a density of 1,000

cells/well, and transfected with 0.4 µM of the indicated oligonucleotides the following day. The

MTS dye reduction was determined by measurement of optical density at 490 nm. Cell viability was

expressed as percent of viable cell mass in mock-treated cultures. Cells treated with JNK1AS and

JNK2AS exhibit statistically significant inhibition of growth compared to all controls (p < 0.001,

Student’s t-test) as well as differ among themselves as early as 24 h post-lipofection (*, p < 0.001).

C, effect of JNKAS treatment on cell number and portion of attached cells. Cells were seeded in 6-

well cluster plates at 25 x 103 cells/cm2 and treated with 0.4 µM oligonucleotides. Attached cells

(open portion of bars) and cells in medium (closed portion of bars) were counted in triplicate


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(Coulter counter) 24, 48, and 72 h after lipofection. Standard errors were <10% in all cases.

Fig. 5. Growth inhibition by JNK1AS and JNK2AS is dose-dependent. T98G cells were seeded

at a density of 1,000 cells/well in 96-well tissue culture plates and treated 24 h later with the

indicated amounts of JNKAS and JNKS oligonucleotides. The total amount of oligonucleotides

added to each well was kept constant (0.4 µM) by the addition of either JNK1Scr or JNK2Scr. Three

days later, the viable cell mass was determined by addition of MTS as described in the Experimental

Procedures section. Broken lines indicate trend-lines for growth inhibition by a corresponding

oligonucleotide.

Fig. 6. Morphology of JNKAS-treated cells. Microscopic (phase contrast) appearance of T98G

cells is shown at various times following their lipofection with 0.4 µM of indicated oligonucleotides.

Untreated, mock- and JNKScr-treated control cultures were indistinguishable at all times tested (24

h post-lipofection data are shown in Left panel). JNKAS-treated cultures (Right panels), particularly

JNK2AS-treated cultures, exhibited morphological alterations accentuated with time. Photographs

were taken at a 400x magnification.

Fig. 7. Apoptosis analysis following treatment with JNKAS. A, detection of apoptotic bodies in

DAPI-stained cells. T98G cells were treated with 0.4 µM of corresponding oligonucleotides, fixed

24 h later on 60-mm tissue culture dishes with 70% ethanol and stained with DAPI. Photographs

were taken at a 400x magnification. B, detection of apoptotic cell fractions using APO-BRDUTM kit

(Pharmingen). Cells were treated with 0.4 µM corresponding oligonucleotides and fixed 24 h later

as described in the Experimental Procedures section. ”Positive Control” and “Negative Control”

insets represent data from assay-control samples provided by the manufacturer. Apoptosis-positive


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cells are located in upper right corner of plots: 22% in “Positive Control” sample; less than 1% in all

other samples.

Fig. 8. Cell cycle analysis following treatment with JNKAS. A, flow cytometric analysis of PI-

stained cells 24 h post-lipofection. T98G cells were treated with 0.4 µM of corresponding

oligonucleotides; both attached and detached fractions were collected 24 h after the treatment, fixed

and stained with propidium iodine and subjected to flow cytometry. Cultures treated with

combination of JNK1Scr and JNK2Scr (0.2 µΜ each) are labeled JNKScr. The percentage of cells

in different phases of the cell cycle was determined using ModFitLT software program. B, detection

of S-phase cells using BrdU pulse incorporation. The cells treated as described above were pulsed

with BrdU labeling reagent for 1 h, fixed and stained as described in the Experimental Procedures

section. Dual-parameter analysis (BrdU vs PI) was used to determine BrdU-positive cells with

intermediate DNA content. C, graphic representation of cell cycle analysis performed using

ModFitLT software program. The average data of three independent experiments were plotted to

show consistency of the numbers obtained by this way of analysis. “Controls” include non-treated,

mock-treated, and JNKScr-treated populations.

Fig. 9. Inhibition of DNA synthesis after treatment with JNKAS. A, T98G cells were seeded

into 96-well cluster plates (1,000 cells/well) and on the following day treated with various

oligonucleotides. Incorporation of [3H]-thymidine into polymeric DNA was determined 24 h post

treatment. Results were normalized to relative amounts of viable cells (Fig. 4A), and expressed as %

of values obtained in the untreated population (3 wells/treatment were analyzed in duplicate).

*Values for JNK1AS- and JNK2AS-treated cells differ significantly from each other (p < 0.001,

Student’s t-test) as well as from control values (p < 0.001). B, recovery of DNA synthesis following


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treatment with JNK1AS but not with JNK2AS. Relative DNA synthesis rates were determined as

described above at different time points after lipofection. Values in mock-treated cultures were set to

100%. The value labeled “Control oligonucleotides” represents the mean of values received from

two sense and two scrambled sequence oligonucleotides treatments. Oligonucleotide treatments

were performed in triplicate, and duplicate samples from each well were assayed for 3H-Thymidine

incorporation.

Fig. 10. Induction of p21Cip1/Waf1 expression in JNK2AS-treated cells and associated inhibition

of cyclin-dependent kinase activities. A, northern analysis of p21Cip1/Waf1 gene expression 24 h

post treatment with 0.4 µM of either JNK1As, JNK2AS, JNK1Scr or JNK2Scr oligonucleotides.

RNA was prepared using Stat-60 reagent as described in the Experimental Procedures section.

Hybridization with a probe recognizing 18S RNA was used to assess the quality of loading and

transfer among samples. B, western blot analysis for expression of cell cycle regulatory proteins in

JNK1AS- and JNK2AS-treated cultures. Cells were harvested 24 h after lipofection and 100 µg of

whole-cell extracts were analyzed. C, inhibition of cdk2/cdc2 kinase activities in JNK1AS- and

JNK2AS-treated cultures. In vitro kinase assays were performed as described in the Experimental

Procedures section after immunoprecipitating complexes containing cdk2 and cdc2 and using

Histone H1 as a substrate. Sample labeled ”–H1” depicts a no-substrate control reaction.


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Mercola and Nikki J HolbrookOlga Potapova, Myriam Gorospe, Frederic Bost, Nicholas M Dean, William A Gaarde, Danc-Jun N-Terminal Kinase is Essential for Growth of Human T98G Glioblastoma Cells

published online May 23, 2000J. Biol. Chem.

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