c-fos is a mediator of the c-myc-induced apoptotic ... · deprived hepatoma cells via p38 map...

c-Fos is a mediator of the c-Myc-induced apoptotic signaling in serum-

deprived hepatoma cells via p38 MAP kinase pathway

Neetu Kalra and Vijay Kumar*

Virology Group, International Centre for Genetic Engineering and Biotechnology,

Aruna Asaf Ali Marg, New Delhi-110067, India.

Running Title: c-Fos as mediator of c-Myc-induced apoptosis

*Corresponding author: Dr. Vijay Kumar

Virology Group, I.C.G.E.B.

P.O. Box 10504

Aruna Asaf Ali Marg

New Delhi – 110067, India

Phone: +91-11-26176680

Fax +91-11-26162316

E-mail: [email protected]

c-Fos as mediator of c-Myc-apoptosis

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JBC Papers in Press. Published on April 12, 2004 as Manuscript M400932200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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SUMMARY

The protooncogene c-Myc encodes a transcription factor that plays a pivotal role in

cell proliferation, differentiation, and apoptosis. The signaling mechanism of c-Myc-

induced apoptosis was investigated on the human hepatoma Huh7 cells under growth

factor-deprived conditions. The apoptotic process did not involve p53. Rather it was

dependent on the expression of c-Fos. Activation of caspases 3 and 9, and down

regulation of Bcl2 were observed in the apoptotic process indicating it to be a

mitochondria-dependent event. An increase in the p38 MAP kinase that was mediated by

a Rac1-dependent and cdc42-independent pathway eventually leading to up-regulation

of c-Fos activity was also observed. Deletion analysis of the promoter region of the c-

fos gene indicated that the ATF2-responsive element conferred the Myc-induced

expression of c-Fos. Co-expression of the dominant negative mutants of c-Fos, p38 and

Rac1 blocked the Myc-mediated apoptosis. SB20358 - a chemical inhibitor of p38

pathway – also specifically blocked the apoptotic signaling by c-Myc. Further, co-

expression of the hepatitis B virus X protein (HBx) along with Myc abrogated the

apoptotic signals. The HBx expression was associated with an increase in the levels of

pAKT and down regulation of c-Fos by Myc. Thus, c-Fos appears be a new mediator of

c-Myc-induced apoptosis.


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INTRODUCTION

Apoptosis or programmed cell death is a physiological process for eliminating cells

that are redundant, damaged or infected (1,2). A fine balance between cell proliferation

and apoptosis is fundamental to the normal growth of an organism. Many cellular genes,

like c-myc, p53, and enzymes of signaling cascades that are involved in cell cycle

progression, are also involved in regulation of apoptosis (3). For example, signals such as

c-Myc that promote proliferation in the presence of growth factors appear to promote

apoptosis under starvation or other forms of stress (4). More often, the outcome of a

signal is specified by a second signal, e.g., c-Myc and Bcl-2 together are proliferative

(5,6) while c-Myc along with p53 induces apoptosis (7). Clearly c-Myc, a key

transcription factor of the bHLHZ family, is instrumental in key decisions of the cell

towards either cell proliferation or programmed cell death (8,9).

Enforced c-Myc expression has been shown to be associated with apoptosis in a wide

variety of cell types including fibroblasts (10,11), renal epithelial cells (12), lymphocytic

B cells (13), leukaemic cells (14), breast cancer cells (15), T cell hybridomas (16),

myeloid cells (17) and hepatocytes (18). c-Myc-dependent apoptosis has also been

reported in target tissues of transgenic mice such as pancreatic β cells (19) and

hepatocytes (20). On the contrary, over-expression of the c-myc gene is implicated in

hepatocellular carcinoma (HCC) in the hepadnavirus infected woodchucks (21) and

ground squirrels (22). Similarly, amplification of proto-oncogenes like c-myc has been


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reported in a variety of tumors and is relatively common in carcinomas and sarcomas in

humans (23-25). Normally, the expression of proto-oncogenes like c-Myc has a survival

value for cells unless exposed to environmental stress. However, our knowledge about

the pro-apoptotic signals and the effecter network of c-Myc is rather limited.

Sensitization of cells to apoptosis by c-Myc is associated with the induction of

several pro-apoptotic target genes such as p19ARF1, p53, Bax and FasL (26-29). It

could also involve release of cytochrome C (30), loss of mitochondrial potential (31),

down-regulation of survival signals like Bcl-2 (32) and NF-kB in TNF alpha-mediated

apoptosis (33), and up-regulation of p38 in cisplatin-induced apoptosis (34). Though the

involvement of immediate early response gene c-fos is well documented in a variety of

apoptotic stress stimuli leading to apoptosis (35-37), its involvement in the c-Myc-

mediated apoptosis is not known. In this paper we have examined the role played by c-

Fos in the c-Myc-induced apoptosis of hepatoma cells and the manner in which viral

HBx interferes with this apoptotic process.

MATERIAL AND METHODS

Chemicals and antibodies – 12-O-tetradecanoylphorbol acetate (TPA) was procured

from Sigma (Missouri, USA) while the p38 MAP kinase inhibitor SB20358 was from

Calbiochem (California, USA). Antibodies for c-Fos, Bcl2, pAKT, total AKT,


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fibronectin, pBAD, p53, pErk and total Erk, pJNK, pJun and c-Myc were purchased

from Santa Cruz Biotech (California, USA) while the antibodies for caspase 3, caspase 9,

pP38, total p38, pATF2 and total ATF2 were bought from Cell Signaling Technology

(Massachusetts, USA).

Expression vectors and reporter gene constructs – We have earlier described the

construction of the HBx expression vector (X0) and its deletion mutants (X5, X6, X7,

X9, X10, X14 and X15) (38). The c-Myc expression vector was constructed by cloning a

4.8 Kb Bam HI–Xba I mouse genomic DNA fragment (39) in the pSG5 plasmid

(Stratagene, USA). For constructing the expression vector for dominant negative (DN)

mutant of c-Fos (Fos-DN), a new polylinker was inserted between the EcoRI and

BamHI sites of pSG5 to create pSGI with the following unique sites: 5’-EcoRI-SmaI-

SacI-EcoRV-KpnI-HindIII-PstI-NotI-BamHI–BglII-3’. Finally, the NdeI-HindIII

fragment (300 bp) from pCMV500-8584 hep fos LZ (MO) (40) was sub cloned into the

pSGI vector after repairing the Nde I site to create Fos-DN. The other DN constructs

used in the present study were: Rac1-DN and cdc42-DN (41), MKK3-DN (42) and

FAK-DN (43).

Details of the chloramphenicaol acetyl transferase (CAT) reporter constructs FC1-BL,

FC2-BL, FC4-BL, FC8-BL can be found elsewhere (44). FC80-BL was derived from

FC2-BL by deleting 1.3 Kb Hind III–NarI region and repair-ligation. For constructing


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the E box-CAT reporter, the Pst I-BamHI fragment (1.7 kb) of pUCAT (45) having the

SV40 minimal promoter with CAT gene was cloned into pBS+ vector (Stratagene,

Germany) to generate pBS-CAT. Then oligonucleotides A and B, having a canonical E

box element, were kinased and oligomerized using T4 DNA ligase and a tertameric form

was cloned at Bgl II site of pBS-CAT to create E box-CAT. The orientation and

integrity of E box elements was verified by DNA sequencing. Sequences of

oligonucleotides were: A, 5’ – GATCCGACCACGTGGTTA – 3’; B, 5’ –

GATCTAACCACGTGGTCG – 3’.

Cell culture and transfection – The human hepatoma Huh7 cells (46) were grown in

Dulbecco-Modified Eagle Medium with 10% fetal bovine serum (Hyclone, USA). Cells

were seeded at a density of 0.7 million/60mm dish and transfected using Lipofectin

(Invitrogen) as per manufacturer’s protocol. For promoter analysis, each fos-CAT

reporter construct (0.5 µg) was co-transfected with expression vectors for c-Myc or HBx

(1 µg). Cells were maintained in serum free medium for 48 h and harvested for CAT

assay. For immunoprecipitation and western blot assay, c-Myc (3 µg) or HBx (2 µg)

expression vectors were co-transfected with selected dominant negative constructs (2

µg). Cells were grown in DMEM with 10% serum for 24 h after transfection followed by

24 h culture in serum-free medium before harvesting.


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CAT assay - Cells extracts were prepared in Tris-Cl buffer (250 mM, pH 7.8). CAT

assay was performed as described earlier (38) using normalized amount of protein for

each sample. CAT activity (%) was determined by densitometry and statistical analysis

was done using student ‘t’ test.

Immunoprecipitation and Western Blotting - Cells were lysed using the lysis buffer (50

mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate,

0.1% SDS) in presence of protease inhibitors cocktail (Amersham Biotech, UK). Protein

content of each sample was determined using Bio-Rad protein assay reagent (Bio-Rad,

USA) and equal amount (500 µg) of protein was immunoprecipated overnight with

different antibodies at 4oC on a rocking platform. The immune complexes were isolated

using protein-A sepharose (Amersham Biotech, UK) and the samples were analyzed by

SDS-PAGE. Following electro-transferring to nitrocellulose membrane (Amersham

Biotech, UK), the protein bands were visualized using the enhanced chemiluminescence

(ECL) reagent (Cell Signaling Technology, USA).

For western blot assay, cells were directly lysed in 2x sample-loading buffer (100

mM Tris-Cl, pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue and 20%

glycerol). After heating in a boiling water bath for 5 min, equal amount (25 µg protein) of

each sample was resolved by SDS-PAGE, transferred onto nitrocellulose membrane and


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incubated with primary and secondary antibodies according to Sambrook and Russell

(47). The protein bands were visualized using the ECL reagent as above.

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) –

The transfected Huh7 cells were grown for 24h in complete medium followed by serum

starvation for 24h. The TUNEL assay on hepatocytes was performed as per supplier’s

instructions (Roche Molecular Biochemicals, Germany) and positive cells were counted

in three different power fields (200x). Results (per cent apoptotic cells) were statistically

evaluated using student ‘t’ test

RESULTS

c-Myc activates the c-Fos expression upon serum withdrawal – The c-Myc-induced

apoptosis was investigated in the human hepatoma Huh7 cells. c-Myc protein (64 KDa)

was overexpressed by nearly 2.3 fold in transfected cells as compared to control (Fig. 1A,

compare lanes 2 and 3). To investigate the role of c-Fos in Myc-mediated apoptosis, the

level of c-Fos protein was monitored in Huh7 cells in the presence of c-Myc. As shown

in Fig. 1B, a three-fold increase in the c-Fos protein level was observed in serum starved

cells (compare lanes 1 and 2). However, upon serum stimulation, a rapid depletion in the

c-Fos level was observed in the presence of c-Myc (lanes 4,6). In contrast, serum


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stimulation of control cells showed a rapid increase (3.3 fold within 45 min) in the c-Fos

protein level (lanes 1, 3 and 5). At 48h, the c-Fos levels in serum-stimulated samples

were 5.5 fold and 3.8 fold greater in absence and presence of c-Myc respectively (lanes 7

and 8) with respect to the serum starved samples at 0 time.

To delineate molecular details of the c-Myc-induced upregulation of c-Fos, the

regulatory behavior of c-fos promoter was investigated using FC1-BL, a CAT reporter

construct. As shown in Fig 2A, a 2.6 fold increase in CAT activity was observed in the

presence of Myc (lane 3,4) as compared to the control (lane 1,2). To localize the c-Myc-

responsive element on the fos promoter, four deletion reporter CAT constructs, viz.,

FC2-BL, FC4-BL, FC8-BL and FC80-BL were analyzed for the transactivation

response (Fig. 2B). Deletion of a large portion the 5’ regulatory region of the c-fos

promoter in FC2-BL and FC4-BL (i.e., -2250 to -400) had only a marginal effect on

the transactivation function of c-Myc. With just –225 promoter region (in FC8-BL), a 62

per cent decrease in the reporter activity was observed. Further deletion in the fos

promoter leaving only the cyclic AMP response element and basal transcriptional

elements (FC80-BL) rendered it non-responsive to regulation by Myc.

c-Fos is a key intermediate in c-Myc-mediated apoptosis - To investigate whether

regulation of c-Fos by c-Myc (under the serum-starved condition) was associated with

apoptosis signaling, levels of canonical markers of cell survival (Bcl2, pAkt and


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Fibronectin) and apoptosis (caspase 3, caspase 9 and pBad) were monitored in the

presence of c-Myc. As shown in Fig 3A, the levels of Bcl2, pAkt and Fibronectin were

down regulated in the presence of Myc (lane 2). The c-Myc-dependent inhibition of the

these survival signals could be restored to normal in the presence of Fos-DN and the

viral regulatory protein HBx (compare lane 1 with 3 and 4). Interestingly, c-Myc also

induced the pro-apoptotic markers caspase 3, and caspase 9 and inhibited the

phosphorylation of Bad (Fig 3B, lane 2). Further, co-expression of Fos-DN or HBx

blocked the activation of both caspases and restored the level of pBad to normal (lane

3,4). Under similar conditions, the level of p53 was not affected (Fig. 3C).

c-Myc specifically activates p38 pathway in the absence of growth factors - Since c-

Myc appeared to induce apoptotic signals via c-Fos, the activation of different signaling

pathways was monitored in the presence of DN mutants of selected pathways and the

viral HBx. We observed that the levels of activated MAP kinases such as Erk1/2,

SAPK/JNK, Jun, CREB, FAK and p90 were not affected in the presence of c-Myc and

HBx (Fig. 4). Further, as reported earlier, HBx alone induced the levels of pJNK and

pJun in these cells (lanes 3 in all panels). An increase in pP90 was also observed in the

presence of HBx. Interestingly, the levels p38 MAP kinase and its downstream mediator

ATF2 were activated by nearly 3 fold in the presence of c-Myc (lane 2) and down-

regulated in the presence of HBx (lane 4). Notably, HBx did not stimulate the p38


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pathway on its own and c-Myc did not activate focal adhesion kinase (FAK).

To further investigate the involvement of p38 MAP kinase pathway in the cMyc-

mediated apoptosis, the DN constructs of different upstream kinases like MKK3, FAK,

cdc42 and Rac1 were co-transfected with c-Myc. MKK3-DN could specifically block

the activation of p38 and also its downstream mediator ATF2 (Fig. 5A, lane 3). Likewise,

Rac1-DN also blocked the activation of p38 and ATF2 (lane 6). No change in the levels

of activated p38 or ATF2 was observed in the presence of FAK-DN and cdc42-DN

(lanes 4 and 5 respectively). Interestingly, the activation of caspase 9 was also blocked in

the presence of DN recombinants of Fos, p38 and Rac1 (Fig. 5B, lanes 3 to 5

respectively) and was not influenced by cdc42-DNA or FAK-DN (lanes 6 and 7

respectively).

p38 MAP kinase mediates expression of c-Fos - To study the involvement of p38

MAP kinase on the regulation of c-fos promoter, the FC4-BL reporter was co-

transfected with c-Myc and different DN constructs. As shown in Fig. 6, the activation of

c-fos promoter by c-Myc (lane 2) was significantly inhibited (p <0.001) in the presence

of MKK3-DN (lane3) and Rac1-DN (lane 6). Under similar conditions, both FAK-DN

(lane 4) and cdc42-DN (lane 5) did not affect the transactivation function of c-Myc.

Moreover, a down-regulation in the levels of c-Fos protein was also observed in the

presence both MKK3-DN and Rac1-DN (Fig. 7A, lanes 4 and 6 respectively). No


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change in the expression of c-Fos was observed with FAK-DN or cdc42-DN (lanes 3

and 5 respectively). Further, the p38 MAP kinase inhibitor SB20358 also blocked the c-

Myc-induced expression of c-Fos (Fig. 7B, compare lanes 2 and 3). Interestingly, the

Myc-induced down-regulation of Bcl2 (Fig. 7C, lane 2) was restored to normal in the

presence of fos-DN (lane 3), MKK3-DN (lane 4) and Rac1-DN (lane 7). The Bcl2

level, however, remained unchanged due to FAK-DN and cdc42-DN (lanes 5 and 6

respectively).

Viral HBx can inhibit the c-Myc-mediated apoptotic signaling – As many viral

proteins are known to influence the apoptotic death of host cells (48), the levels of c-Fos

and transactivation of the c-fos promoter were evaluated in presence of HBx. As shown

in Fig. 8A, the Myc-induced expression of c–Fos (lane 2) was inhibited considerably in

the presence of HBx (lane 3). As expected, the c-Myc-dependent transactivation of c-

fos (Fig. 8B, lane 2) was also inhibited in the presence of HBx (lane 3). Functional

analysis with the HBx deletion mutants revealed that its amino-terminal B and carboxy-

terminal F regions (in X6 and X14 respectively) were involved in the inhibition function.

As expected, mutant X15 lacking the amino terminal A and B, and carboxy-terminal F

regions, did not show any inhibition. Other regions of HBx were not involved in this pro-

survival activity and therefore, significantly inhibited the Myc activity (P <0.001).

Interestingly, however, the c-Myc-mediated transactivation of the E-box CAT reporter


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was not inhibited by HBx (Fig.8C).

Abrogation of apoptosis by interference in the c-Fos and p38 pathways – The

involvement of p38 MAP kinase pathway in the Myc-induced apoptotic cell death was

finally confirmed using TUNEL assay. As shown in Fig.9, c-Myc could induce the

apoptosis of hepatoma cells under the experimental conditions used by us. Specific

inhibitor of the p38 pathway SB20358 and Rac1-DN completely reversed the process of

apoptosis (p <0.001). Similarly, Fos-DN and HBx could also abrogate the Myc-induced

apoptosis of hepatoma cells (p <0.001). As expected, cdc42-DN did not affect the

apoptosis.

DISCUSSION

Apoptosis is well established as a vital biological phenomenon of crucial importance

in the maintenance of cellular homeostasis (1). An examination of several studies reveals

that the decision of a cell to undergo apoptosis is mediated by a complex integration of

signal transduction pathways. Three groups of protooncogenic proteins, viz. c-Myc/Max,

c-Fos/c-Jun and Bcl2/Bax are known to exert a synergistic effect to enhance their roles

in the pro- or anti-apoptotic action (49). Repression or overproduction of these proteins

is usually associated with induction of apoptosis, an event in which the cell type


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specificity and the nature of the apoptotic stimuli play an important role. There are

several reports suggesting oncogenic co-operation of c-Myc with Ras, v-abl, Bmi1 and

Bcl2 in cell proliferation and transformation (reviewed in 6). However, there is no

evidence of co-operation between c-Myc and c-Fos leading to apoptosis. Our study has

identified c-Fos as a key player in the c-Myc-mediated apoptosis.

We observed that ectopic expression of c-Myc in the hepatoma Huh7 cells under

serum-deprived conditions led to the induction of c-fos promoter (Fig.2) that is

eventually reflected in the upregulation of intracellular c-Fos levels (Fig.1). Since

enforced expression of c-Myc is well known to induce apoptosis, we wanted to explore

the role played by c-Fos under these conditions. We observed a rapid depletion of c-Fos

upon stimulation by serum (Fig. 1B). This may be an intrinsic mechanism employed by

the Myc-expressing cells to re-enter the cell cycle after sensitization to apoptosis. Thus,

we recognized acute accumulation of c-Fos protein in hepatoma cells as a signature of

apoptosis, a phenomenon observed with a variety of cell types including photoreceptor

cells (50), HeLa (51), rat spermatocytes (52) and germinal center B cells (53). However,

the role of c-Fos induction in the Myc-mediated apoptosis has not been recognized.

The c-Myc-induced apoptosis was first observed in a myeloid progenitor cell

line (32D) harboring a constitutive c-Myc expression vector. Here IL-3 deprivation led

to rapid initiation of apoptosis (17). Later, serum-deprived Rat1 fibroblasts with enforced

expression of c-Myc or the Myc-estrogen receptor fusion protein were shown to undergo


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dramatic apoptosis with eventual loss of the entire cell population (10). Myc-induced

apoptosis was dependent on wild-type p53 (27,54) or required specific autocrine

interaction with CD95 and its ligand (55). However, in glucose-deprived cells, the c-

Myc-induced apoptosis is dependent on lactate dehydrogenase A rather than on p53 (11).

The Myc-responsive genes such as ornithine decarboxylase (56) and the cell cycle

phosphatase cdc25A (57) that have dual roles in cell cycle progression as well as

apoptosis, leave scope for the identification of additional target genes. The present studies

revealed that the c-fos promoter was under the regulatory control of c-Myc. Mutational

analysis of the c-fos promoter indicated that Myc-responsive elements were present

within the proximal promoter region (-400 to -225 bp) that included response elements

for serum (SRE), cAMP (CRE) and AP1. A non-canonical ‘E box’ element (5’-

CATGTG 3’) was also detected in this promoter. However, the up-regulation of c-fos by

c-Myc seems unlikely to be due to a direct binding of Myc to this E box element since no

DNA-protein interaction was observed in the electrophoretic mobility shift assay (data

not shown). Since the p38 MAPK pathway is also activated by c-Myc, it is possible that

activation of c-fos promoter occurs via DNA elements responsive to p38 signaling, such

as AP-1 elements that bind ATF2, and SRE that binds Elk1.

Though, the transcription factors of the AP-1 family have well documented role

in cell proliferation and cell cycle progression, the induction of immediate early genes

like c-fos/c-jun has been associated with apoptosis as well (35). We observed that in


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presence of c-Myc, the key markers of cell survival, viz., Bcl2, Akt and Fibronectin were

down-regulated, concomitant with activation of pro-apoptotic signals like caspase 3 and

caspase 9 and inhibition of the phosphorylation of Bad. Importantly, the profile of these

survival and apoptotic markers was reversed in the presence of a dominant negative

mutant of c-Fos (Fig. 3). The negative regulation of Fibronectin is known to be mediated

by the G10BP-1 gene. G10BP-1 in turn is upregulated by Myc and Jun/Fos via ‘E’ Box

and AP-1 elements respectively (58). It is possible that the G10BP-1 repressor protein

plays a role in Myc-mediated apoptosis in hepatocytes. The down-regulation of Bcl2

and activation of caspase 9 suggested that the Myc-mediated apoptosis of hepatoma cells

might be a mitochondria-dependent event as also observed in rat fibroblasts (30).

Further, suppression in the phosphorylation of the serine-threonine kinase Akt (Fig. 3) by

c-Myc implies that cells are on the path of apoptotic cell death (59). No change in the

p53 level (Fig. 3) suggested that c-Myc did not engage the p53 pathway for apoptotic

signaling.

Similar to our observations in Huh7 cells, the cellular level of Fos/Jun is reported

to be elevated by Rho like GTPases in sympathetic neurons upon neuronal growth factor

withdrawal induced apoptosis (60) or by shear stress in vascular endothelial cells (61).

The Rho GTPases (Rho, Rac1 and cdc42) that mediate stimulus-dependent proapoptotic

and survival signals belong to GTP binding proteins of the Ras family which are

instrumental in a wide variety of cellular responses like cytoskeleton reorganization, cell


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cycle progression and adhesion (62). While cdc42 is a major player in neuronal cell death

and involves the up-regulation of JNK pathway (60), Ras and Rac1 are essential for Fas-

mediated apoptotic death of Jurkat cells (63). The ability of these GTP binding proteins to

induce apoptosis originates from their ability to activate downstream stress activated

kinases like p38 and JNK (64). In agreement with these reports, the present study

revealed that a Rac1-dependent and cdc42-independent activation of p38 signaling

cascade was involved in the c-Myc-mediated transcriptional regulation of c-Fos.

Therefore as expected, the DN mutants of MKK3 and Rac1 interfered with the activation

of p38 and ATF2 (Fig. 5A), expression of c-Fos (Fig. 7A) and the transactivation

function of c-Myc (Fig. 6). More importantly, the terminal pro-apoptotic marker caspase

9 was also inhibited in the presence of these DNA mutants (Fig. 5B). Notably, the Bcl2

levels could be also restored to normal in the presence of these mutants (Fig.7C).

Activation of p38 in the rat fibroblasts is known to be associated with Myc-dependent

apoptosis when exposed to cisplatin (34). Further, a significantly lowered activity of p38

and MKK6 has been reported in HCC as compared to normal tissue suggesting the

importance of this pathway in controlling unrestricted cellular proliferation (65). Though

Rac1 and cdc42 belong to the same family of Rho GTPase we have observed the

involvement of only Rac1 in activation of p38 pathway. This might be possible through

regulation of some specific targets/signals by Myc upstream of Rho GTPase leading to

specific activation of Rac1 and not cdc42. Since a down-regulation in the levels of extra


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cellular matrix protein Fibronectin was also observed, we investigated the regulation of

FAK signaling under these conditions. No change in the levels of FAK phosphorylation

was seen (Fig. 4) and the expression of alpha-5 integrin was also not affected (data not

shown).

To promote self-replication and/or persistence, many viral pathogens including

Hepatitis B virus have developed mechanisms to inhibit the death of host cells (48). It

was interesting to note that the hepatitis B virus X protein inhibited not only the c-Fos

expression but also the transactivation of c-fos promoter (Fig.8 A and B). The relatively

less conserved amino-terminal region B and the carboxy-terminal region F of HBx (38)

were found to be important for this inhibitory function (Fig. 8B). Accordingly, X15 - a

truncated mutant of HBx lacking both these regions, failed to inhibit the apoptotic

functions of c-Myc. Interestingly, the failure of HBx to inhibit the Myc-mediated

transactivation through E box (Fig. 8C) suggests that it may not directly antagonize the

transactivation potential of c-Myc. Rather, the inhibitory effects could be through yet

uncharacterized transrepression mechanism. The reversal of suppression of the Akt/

phosphatidylinositol 3-kinase (PI3K) pathway and its downstream target Bad by HBx

(Fig. 3) suggested that it promotes cell survival in hepatoma cells. Further, the abrogation

of activation of hallmarks of apoptosis like caspase 3 and 9 and restoration of Bcl2 and

fibronectin levels suggested the anti-apoptotic role played by HBx under these

conditions (Fig. 3). Earlier, HBx has been shown to block the p53-mediated apoptosis


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either by reducing p21WAF1 expression or interfering with binding of p53 to the TFIIH

transcription-nucleotide excision repair complex (66). HBx is also known to protect

Hep3B hepatoma cells from TGFβ-induced apoptosis by inducing a survival signal that

links Src to PI3K/Akt signaling pathway (67). Furthermore, HBx has been shown to

inhibit the activity of caspase-3 (68). Thus, abrogation of the antiproliferative and

apoptotic effects of c-Myc by HBx might tune the hepatocytes to uncontrolled growth

and contribute to multi-step hepatocarcinogenesis associated with HBV infection.

Finally, using TUNEL assay, we have established the involvement of immediate

early gene c-Fos, and the p38 MAP kinase pathway in the c-Myc-induced apoptosis of

serum deprived hepatoma cells (Fig. 9). This process involves not only the activation of

p38 MAP kinase cascade but also the transduction of apoptotic signals through the

regulation of c-Fos (Fig. 10). The use of physiological and chemical inhibitors has amply

substantiated these points and the possible mechanisms of anti-apoptotic activities of a

viral protein like HBx have been addressed. Further investigations will be necessary to

understand the secret of elevated level of c-Fos and regulation of its downstream targets

in cells sensitized to apoptosis by c-Myc.

Acknowledgements: We are thankful to Dr. D. Sahal for critical reading of the

manuscript and Drs. S. Jameel and S.K. Lal for material help. We are grateful to the


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following scientists for generous gift of different recombinants: P. Chambon (IGBMC,

France) for the mouse c-Myc genomic construct, A. Weisz (University of Napoli, Italy)

for the fos-CAT reporter constructs FC1-BL, FC2-BL, FC4-BL and FC8-BL, C.

Vinson (National Institutes of Health, USA) for Fos-DN, W. G. Cance (University of

North Carolina, USA) for FAK-DN (PLXXSN-HA-FRNK), U. Hibner (Institut de

Genetique Moleculaire, Montpellier, France).for Rac1-DN and cdc42-DN, J. R.

Woodgett (Ontario Cancer Institute, Canada) for MKK3-DN, R. Tjian (University of

Californian USA) for pUCAT. Technical help from R. Kumar in cell culture maintenance

and propagation is acknowledged. The work was supported by core grants from the

International Centre for Genetic Engineering and Biotechnology, New Delhi. NK is a

recipient of Senior Research Fellowship from the Council of Scientific and Industrial

Research, New Delhi.


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FOOTNOTES

Key words: Apoptosis, Serum deprivation, Dominant negative mutant, promoter

activation, CAT assay, X protein

Abbreviations: CRE, cyclic AMP response element; DN, dominant negative; HCC,

hepatocellular carcinoma; HBx, hepatitis B virus X protein; PAGE, polyacrylamide gel

electrophoresis; PI3K, phosphatidylinositol 3-kinase; SRE, serum responsive element;

TNF, tumor necrosis factor; TPA, 12-O-tetradecanoylphorbol acetate.


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FIGURE LEGEND

Fig.1. c-Myc expression and regulation of c-fos under serum-deprived conditions. A,

Huh7 cells were transfected with the expression vector for c-Myc (3µg) and analyzed by

western blotting using anti-Myc antibody. Arrowhead shows the position of c-Myc

protein. B, c-Myc transfected cells were grown without serum for 24h followed by serum

stimulation for indicated time periods and the levels of c-fos expression were measured

by western blotting using equal amount of protein. Expression levels are shown as fold

stimulation compared to control in lane 1. TPA-stimulated positive control (at 200 nM

for 30 min) is shown in lane 9. Bottom panel shows total Erk as sample loading control.

Fig. 2. Regulation of the c-fos promoter by c-Myc. Cells were transfected with the c-

Myc expression vector and either the full-length c-fos promoter-CAT construct FC1-

BL (A) or separately with its deletion mutants FC2-BL, FC4-BL, FC8-Bl and FC80-

BL (B). CAT activity was measured in the cell extracts and values are represented as fold

(A) or per cent transactivation (B).

Fig. 3. Involvement of c-fos and HBx in the myc-mediated apoptotic signaling. Cells

were transfected with either expression vectors for c-Myc alone or along with Fos-DN

or HBx. Cell extracts were immunoprecipitated for cell survival markers Bcl2, pAkt and


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Fibronectin (A), the pro-apoptotic markers caspase 3, caspase 9 and pBad (B), or the key

mediator of apoptosis p53 (C) and analyzed by western blotting using equal amounts of

protein. Bottom panel shows total Erk as sample loading control.

Fig. 4. Specific activation of p38 and ATF2 by c-Myc and its inhibition by viral HBx.

Huh7 cells were transfected with the expression vectors for c-Myc (lane 2), HBx (lane 3)

or both (lane 4). Finally, the cell extracts were immunoprecipitated for the activated

forms of mediators of different signaling pathways like Erk1/2, pJNK, pJun, pCREB,

p90, p38, pATF2 and pFAK. Lane 1, vector control. Bottom panel shows total Erk as

sample loading control.

Fig. 5. Involvement of the p38 pathway in the Myc-mediated apoptotic signaling. Huh7

cells were transfected with either c-Myc alone (lane 2) or separately with the expression

vectors for DN mutants of the cell signaling pathways like Fos, MKK3, FAK, cdc42 and

Rac1 (lane 3-7). The cell extracts were immuno-blotted for phosphorylated as well as

total p38 and ATF2 (A) or caspase 9 (B) using normalized amounts of protein. Lane 1,

vector control.

Fig. 6. Involvement of p38 pathway in the Myc-dependent transcriptional regulation of

c-fos. Huh7 cells were transfected with the reporter constructs FC4-BL in the presence

of Myc expression plasmid alone or separately with the DN mutants of MKK3, FAK,


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Rac1 and cdc42. CAT assay was performed as shown in top panel and percent CAT

activity was determined by densitometry (bottom panel). Bars in the lower panel

represent mean ± standard deviation of three observations. *, p <0.001.

Fig. 7. Role of p38 in the expression of c-fos. Huh7 Cells were transfected with either c-

Myc alone or separately with the DN expression vectors for FAK, MKK3, cdc42, Rac1

and c-fos. After incubation as described in the method section, cell extracts were

immunoprecipated for c-Fos (A, B) and Bcl2 (C). In panel B, cells were incubated

without or with SB20358 (10 µM) for 5 h before harvesting. Total Erk is shown as

loading control for samples in panels A and B.

Fig. 8. Inhibition of c-Myc activity by HBx. For analyzing the effect HBx on the c-Myc

mediated expression of c-fos, Huh7 cells were co-transfected with the expression vector

of c-Myc and X0, and immunoprecipitated using anti-fos antibody (A). For analyzing

the differential effects of HBx and its mutants (X5, X6, X7, X9, X10, X14 and X15) on

c-Myc transactivation function, cells were co-transfected with the expression vectors of

c-Myc and X0 or its mutants along with reporter construct FC1-BL (B) or E Box-CAT

(C). Finally, the cell extracts were analyzed for CAT activity represented as per cent

transactivation (mean ± standard deviation of three observations). Total Erk is shown also

shown as loading control in panel A. V, pSG5 vector control. *, p <0.001; **, p = 0.27.


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Fig. 9. Inhibition of the c-Myc-induced apoptosis. Huh7 cells were transfected with

either the c-Myc expression vector alone or along with X0, Fos-DN, Rac1-DN of

cdc42-DN. The Myc-transfected cells were also grown in the presence of SB20358 (10

µM) for 5h before the termination of experiment. TUNEL assay was performed to count

the number of apoptotic cells. Data (per cent apoptotic cells) is shown as mean ± standard

deviation of three observations. *, p <0.001.

Fig. 10. Possible mechanism of apoptotic signaling by c-Myc. c-Myc sends proliferative

signals in the presence of growth factors. Under these conditions, Myc is capable of

transforming cells by oncogenic co-operativity with other signaling molecules such as

Ras and Bcl2. However, in the absence of growth factors, c-Myc could induce apoptotic

cell death by inducing the immediate early response genes like c-fos via the p38 MAP

kinase pathway. Viral proteins like HBx could abrogate the apoptotic process by

bypassing the requirements of growth factors.


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Neetu Kalra and Vijay Kumarcells via p38 MAP kinase pathway

c-Fos is a mediator of c-Myc-induced apoptotic signaling in serum-deprived hepatoma

published online April 12, 2004J. Biol. Chem.

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