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[Cancer Research 64, 3772-3779, June 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Reversible Regulation of the Transformed Phenotype of Ornithine Decarboxylase- and Ras-Overexpressing Cells by Dominant-Negative Mutants of c-Jun

Mari Kielosto1, Pirjo Nummela1, Riikka Katainen1, Virna Leaner2, Michael J. Birrer2 and Erkki Hölttä1

1 Haartman Institute and Helsinki University Central Hospital, Department of Pathology, University of Helsinki, Helsinki, Finland, and 2 Cancer and Cell Biology Department, Center for Cancer Research, National Cancer Institute, Rockville, Maryland


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
c-Jun is an oncogenic transcription factor involved in the regulation of cell proliferation, apoptosis and transformation. We have previously reported that cell transformations induced by ornithine decarboxylase (ODC) and c-Ha-ras oncogene, commonly activated in various cancer cells, are associated with constitutively increased phosphorylation of c-Jun on Ser residues 63 and 73. In the present study, we examined the significance of c-Jun phosphorylation and activation on the phenotype of the ODC- and ras-transformants, by using specific inhibitors and dominant-negative (DN) mutants to c-Jun NH2-terminal kinase (JNK) and its upstream kinase, SEK1/MKK4 (mitogen-activated protein kinase kinase 4), and to c-Jun. The transformed morphology of both the ODC- and ras-expressing cells was reversed partially by JNK inhibitors and DN JNK1, more effectively by DN SEK1/MKK4 and phosphorylation-deficient c-Jun mutants (c-JunS63,73A, c-JunS63,73A,T91,93A) and most potently by a transactivation domain deletion mutant of c-Jun (TAM67). Moreover, tetracycline-inducible TAM67 expression in ODC- and ras-transformed cells showed that the transformed phenotype of the cells is reversibly regulatable. TAM67 also inhibited the tumorigenicity of the cells in nude mice. These inducible cell lines, together with their parental cell lines, provide good models to identify the genes and proteins relevant to cellular transformation.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
c-Jun is an oncogenic transcription factor that is present as a major component of the AP-1 (activator protein 1) transcription factor complexes. It has an NH2-terminal transactivation domain, a DNA-binding domain, and a leucine zipper domain in the COOH-terminal domain, through which it can dimerize (1 , 2) . c-jun is an immediate early gene transcribed rapidly and transiently after stimulation of normal quiescent cells with different kinds of mitogens and tumor promoters. The expression of c-Jun is constitutively increased in many transformed cell lines (1 , 2) and human cancers (3) , and overexpression of c-Jun alone can induce the transformation of immortalized rodent fibroblasts (4) and chicken embryo fibroblasts (5 , 6) .

The regulation of c-Jun occurs both transcriptionally and translationally, and its activity can be regulated through phosphorylation and dimerization with different partners. c-Jun is phosphorylated by specific kinases called c-Jun NH2-terminal kinases (JNKs) and in some cell types also by mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK)-mediated mechanisms (7 , 8) . JNKs (including 10 known isoforms) phosphorylate c-Jun within its NH2-terminal activation domain at Sers 63 and 73 (9) , which results in an increase in its transcriptional activity and stability (10, 11, 12) . JNKs (mainly the ubiquitous isoforms 1 and 2) are known to be potently activated by cell exposure to UV radiation, pro-inflammatory cytokines, and environmental stress (13, 14, 15) but may also be activated during apoptosis, differentiation, morphogenesis, and oncogenesis (9) . JNK activation is triggered by dual phosphorylation on distinct Thr and Tyr residues by two specific MAPK kinases, MKK4 (SEK1) and MKK7 (9) . Finally, c-Jun can form homodimers and/or heterodimers, the latter preferred, with other Jun family members (JunB and JunD), Fos family members (c-Fos, FosB, Fra1, and Fra2), activating transcription factor proteins (ATF-2) and other proteins (e.g., Maf). The different complexes formed can then bind either to a 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive element (TRE; Refs. 16 , 17 ) or to a cAMP-responsive element (CRE; Ref. 17 ) in the promoters of genes that regulate cell proliferation and differentiation.

The activation of c-Jun seems to be specifically required for progression through the G1 phase of the cell cycle (18, 19, 20) . Interestingly, in fibroblasts, c-Jun has recently been shown to control the cell cycle by acting as a direct negative regulator of p53 expression (21) . However, c-Jun may also act independently of p53 (22) . c-Jun has also been shown to be involved in the control of apoptosis (19 , 23, 24, 25) . Not much is yet known about the molecular mechanisms involved, but there is some indication that the regulation of cell proliferation and apoptosis by c-Jun may be governed by at least two distinct pathways (19 , 24) . Which pathway is selected may depend on the availability and formation of specific c-Jun dimerization complexes or on the other signaling pathways coincidentally activated. In addition, growing evidence in tissue culture and animal tumor models indicate that c-Jun is a central player in the control of cellular transformation. Indeed, the transforming activity of many known and putative oncogenes, such as activated receptor Tyr kinases, intracellular Tyr and Ser kinases, ras, S-adenosylmethionine decarboxylase (AdoMetDC), and nuclear oncoproteins c-Myc and SV40T, appear to be dependent on c-Jun activation (22 , 26, 27, 28, 29, 30, 31, 32) .

Recent studies have shown that ornithine decarboxylase (ODC), a key regulatory enzyme in the biosynthesis of polyamines (putrescine, spermidine, and spermine) is also a protein that, like c-Jun, is essential for mammalian cell proliferation (33, 34, 35, 36, 37) and may have role in cell transformation. Overexpression of ODC alone is sufficient to transform immortalized rodent cell lines (38 , 39) , and, with the ras oncogene, it is able to transform primary cells (40) . ODC is also known to be highly activated in cells transformed by various carcinogens and oncogenes, such as v-src, neu, myc, and ras, as well as in a variety of clinical cancers (34, 35, 36, 37 , 41, 42, 43) . Furthermore, inhibition of ODC activity with specific inhibitors or by dominant-negative mutants results in inhibition or reversion of transformation by v-src (39 , 44) and ras (45 , 46) . As to the possible mechanisms involved in ODC-induced transformation, we have previously detected constitutive activation of JNK and constitutively increased phosphorylation of the c-Jun transcription factor on Sers 63 and 73 in these transformants (41 , 47) . These findings are similar to those obtained with ras, v-sis, v-src, raf-1, and polyomavirus middle-sized tumor antigen (13 , 48, 49, 50, 51, 52, 53, 54) , implying an important role specifically for c-Jun phosphorylation in the regulation of transformation.

In the present study, we examined the significance of c-Jun phosphorylation and activation in ODC- and Ha-ras-induced cell transformation. We studied the effects of dominant-negative mutants of SEK1/MKK4 and JNK1, specific JNK inhibitors, two phosphodeficient mutants of c-Jun and a transactivation domain deletion mutant of c-Jun (TAM67) on the transformed phenotype of the cells. We also generated tetracycline-regulatable TAM67-expressing cell lines from the ODC- and ras-transformants, and show that the state of transformation and tumorigenicity of these cells can be effectively regulated by the expression of TAM67. These inducible cell lines provide good models to identify the molecular mechanisms relevant to cell transformation.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
NIH3T3 cells stably transfected with the human ODC cDNA (Odc; Ref. 39 ), the c-Ha-rasVal12 oncogene (pGEJ6.6; E4; Ref. 55 ) and an ODC-transformant derived from the Odc cell line-induced tumors in nude mice (Odc-n; Ref. 56 ) have been described previously. The cells were grown in DMEM containing antibiotics (penicillin, streptomycin, and gentamicin) and 5% fetal bovine serum (FBS; Bioclear) at 37°C in a 5% CO2 atmosphere. The normal NIH 3T3 cells were grown in DMEM with antibiotics and 5% FBS or newborn calf serum (Life Technologies, Inc.).

The Odc, Odc-n, E4, and normal NIH 3T3 cells, stably transfected with a tetracycline-inducible expression system of c-Jun deletion mutant TAM67 (see below) were cultured as above or in MEM {alpha} ({alpha}-MEM) supplemented with gentamicin (50 µg/ml) and 5% FBS, TET System Approved FBS (Invitrogen), or newborn calf serum.

Plasmids and Transfections.
Empty pcDNA3-vector (Invitrogen), dominant-negative (DN) JNK1 (FLAG-JNK1[APF]; Ref. 13 ), DN SEK1 (SEK1[AL]; Ref. 57 ), DN MKK4 (Flag-MKK4[Ala]; Ref. 58 ), pMT 108 (c-Jun), pMT111 (c-Jun with Ser 63 and Ser 73 mutated to alanines), pMT 161 (c-Jun with Ser 63, Ser 73, Thr 91, and Thr 93 mutated to alanines; Refs. 59 , 60 ), and pCMV-TAM67 (Ref. 26 ; 1 µg) were transfected together with a pBabe puro selection marker (Ref. 61 ; 0.1 µg) into the cells (grown on six-well plates) using the LipofectAMINE Plus method according to the manufacturer’s instructions (Life Technologies, Inc.). The transfections were performed without serum for 3 h. The cells were selected for resistance to puromycin (1.5 µg/ml), which was added 2 days after transfection. Pools of transfectants and multiple individual clones (between 5 and 33) were isolated and selected for further analysis.

Generation of Cell Lines Carrying a Tetracycline-Inducible Expression System of the Transactivation Domain Deletion Mutant of c-Jun (pLRT-TAM67).
The TAM67 mutant of c-Jun, lacking the transactivation domain, was released from pGEM3-T67 with EcoR1, and the fragment was cloned into pBluescript. Fragments in the sense orientation were released by XhoI/NotI digestion and cloned into XhoI/NotI-digested reverse tetracycline-regulated retroviral vector (62) . The pLRT-TAM67 plasmid (1 µg) was transfected into the Odc, Odc-n, E4, and normal NIH 3T3 cells (on six-well plates) using LipofectAMINE Plus. Two days after transfection, selection was started with 5 µg/ml blasticidin (Invitrogen) and continued for 1–2 weeks. The stable transfectants were maintained in 1 µg/ml blasticidin. Several clones (transformed foci) were picked up by cylinder cloning for initial screening of the regulation of TAM67 expression, and the best ones were further subjected to single-cell cloning in 96-wells. The cloned cells were tested for their inducibility of TAM67 expression by adding 1 µg/ml doxycycline (Sigma) 1 day after plating and by analyzing 2–3 days thereafter, the expression of TAM67 by immunoblotting and the morphological changes by microscopy.

Cell Lysates.
Cells were harvested by scraping and centrifugation, and were washed 1–2 times with PBS. If not processed immediately, the cell pellets were kept frozen at –70°C until use. For whole cell extracts, the cells were lysed directly in a hot Laemmli sample buffer without 2-mercaptoethanol [62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 0.01% bromphenol blue]. The samples were sonicated on ice for 10 s, and the lysates were cleared by centrifugation. The protein concentration was determined using the BCA Protein Assay Reagent Kit (Pierce Chemical, Rockford, IL). Finally, 2-mercaptoethanol was added to a final concentration of 5% and the samples were boiled for 5 min.

Western Blotting.
The whole cell lysates (20–50 µg) were resolved by SDS-PAGE (10% acrylamide) and were transferred onto 0.2-µm nitrocellulose membrane (Bio-Rad trans-Blot transfer medium). The membranes were incubated in blocking buffer [25 mM Tris (pH 8.0), 125 mM NaCl, 0.1% Tween, 2% BSA, and 0.1% NaN3] for 4–16 h and then with specific antibodies for 2–4 h at room temperature or overnight at 4°C. The membranes were washed five times with the TBS-NP/T buffer [10 mM Tris (pH 8.0), 150 mM NaCl, 0.05% NP40, and 0.05% Tween] and were incubated 30 min at room temperature with horseradish peroxidase-conjugated swine antirabbit IgGs (DAKO), rabbit antimouse IgGs (DAKO) or goat antimouse IgG/IgM (Chemicon) in the TBS-NP/T buffer. Phospho-p38 MAPK antibody was bridged with biotinylated antirabbit IgG (Dako) and streptavidin-biotinylated horseradish peroxidase complex. The membranes were washed five or six times with the TBS-NP/T buffer, 15–30 min in high-salt buffer [10 mM Tris (pH 8.0), and 300 mM NaCl), and, finally, three times in TBS (10 mM Tris (pH 8.0) and 150 mM NaCl). The protein bands were visualized using enhanced chemiluminescence (ECL) detection system and exposition to FUJI Rx film. Equal loading was assessed by staining the membranes with Ponceau S solution (Sigma) and blotting with actin (see below).

Antibodies.
c-Jun and the transactivation domain deletion mutant of c-Jun (TAM67) were detected using c-Jun/AP-1 (Ab-1) polyclonal antibody (Oncogene Research Products, Calbiochem) and the HA-tagged c-Jun (pMT108) and the phosphorylation-site mutants of c-Jun (pMT 111, pMT 161) with mouse monoclonal antibody to HA (clone 12CA5; Boehringer Mannheim). DN SEK1/MKK4 (SEK1 [AL], Flag-MKK4 [Ala]), and DN JNK1 (FLAG-JNK1 [APF]) were analyzed with rabbit polyclonal antibody to MKK4/SEK1/JNKK1 (Sigma) and with mouse monoclonal antibody to the FLAG-tag, FLAG M2 (Sigma), respectively. Phosphorylated and total p38 MAPK were detected using polyclonal rabbit antibody to phospho-p38 (Thr180/Tyr182; Cell Signaling Technology, Inc.) and p38 (C-20; Santa Cruz Biotechnology), respectively. As a loading control, actin was probed with mouse monoclonal antibody to actin (Ab-1; Oncogene Research Products).

Analysis of Cell Growth.
We studied the effect of TAM67 expression on the growth of the Odc, E4, and normal NIH 3T3 cells carrying the tetracycline-inducible expression system by recording the increase in cell number. The cells (5 x 104) were plated in complete medium ({alpha}-MEM or DMEM with 5% FBS or newborn calf serum) on 3-cm-diameter dishes in triplicates in the absence or presence of doxycycline (1 µg/ml). During the next 4 days of culture, the cell numbers were determined every 24 h by Coulter counting.

Use of JNK Inhibitors in Cell Culture.
L-Stereoisomer of JNK peptide inhibitor 1 (L-JNK Inhibitor 1) and L-stereoisomer of TAT control peptide (L-TAT; Alexis Biochemicals) were used at 0, 1, 5, 15, and 25 µM concentrations. The peptides were added daily to the cultures, and the medium was changed every other day. JNK Inhibitor II (SP600125; Calbiochem), a novel, potent catalytic inhibitor (10 mM stock solution made in DMSO), was used at concentrations of 0, 0.1, 0.25, 0.5, 1, 2.5, 5, and 10 µM and 0.1% DMSO as a control. The medium was changed every other day.

Soft Agar Growth.
Cells (1 x 104) in growth medium supplemented with 10% serum were mixed with agar (Noble agar; Difco) to yield a 0.35% agar mixture. The mixtures were then overlaid onto 0.7% bottom agar in 24 well plates. Both agar layers were made with or without doxycycline 1 µg/ml. After polymerization, growth medium with or without doxycycline was added, and the medium was replenished twice a week. The colony formation was followed for 2–3 weeks.

Matrigel Invasion Assay.
Twenty-four-well plates (Greiner) were coated with 300 µl of Growth Factor Reduced Matrigel (Becton Dickinson Biosciences, Bedford, MA) diluted 1:3 in DMEM (supplemented or not with doxycycline, 1 µg/ml). Matrigel was polymerized for 30 min at 37°C. Thereafter, 10 000 cells were plated on top of the Matrigel in 100 µl of DMEM and allowed to adhere for 1 h at 37°C. Excess DMEM was removed, and 250 µl Matrigel layered above the cells. Finally, 500 µl of DMEM containing 10% FCS, without or with doxycycline (1 µg/ml), was added on top of the Matrigel matrix. The growth medium was replenished every third day. The growth pattern of the cells in Matrigel was followed daily by microscopy and photography.

Tumorigenicity Assay.
Six-week-old nude mice (HsdCpb:NMRI-nu) were obtained from Harlan Netherland, the Netherlands. Odc cells with inducible TAM67 expression system (1.5 x 106 cells in 125 µl of growth medium) were injected s.c. into both flanks of the mice. The mice received or not doxycycline (1 mg/ml) in the drinking solution (2% sucrose to mask the bitter taste of tetracycline), started 2 days before the injections and replaced at 2-day intervals. The mice were sacrificed before tumors reached 1 cm3 in size or ulcerated. The tumors were weighed, and one-half of them were frozen in liquid nitrogen for molecular biological analyses and one-half were fixed with formalin and embedded in paraffin for histological analyses.

ODC Assay.
ODC activity was assayed by measuring the release of 14CO2 from L-(1-14CO2) ornithine (55) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Dominant-Negative Mutants of SEK1/MKK4 and JNK1 Reverse, with Different Efficacies, the Transformed Morphology of the ODC- and ras-Overexpressing NIH 3T3 Fibroblasts.
We have previously shown that NIH 3T3 cells, transformed by overexpression of human ODC, display constitutive activation of JNK and phosphorylation of c-Jun at Sers 63 and 73 (41) . To determine the significance of the JNK activation, we transfected ODC-overexpressing cells with dominant-negative mutants of SEK1/MKK4 and JNK1. Expression of MKK4 [Ala] and DN SEK1/MKK4 (SEK1 [AL]) resulted in reversion of the morphology of ODC-transformed cells (Odc; Fig. 1A, c and dCitation ). Similar results were obtained in cells derived from the ODC-overexpressor-induced tumors in nude mice (Odc-n; Fig. 1B, bCitation ). DN JNK1 expression resulted in less prominent changes, causing partial reversion of the ODC-induced morphology (Fig. 1, A,e and B,c)Citation . It is possible that the amount of DN JNK1 expression is not sufficient to attain complete inhibition of JNK1 and JNK2, of which the latter may be of more importance for transformation (63 , 64) . We were, therefore, interested to study also the effects of the recently introduced cell-permeable inhibitors of JNKs: L-JNK Inhibitor 1, which binds to JNK1 and JNK2 with similar affinity and blocks their interaction with c-Jun (65) , and JNK Inhibitor II, which potently blocks (Ki, 0.19 µM) the activity of all of the JNKs (66) . Addition of increasing concentrations of L-JNK Inhibitor 1 (0. 1, 5, 15, and 25 µM) to the Odc cell cultures resulted in slight flattening of the morphology of the cells at concentrations higher than 5 µM, whereas the control peptide L-TAT did not have any appreciable effects at the same concentrations (Fig. 1C)Citation . Similarly, JNK Inhibitor II caused a partial reversion of the transformed morphology of Odc cells at concentrations higher than 0.5 µM, whereas bare 0.1% DMSO (the solvent) did not have any effect on the cell morphology (Fig. 1C)Citation . For comparison, we also studied the effects of DN SEK1/MKK4 and DN JNK1 on the Ha-rasVal12-oncogene-transfected NIH3T3 cells (E4; Fig. 1DCitation ). As with ODC-induced transformation, in the ras-transformed E4 cells, the dominant-negative mutants of SEK1/MKK4 (SEK1 [AL] and MKK4 [Ala]) caused a clear morphological reversion (Fig. 1D, b and c)Citation , whereas DN JNK1 had again a less prominent effect on the morphology (Fig. 1D,d)Citation .



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  		Fig. 1. A, the morphology of normal NIH 3T3 cells (a) and of the ornithine decarboxylase (ODC)-transformed cells (Odc) transfected as follows: (b) with an empty vector [Odc +pcDNA3]; (c) with dominant-negative (DN) SEK1/mitogen-activated protein kinase kinase 4 [Odc +MKK4 (Ala)]; (d) with SEK1 (AL) [Odc +SEK1(AL)]; (e) with DN c-Jun NH2-terminal kinase1 (Odc +DNJNK1); (f) with wild-type c-Jun (Odc +pMT108); (g) with c-JunS63,73A (Odc + pMT111); (h) with c-JunS63,73A,T91,93A (Odc + pMT161); and (i) with a transactivation domain deletion mutant of c-Jun (Odc +TAM67). B, the morphology of cells derived from the ODC-overexpressing NIH 3T3 cell-induced tumors in nude mice (Odc-n) transfected as follows: (a) with an empty vector (+pcDNA3); (b) with SEK1 (AL) [SEK1 (AL)]; (c) with DN JNK1 (+DNJNK1); and (d) with TAM67 (+TAM67). C, the morphology of the Odc cells after incubation of 3 days as follows: (a) without inhibitor (0 µM); (b) with 5 µM L-stereoisomer of JNK peptide inhibitor 1 (L-JNKI 1; 5 µM); (c) with 15 µM L-JNKI 1 (15 µM); (d) with 15 µM L-TAT peptide as a control (L-TAT 15 µM); (e) without inhibitor (0 µM); (f) with 0.5 µM JNK Inhibitor II (0.5 µM); (g) with 1.0 µM JNK Inhibitor II (1.0 µM); or (h) with 0.1% DMSO (the solvent) as a control (DMSO 0.1%). D, the morphology of NIH 3T3 cells transformed by c-Ha-rasVal12 oncogene (E4) after transfection with the following: (a) with an empty vector (+pcDNA3); (b) with DN SEK1/MKK4 [MKK4 (Ala)]; or (c) with SEK1 (AL) [+SEK1 (AL)]; (d) with DN JNK1 (+DN JNK1); (e) with pMT 108 (+pMT 108); (f) with pMT 111 (+pMT 111); (g) with pMT 161 (+pMT 161); and (h) with TAM67 (+TAM67).

 
The NH2-Terminal Phosphorylation Site Mutants and Transactivation Domain Deletion Mutant (TAM67) of c-Jun Reverse the Morphology of the ODC- and ras-Transformed Cells.
To investigate the role of c-Jun phosphorylation and activation in the ODC- and ras-induced transformations, we used two phosphodeficient c-Jun mutants, c-JunS63,73A (pMT 111) and c-JunS63,73A,T91,93A (pMT 161) and a transactivation incompetent deletion mutant of c-Jun (TAM67) having most of the transactivation domain (amino acids 3–122) deleted (26) . A control transfection with wild-type c-Jun (pMT 108) retained the transformed morphology (Fig. 1, A, f and D,e)Citation . The expression of the c-Jun mutants pMT 111 and pMT 161, instead, resulted in reversion of the ODC- (Fig. 1A, g and h)Citation and ras- (Fig. 1D, f and g)Citation induced morphology. Finally, in repeated experiments, the expression of the transcriptionally inactive c-Jun mutant TAM67 caused the most significant reversion of both the ODC- (Fig. 1 A, i and B,d)Citation and the ras- (Fig. 1D,h)Citation transformed phenotype.

The expression of DN SEK1/MKK4, DN JNK1, wild-type c-Jun (pMT 108), the phosphorylation defective c-Jun mutants (pMT 111 and pMT 161), and TAM67 in the above experiments was verified by Western blot analyses (Fig. 2A, B, and D–F)Citation ; it was verified as well that DN SEK1/MKK4 is not affecting p38 MAPK activation in Odc cells (Fig. 2C)Citation . Of note is that the expression of TAM67 was associated with a concomitant down-regulation of the endogenous c-Jun expression (Fig. 2, E and F)Citation . This is likely caused by a transcriptional repression of the c-Jun gene, which is known to have a variant c-Jun response element in its promoter region (16) .



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  		Fig. 2. Western blot analyses of the expression of dominant-negative (DN) SEK1 [Odc+DN SEK1 (AL)] (A), and DN c-Jun NH2-terminal kinase (Odc+DN JNK1; B) in the ornithine decarboxylase (ODC)-transformed cells (Odc). C, Western blot analysis of the effect of DN SEK1 on p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation. Positive control for phospho-p38 (P-p38) is from NIH 3T3 cells exposed to UV light at 50 J/m2 for 1 h (it is loaded less). D, Western blot analyses of the expression of wild-type c-Jun pMT 108, the phospho-acceptor site c-Jun mutants pMT 111 and pMT 161 in the ODC-transformed cells. E and F, Western blot analyses of the expression of TAM67 in the ODC-transformed (Odc+TAM67) and ras-transformed E4 (E4+TAM67) cells. DN SEK1, p38 MAPK, and TAM67 were detected by antibodies raised against the respective proteins; DN JNK1 was detected by antibodies to the Flag-tag; and pMT108, pMT111, and pMT161 were detected by antibodies to the HA-tag. The endogenous c-Jun expression is reduced in the cells expressing TAM67. Actin expression was used as a loading control. kDa, Mr in thousands.

 
The Transformed Phenotype of the ODC- and ras-Transformed Cells Is Reversibly Regulated by Tetracycline-Inducible Expression System of TAM67.
Because we did not obtain stable cell lines expressing TAM67 (at high levels) because of a counterselection (growth inhibition; see below), and to obtain more definitive evidence that expression of TAM67 can reverse the ODC- and ras-induced morphology, we generated ODC- and ras-transformed cell lines in which TAM67 could be conditionally expressed. TAM67 was cloned into a reverse tetracycline-regulatable retroviral expression vector (62) and transfected into the ODC- and ras-transformed cells, and multiple (>25) stable cell clones were isolated. In all of the clones, the expression of TAM67 was tightly regulated by doxycycline addition (with no, or only tiny, leakage under the noninduced conditions) as evidenced by immunoblotting (Fig. 3)Citation . As in transient transfection experiments, the induction of TAM67 expression resulted in down-regulation of the endogenous c-Jun expression in both the ODC- (Fig. 3, A and B)Citation and the ras-transformed (Fig. 3C)Citation cells. To study specifically the transformation reversal and to avoid interfering with the serum growth-factor-induced early changes in gene expression, we chose to add doxycycline (induce TAM67) 1 day after passaging the cells. However, with regard to the data presented here, there was no difference if doxycycline was added immediately after the plating. The induction of TAM67 expression resulted in reversion of the transformed morphology to a more flattened appearance within 1–3 days of doxycycline treatment in both Odc- and E4-pLRT-TAM67 transformants (Fig. 4A, a and b and B, a and b)Citation . Similar results were obtained with all of the clones analyzed. Titration with different concentrations of doxycycline (0.1–2.0 µg/ml) revealed that the expression level of TAM67 correlated with the degree of reversal of the transformed morphology. After withdrawal of doxycycline, the cells returned to the transformed phenotype in 2–3 days, showing that the cellular transformation process can be reversibly regulated. The inducible expression of TAM67 made it possible to study specifically the effects of TAM67 on the ability of the cells to grow anchorage independently in soft agar and Matrigel. The induction of TAM67 in Odc cells markedly inhibited (>90%) cell growth in soft agar, and the colonies were reduced both in number and size when compared with the noninduced cells (Fig. 4A, c and d)Citation . Similarly, the invasive growth potential of the ODC- and ras-transformed cells in Matrigel was highly reduced by TAM67 expression (Fig. 4A, e and f, and B, c and d)Citation .



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  		Fig. 3. Western blot analyses of the expression of TAM67 in the ornithine decarboxylase (ODC)-transformed Odc (A) and Odc-n (B), and ras-transformed E4 cells (C) transfected with a tetracycline inducible TAM67 construct. The cells were passaged and left untreated [without doxycycline (–dox)] or treated with 1 µg/ml doxycycline (+dox) as described in "Materials and Methods". Again, TAM67 expression was accompanied by down-regulation of the endogenous c-Jun expression. Odc-n, ODC-transformant derived from the Odc cell line-induced tumors in nude mice. kDa, Mr in thousands.

 


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  		Fig. 4. The effect of inducible TAM67 on the transformed phenotype and invasiveness of ornithine decarboxylase (ODC)- (A) and ras-transformed E4 cells (B). Induction of TAM67 expression by doxycycline reversed the transformed morphology of Odc-pLRT TAM67 (A, a and b) and E4-pLRT TAM67 cells (B, a and b). TAM67 expression also inhibited the growth of the ODC-transformants in soft agar (A, c and d) and in Matrigel (A, e and f, and B, c and d). Doxycycline (dox) was added to the cells 2 days before they were transferred to soft agar and Matrigel. The morphology of the cells grown in the absence (–dox) and presence (+dox) of doxycycline was documented by phase-contrast micrcoscopy and photography after 2 days, the number of soft agar colonies after 3 weeks, and the growth pattern in Matrigel after 3 days (Odc-pLRT TAM67) and 5 days (E4-pLRT TAM67) of culture.

 
In addition to the morphological reversion, the expression of TAM67 resulted in the inhibition of growth of the ODC- (Fig. 5A)Citation and ras-transformed cells. Interestingly, this was not the case with the normal NIH 3T3 cells, in which inducible expression of TAM67 had no significant effect on cell growth (Fig. 5B)Citation . Finally, the activity of ODC was found to remain high in the TAM67-transfected Odc cells, both in the transiently transfected cells and in the cells carrying the inducible pLRT TAM67. After induction of TAM67 expression for 2 days, the ODC activity (which correlates with the amount of enzyme protein) was only minimally affected (24% decreased) relative to the noninduced control (Fig. 6)Citation . This small decrease in the ODC activity is likely secondary to the TAM67-induced growth inhibition. These data indicate that the reversal of transformation by TAM67 was not due to its blocking the ODC expression but to the inhibition of AP-1 mediated cellular events.



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  		Fig. 5. The effect of inducible TAM67 on the growth of ornithine decarboxylase (ODC)-transformed (Odc; A) and normal NIH 3T3 cells (B). Cells (5 x 104) were seeded on 6-well plates in triplicates and 1 µg/ml doxycycline (dox) was added to one-half of the cultures immediately after plating. The cells were counted at 24-h intervals by Coulter counter, and growth curves plotted ± SD.

 


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  		Fig. 6. Ornithine decarboxylase (ODC) activity in the ODC-transformants carrying a tetracycline inducible expression system of TAM67. Odc cells with inducible TAM67 expression system were grown with (+dox) or without (–dox) doxycycline for two days. The values (nmol/mg protein/h) are means ± SD of four or five cultures from two experiments. The activity of ODC in the normal NIH 3T3 cells is shown for a reference.

 
Expression of TAM67 Inhibits Tumor Formation in Nude Mice.
Inoculation of Odc-pLRT TAM67 cells s.c. at both flanks of the mice having the control drinking solution (2% sucrose) resulted, in all cases, in rapidly growing tumors, fibrosarcomas with ulcerative tendency (Table 1Citation , Fig. 7ACitation ), whereas the mice receiving doxycycline for TAM67 expression in the drinking solution developed only very small or no tumors during the treatment period of 17 days (Table 1)Citation . Similarly to that, we have earlier reported for the ODC-transformed parental cells (56) , the uninduced Odc-pLRT TAM67 cells (in mice not having doxycycline) could invade locally to striated muscle and fat tissues (Fig. 7B)Citation , whereas the small tumors, arisen from the Odc-pLRT TAM67 cells induced to express TAM67 by doxycycline supplementation, showed reduced invasive capacity (Fig. 7C)Citation .


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  		Table 1 Inducible expression of TAM67 inhibits the tumorigenicity of Odc-pLRT TAM67 cells in nude mice

Odc cells with inducible TAM67 expression system were injected s.c. into both flanks of nude mice, which were then given (+dox) or not (–dox) doxycycline in their drinking solution (see "Materials and Methods"). The mice were killed after 17 days, and the tumors arisen were weighed.

 


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  		Fig. 7. Odc-pLRT TAM67 cells induce ulcerated and invasive tumors in nude mice in a TAM67 expression-sensitive manner. The experiment was performed as described in the legend of Table 1Citation . A, the tumors formed were fixed in formalin, embedded in paraffin, cut in 5-µm sections and stained with H&E. Odc-pLRT TAM67 cell-induced tumor with ulceration in a mouse not having doxycycline to drink (–dox). There is a lack of the normal epidermis in the ulcer (arrow). B, invasion of the tumor cells into striated muscle (arrow) and fat tissue in the untreated (–dox) mouse. C, reduced invasion of the tumor cells into striated muscle (bottom right) in a mouse treated with doxycycline (+dox). A, x100; B and C, x200.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In our study, dominant-negative mutants of SEK1/MKK4 were found to reverse the morphology of the ODC-transformed cells, implicating that the JNK pathway is relevant to cellular transformation associated with overexpression of ODC. In support of this notion, transfection of DN JNK1 also resulted in a morphological change, but did not fully revert the transformed phenotype. It is possible that DN JNK1 is ineffective at blocking the functions of both JNK1 and JNK2. It is also conceivable that DN SEK1 not only blocks JNK function but also other kinases, resulting in the significant effect observed. The two different JNK inhibitors (L-JNK Inhibitor 1 and JNK Inhibitor II), inhibiting both JNK1 and JNK2 (65 , 66) , resulted also in partial reversion of the transformed morphology; this supports the latter possibility of JNKs not being the only kinases involved in ODC-induced cell transformation. We also found a minor increase in p38 MAPK activation (phosphorylation) in Odc cells, but DN SEK had no effect on the phosphorylation of p38 MAPK, suggesting that DNSEK may still have other unknown sites of action. Similar findings were made in the ras-transformed E4 cells, i.e., that DN SEK1/MKK4 was more effective than DN JNK1 in reversing the transformed morphology. Previous studies in our laboratory have shown that, unlike the ODC-transformants, the ras-transformed E4 cells do not display constitutive activation of JNK, and that the MAPKs extracellular signal-regulated kinase 1 and 2 are not activated in either of these cell lines (41 , 47) . Altogether, these data suggest that a yet unidentified kinase(s) may additionally be involved in the maintenance of transformation of these cells. Considering this possibility, we note that, in mice, the deletion of SEK1/MKK4 causes a more severe phenotype than the combined knocking out of JNK1 and JNK2 (12) , which also suggests a broader spectrum of downstream targets for SEK1/MKK4.

In any case, both the ODC- and ras-transformed cells show a constitutive increase in the phosphorylation of c-Jun on the Ser residues 63 and 73 (41 , 47) . In this study, we show that transfection of the ODC- and ras-transformants with the phosphorylation site mutants of c-Jun (c-JunS63,73A and c-JunS63,73A,T91,93A) could reverse the transformed morphology. These data suggest an important role for c-Jun phosphorylation in the maintenance of transformation of the ODC- and ras-transformed cells. In concert with this data, Behrens et al. (51) have recently shown that the phosphorylation of c-Jun at Sers 63 and 73 is essential for the induction of ras transformation in genetically manipulated mice. Thus, these c-Jun mutants may provide viable tools for cancer therapeutic approaches.

However, TAM67, the transactivation domain deletion mutant of c-Jun, had the most potent transformation reversing effect of all of the c-Jun mutants tested, both in the case of ODC- and ras-induced transformation. This indicates that in addition to phosphorylation there are other ways by which c-Jun plays a role in the maintenance of these transformations. Noteworthy, a previous study has shown that c-Jun is also important for the induction of cell transformation by ras, as evidenced by the finding that cells lacking c-Jun are unresponsive to oncogenic forms of ras (28) . Of significance is that, although the signaling pathways through the different MAPK modules may vary depending on the cell type and growth conditions, all of these signalings appear to converge on c-Jun/AP-1 activity among others, making c-Jun/AP-1 an ideal candidate for therapeutic interventions.

Our results show that the expression of TAM67 was accompanied by down-regulation of endogenous c-Jun, presumably as a result of transcriptional repression through the variant AP-1 site in its promoter (16) . However, the TAM67 expression has also been reported not to interfere with endogenous c-Jun expression (67) . It is possible that the reduction of c-Jun in our study is due to achieving higher expression levels of TAM67. This may result in significant alteration of the composition of the AP-1 complexes.

Curiously, previous reports on the effects of TAM67 on ras-induced transformation have been conflicting. Whereas Brown et al. (26) and Rapp et al. (30) show that TAM67 affects the transformed morphology (rise of transformed cells on ras cotransfection), Janulis et al. (68) do not see these effects. These discrepancies may reflect differences in NIH 3T3 cell lines or may result, e.g., from different transfection systems and different TAM67 expression levels achieved. Indeed, our studies have revealed that the transfection protocol may greatly affect the results of transient transfection experiments, and that there may be a strong selection against the high TAM67 expressers, complicating the analyses. Clearly, the transient transfection assays should be interpreted with caution.

It is also important to note, that most of the previous studies have dealt with the effects of TAM67 on the induction of transformation and not on the established transformed cell lines studied here. Using a reverse tetracycline-inducible expression system, we could definitively establish that TAM67 can normalize the phenotype of the ODC- and ras-transformed cells. TAM67 not only reversed the transformed morphology but also inhibited the proliferation of the ODC- and ras-transformed cells, whereas the growth of the normal cells was not significantly affected by TAM67. TAM67 also blocked the tumorigenic activity of the cells in nude mice. This raises an intriguing possibility that the inhibition of c-Jun/AP-1 by TAM67 could be effectively exploited in cancer gene therapy without having much adverse effect on the normal cells. The finding that TAM67 superceded the effectiveness of DN MKK4/SEK1, DN JNK, JNK inhibitors and phosphodeficient c-Jun mutants in transformation reversal is also a persuasive argument for TAM67 exploitation in future cancer therapeutic approaches. Moreover, our studies show that TAM67 can block the invasive capacity of the ODC- and ras-transformed cells. Finally, as TAM67 expression and the transformation state of the cells can be conditionally regulated, the ODC-pLRT TAM67 and ras-pLRT TAM67 cell lines together with their parental transformed and normal cell counterparts, offer powerful tools to explore and characterize the genes crucial for cellular transformation. As an indication of this, our initial screening of about 9500 genes in these cell lines by cDNA microarray analyses have revealed that there is only a surprisingly small number of potential transformation relevant genes.3


   ACKNOWLEDGMENTS
 
We are grateful to Drs. R. J. Davis (University of Massachusetts Medical School, Worcester, MA), D. Bohmann (University of Rochester Medical Center, Rochester, NY), and J. R. Woodgett (Ontario Cancer Institute, Toronto, Ontario, Canada) for providing plasmids. We also thank Dr. Yulong He (University of Helsinki, Helsinki, Finland) for the advice and help with nude mice injections.


   FOOTNOTES
 
Grant support: E. Hölttä was supported by grants from the University of Helsinki, the Finnish Cancer Organizations, the Academy of Finland, the Sigrid Juselius Foundation, Helsinki University Central Hospital Research Funds, and M. Kielosto and P. Nummela were supported by the Ida Montin Foundation. M. Kielosto and P. Nummela are predoctoral fellows of the Helsinki Biomedical Graduate School.

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.

Requests for reprints: Erkki Hölttä, Haartman Institute and Helsinki University Central Hospital, Department of Pathology, P. O. Box 21, 00014 University of Helsinki, Finland. Phone: 358-9-19126516; Fax: 358-9-19126675; E-mail: erkki.holtta{at}helsinki.fi

3 M. Kielosto, P. Nummela, E. Hölttä, unpublished data. Back

Received 11/ 7/02. Revised 3/ 1/04. Accepted 4/ 1/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bohmann D, Bos TJ, Admon A, Nishimura T, Vogt PK, Tjian R Human proto-oncogene c-jun encodes a DNA binding protein with structural and functional properties of transcription factor AP-1. Science (Wash DC), 238: 1386-92, 1987.[Abstract/Free Full Text]
  2. Vogt PK, Tjian R Jun: a transcriptional regulator turned oncogenic. Oncogene, 3: 3-7, 1988.
  3. Tessari G, Ferrara C, Poletti A, et al The expression of proto-oncogene c-jun in human pancreatic cancer. Anticancer Res, 19: 863-7, 1999.[Medline]
  4. Schutte J, Minna JD, Birrer MJ Deregulated expression of human c-jun transforms primary rat embryo cells in cooperation with an activated c-Ha-ras gene and transforms rat-1a cells as a single gene. Proc Natl Acad Sci USA, 86: 2257-61, 1989.[Abstract/Free Full Text]
  5. Castellazzi M, Dangy JP, Mechta F, et al Overexpression of avian or mouse c-jun in primary chick embryo fibroblasts confers a partially transformed phenotype. Oncogene, 5: 1541-7, 1990.[Medline]
  6. Wong WY, Havarstein LS, Morgan IM, Vogt PK c-Jun causes focus formation and anchorage-independent growth in culture but is non-tumorigenic. Oncogene, 7: 2077-80, 1992.[Medline]
  7. Karin M, Hunter T Transcriptional control by protein phosphorylation: signal transmission from the cell surface to the nucleus. Curr Biol, 5: 747-57, 1995.[CrossRef][Medline]
  8. Whitmarsh AJ, Davis RJ Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J Mol Med, 74: 589-607, 1996.[CrossRef][Medline]
  9. Ip YT, Davis RJ Signal transduction by the c-Jun N-terminal kinase (JNK)—from inflammation to development. Curr Opin Cell Biol, 10: 205-19, 1998.[CrossRef][Medline]
  10. Hibi M, Lin A, Smeal T, Minden A, Karin M Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes Dev, 7: 2135-48, 1993.[Abstract/Free Full Text]
  11. Musti AM, Treier M, Bohmann D Reduced ubiquitin-dependent degradation of c-Jun after phosphorylation by MAP kinases. Science (Wash DC), 275: 400-2, 1997.[Abstract/Free Full Text]
  12. Davis RJ Signal transduction by the JNK group of MAP kinases. Cell, 103: 239-52, 2000.[CrossRef][Medline]
  13. Derijard B, Hibi M, Wu IH, et al JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell, 76: 1025-37, 1994.[CrossRef][Medline]
  14. Minden A, Lin A, Smeal T, et al c-Jun N-terminal phosphorylation correlates with activation of the JNK subgroup but not the ERK subgroup of mitogen-activated protein kinases. Mol Cell Biol, 14: 6683-8, 1994.[Abstract/Free Full Text]
  15. Kyriakis JM, Banerjee P, Nikolakaki E, et al The stress-activated protein kinase subfamily of c-Jun kinases. Nature (Lond), 369: 156-60, 1994.[CrossRef][Medline]
  16. Angel P, Karin M The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta, 1072: 129-57, 1991.[Medline]
  17. Karin M, Liu Z, Zandi E AP-1 function and regulation. Curr Opin Cell Biol, 9: 240-6, 1997.[CrossRef][Medline]
  18. Bakiri L, Lallemand D, Bossy-Wetzel E, Yaniv M Cell cycle-dependent variations in c-Jun and JunB phosphorylation: a role in the control of cyclin D1 expression. EMBO J, 19: 2056-68, 2000.[CrossRef][Medline]
  19. Wisdom R, Johnson RS, Moore C c-Jun regulates cell cycle progression and apoptosis by distinct mechanisms. EMBO J, 18: 188-97, 1999.[CrossRef][Medline]
  20. Ryseck RP, Hirai SI, Yaniv M, Bravo R Transcriptional activation of c-jun during the G0/G1 transition in mouse fibroblasts. Nature (Lond), 334: 535-7, 1988.[CrossRef][Medline]
  21. Schreiber M, Kolbus A, Piu F, et al Control of cell cycle progression by c-Jun is p53 dependent. Genes Dev, 13: 607-19, 1999.[Abstract/Free Full Text]
  22. Li JJ, Cao Y, Young MR, Colburn NH Induced expression of dominant-negative c-jun downregulates NFkappaB and AP-1 target genes and suppresses tumor phenotype in human keratinocytes. Mol Carcinog, 29: 159-69, 2000.[CrossRef][Medline]
  23. Behrens A, Sibilia M, Wagner EF Amino-terminal phosphorylation of c-Jun regulates stress-induced apoptosis and cellular proliferation. Nat Genet, 21: 326-9, 1999.[CrossRef][Medline]
  24. Bossy-Wetzel E, Bakiri L, Yaniv M Induction of apoptosis by the transcription factor c-Jun. EMBO J, 16: 1695-709, 1997.[CrossRef][Medline]
  25. Leppa S, Bohmann D Diverse functions of JNK signaling and c-Jun in stress response and apoptosis. Oncogene, 18: 6158-62, 1999.[CrossRef][Medline]
  26. Brown PH, Alani R, Preis LH, Szabo E, Birrer MJ Suppression of oncogene-induced transformation by a deletion mutant of c-jun. Oncogene, 8: 877-86, 1993.[Medline]
  27. Brown PH, Chen TK, Birrer MJ Mechanism of action of a dominant-negative mutant of c-Jun. Oncogene, 9: 791-9, 1994.[Medline]
  28. Johnson R, Spiegelman B, Hanahan D, Wisdom R Cellular transformation and malignancy induced by ras require c-jun. Mol Cell Biol, 16: 4504-11, 1996.[Abstract]
  29. Rutberg SE, Adams TL, Glick A, Bonovich MT, Vinson C, Yuspa SH Activator protein 1 transcription factors are fundamental to v-rasHa-induced changes in gene expression in neoplastic keratinocytes. Cancer Res, 60: 6332-8, 2000.[Abstract/Free Full Text]
  30. Rapp UR, Troppmair J, Beck T, Birrer MJ Transformation by Raf and other oncogenes renders cells differentially sensitive to growth inhibition by a dominant negative c-jun mutant. Oncogene, 9: 3493-8, 1994.[Medline]
  31. Domann FE, Levy JP, Birrer MJ, Bowden GT Stable expression of a c-JUN deletion mutant in two malignant mouse epidermal cell lines blocks tumor formation in nude mice. Cell Growth Differ, 5: 9-16, 1994.[Abstract]
  32. Paasinen-Sohns A, Kielosto M, Kaariainen E, et al c-Jun activation-dependent tumorigenic transformation induced paradoxically by overexpression or block of S-adenosylmethionine decarboxylase. J Cell Biol, 151: 801-10, 2000.[Abstract/Free Full Text]
  33. Persson L, Wallstrom EL, Nasizadeh S, et al Regulation of mammalian ornithine decarboxylase. Biochem Soc Trans, 26: 575-9, 1998.[Medline]
  34. Pegg AE Recent advances in the biochemistry of polyamines in eukaryotes. Biochem J, 234: 249-62, 1986.[Medline]
  35. Heby O, Persson L Molecular genetics of polyamine synthesis in eukaryotic cells. Trends Biochem Sci, 15: 153-8, 1990.[CrossRef][Medline]
  36. Tabor CW, Tabor H Polyamines. Annu Rev Biochem, 53: 749-90, 1984.[CrossRef][Medline]
  37. Cohen SS . A guide to the polyamines, p. 595 Oxford University Press New York 1998.
  38. Moshier JA, Dosescu J, Skunca M, Luk GD Transformation of NIH/3T3 cells by ornithine decarboxylase overexpression. Cancer Res, 53: 2618-22, 1993.[Abstract/Free Full Text]
  39. Auvinen M, Paasinen A, Andersson LC, Holtta E Ornithine decarboxylase activity is critical for cell transformation. Nature (Lond), 360: 355-8, 1992.[CrossRef][Medline]
  40. Clifford A, Morgan D, Yuspa SH, Soler AP, Gilmour S Role of ornithine decarboxylase in epidermal tumorigenesis. Cancer Res, 55: 1680-6, 1995.[Abstract/Free Full Text]
  41. Holtta E, Paasinen-Sohns A, Povelainen M, Jarvinen K, Ravanko K, Kangas A Cell transformation by ornithine decarboxylase is associated with phosphorylation of the transactivation domain of c-Jun. Biochem Soc Trans, 26: 621-7, 1998.[Medline]
  42. Pegg AE Polyamine metabolism and its importance in neoplastic growth and a target for chemotherapy. Cancer Res, 48: 759-74, 1988.[Abstract/Free Full Text]
  43. Meyskens FL, Jr, Gerner EW Development of difluoromethylornithine (DFMO) as a chemoprevention agent. Clin Cancer Res, 5: 945-51, 1999.[Abstract/Free Full Text]
  44. Holtta E, Auvinen M, Andersson LC Polyamines are essential for cell transformation by pp60v-src: delineation of molecular events relevant for the transformed phenotype. J Cell Biol, 122: 903-14, 1993.[Abstract/Free Full Text]
  45. Shantz LM, Coleman CS, Pegg AE Expression of an ornithine decarboxylase dominant-negative mutant reverses eukaryotic initiation factor 4E-induced cell transformation. Cancer Res, 56: 5136-40, 1996.[Abstract/Free Full Text]
  46. Shantz LM, Pegg AE Ornithine decarboxylase induction in transformation by H-Ras and RhoA. Cancer Res, 58: 2748-53, 1998.[Abstract/Free Full Text]
  47. Paasinen-Sohns A, Holtta E Cells transformed by ODC, c-Ha-ras and v-src exhibit MAP kinase/Erk-independent constitutive phosphorylation of Sos, Raf and c-Jun activation domain, and reduced PDGF receptor expression. Oncogene, 15: 1953-66, 1997.[CrossRef][Medline]
  48. Binetruy B, Smeal T, Karin M Ha-Ras augments c-Jun activity and stimulates phosphorylation of its activation domain. Nature (Lond), 351: 122-7, 1991.[CrossRef][Medline]
  49. Davis RJ MAPKs: new JNK expands the group. Trends Biochem Sci, 19: 470-3, 1994.[CrossRef][Medline]
  50. Westwick JK, Cox AD, Der CJ, et al Oncogenic Ras activates c-Jun via a separate pathway from the activation of extracellular signal-regulated kinases. Proc Natl Acad Sci USA, 91: 6030-4, 1994.[Abstract/Free Full Text]
  51. Behrens A, Jochum W, Sibilia M, Wagner EF Oncogenic transformation by ras and fos is mediated by c-Jun N-terminal phosphorylation. Oncogene, 19: 2657-63, 2000.[CrossRef][Medline]
  52. Smeal T, Binetruy B, Mercola DA, Birrer M, Karin M Oncogenic and transcriptional cooperation with Ha-Ras requires phosphorylation of c-Jun on serines 63 and 73. Nature (Lond), 354: 494-6, 1991.[CrossRef][Medline]
  53. Smeal T, Binetruy B, Mercola D, et al Oncoprotein-mediated signalling cascade stimulates c-Jun activity by phosphorylation of serines 63 and 73. Mol Cell Biol, 12: 3507-13, 1992.[Abstract/Free Full Text]
  54. Srinivas S, Schonthal A, Eckhart W Polyomavirus middle-sized tumor antigen modulates c-Jun phosphorylation and transcriptional activity. Proc Natl Acad Sci USA, 91: 10064-8, 1994.[Abstract/Free Full Text]
  55. Holtta E, Sistonen L, Alitalo K The mechanisms of ornithine decarboxylase deregulation in c-Ha-ras oncogene-transformed NIH 3T3 cells. J Biol Chem, 263: 4500-7, 1988.[Abstract/Free Full Text]
  56. Auvinen M, Laine A, Paasinen-Sohns A, et al Human ornithine decarboxylase-overproducing NIH3T3 cells induce rapidly growing, highly vascularized tumors in nude mice. Cancer Res, 57: 3016-25, 1997.[Abstract/Free Full Text]
  57. Yan M, Dai T, Deak JC, et al Activation of stress-activated protein kinase by MEKK1 phosphorylation of its activator SEK1. Nature (Lond), 372: 798-800, 1994.[Medline]
  58. Whitmarsh AJ, Yang SH, Su MS, Sharrocks AD, Davis RJ Role of p38 and JNK mitogen-activated protein kinases in the activation of ternary complex factors. Mol Cell Biol, 17: 2360-71, 1997.[Abstract]
  59. Treier M, Bohmann D, Mlodzik M JUN cooperates with the ETS domain protein pointed to induce photoreceptor R7 fate in the Drosophila eye. Cell, 83: 753-60, 1995.[CrossRef][Medline]
  60. Papavassiliou AG, Treier M, Bohmann D Intramolecular signal transduction in c-Jun. EMBO J, 14: 2014-9, 1995.[Medline]
  61. Morgenstern JP, Land H Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res, 18: 3587-96, 1990.[Abstract/Free Full Text]
  62. Watsuji T, Okamoto Y, Emi N, Katsuoka Y, Hagiwara M Controlled gene expression with a reverse tetracycline-regulated retroviral vector (RTRV) system. Biochem. Biophys Res Commun, 234: 769-73, 1997.[CrossRef][Medline]
  63. Chen N, Nomura M, She QB, et al Suppression of skin tumorigenesis in c-Jun NH(2)-terminal kinase-2-deficient mice. Cancer Res, 61: 3908-12, 2001.[Abstract/Free Full Text]
  64. Bost F, McKay R, Bost M, Potapova O, Dean NM, Mercola D The Jun kinase 2 isoform is preferentially required for epidermal growth factor-induced transformation of human A549 lung carcinoma cells. Mol Cell Biol, 19: 1938-49, 1999.[Abstract/Free Full Text]
  65. Dickens M, Rogers JS, Cavanagh J, et al A cytoplasmic inhibitor of the JNK signal transduction pathway. Science (Wash DC), 277: 693-6, 1997.[Abstract/Free Full Text]
  66. Bennett BL, Sasaki DT, Murray BW, et al SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc Natl Acad Sci USA, 98: 13681-6, 2001.[Abstract/Free Full Text]
  67. Hennigan RF, Stambrook PJ Dominant negative c-jun inhibits activation of the cyclin D1 and cyclin E kinase complexes. Mol Biol Cell, 12: 2352-63, 2001.[Abstract/Free Full Text]
  68. Janulis M, Silberman S, Ambegaokar A, Gutkind JS, Schultz RM Role of mitogen-activated protein kinases and c-Jun/AP-1 trans-activating activity in the regulation of protease mRNAs and the malignant phenotype in NIH 3T3 fibroblasts. J Biol Chem, 274: 801-13, 1999.[Abstract/Free Full Text]



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