CG&D
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cell Growth & Differentiation
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gjerset, R. A.
Right arrow Articles by Mercola, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gjerset, R. A.
Right arrow Articles by Mercola, D.
Cell Growth & Differentiation Vol. 10, 545-554, August 1999
© 1999 American Association for Cancer Research

Inhibition of the Jun Kinase Pathway Blocks DNA Repair, Enhances p53-mediated Apoptosis and Promotes Gene Amplification1

Ruth A. Gjerset2, Svetlana Lebedeva, Ali Haghighi, Sally T. Turla and Dan Mercola

Sidney Kimmel Cancer Center, San Diego, California 92121


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
We have previously shown, by expression of a nonphosphorylatable dominant inhibitor mutant of c-Jun [cJun(S63A,S73A)], that activation of the NH2-terminal Jun kinase/stress-activated protein kinase by genotoxic damage is required for DNA repair. Here, we examine the consequences of inhibition of DNA repair on p53-induced apoptosis in T98G cells, which are devoid of endogenous wild-type p53. Relative to parental or wild-type c-Jun-expressing control cells, mutant Jun-expressing T98G clones show similar growth rates and plating efficiencies. However, these cells are unable to repair DNA (PCR-stop assays) and exhibit up to an 80-fold increased methotrexate-induced colony formation due to amplification of the dihydrofolate reductase gene. Moreover, the mutant c-Jun clones exhibit increased apoptosis and elevated bax:bcl2 ratios on expression of wild-type p53. These results indicate that inhibition of DNA repair leads to accumulation of DNA damage in tumor cells with unstable genomes and this, in turn, enhances p53-mediated apoptosis.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
Tumor progression involves the rapid accumulation of genetic alterations, some of which promote cell proliferation and enable cell survival in changing environments. Genomic instability, one of the unique features of cancer, may provide the driving force for progression by facilitating these rapid genetic alterations (see Ref. 1 ). Nevertheless, this instability generates strand breaks and other forms of DNA damage that could be deleterious to cell survival. There is, therefore, selective pressure on the cancer cell to modulate its DNA damage response to insure survival while accommodating this increased genetic instability.

One way in which a cancer cell may modulate its DNA damage response is loss of the tumor suppressor p53. p53 mediates apoptosis in response to DNA damage, possibly as a result of its ability to recognize and bind to damaged DNA, including DNA containing single-stranded ends (2) and DNA in abnormal structures known as insertion-deletion loops (3) . Stabilization of p53 protein occurs after DNA damage in a process that involves DNA-PK3 /ATM as a key mechanism (4 , 5) . Numerous studies correlate loss of p53 with increased genome instability (6, 7, 8, 9) , aneuploidy (10 , 11) , and tumor progression (12) , suggesting that loss of p53 renders cells permissive for further genome destabilizing events that accompany and promote tumor progression, such as gene amplification and deletion. Restoration of p53 function in tumor cells that no longer express wild-type p53 restores the DNA damage recognition pathways and leads to G1 arrest or apoptosis (see Ref. 13 , review).

DNA damage also leads to activation of the JNK/stress-activated PK (14) . JNK phosphorylates the c-Jun component of the AP-1 complex and related transcription complexes on serines 63 and 73 in the NH2-terminal domain, thereby greatly activating transcriptional transactivation by AP-1 and related c-Jun-containing complexes, such as the c-Jun/ATF2 heterodimer. JNK activity is strongly induced in response to a variety of DNA damaging treatments, such as UV irradiation (15) , cisplatin (15 , 16) , camptothecin (17) , and etoposide (18) . We have previously shown that activation of the JNK pathway that follows DNA damage is required for DNA repair, suggesting an essential role of JNK in regulating the DNA repair process (16) . Phosphorylation of c-Jun is also induced by certain oncogenes (19) and is required for c-Jun plus Ha-ras cotransformation of rat embryo fibroblasts (19 , 20) . Complete loss of c-Jun in transgenic mouse embryo fibroblasts results in proliferation defects leading to prolonged passage through crisis and delay of spontaneous immortalization (21) .

To more fully understand the role of the JNK pathway and c-Jun phosphorylation in cellular transformation, tumorigenesis, and DNA repair, we have recently selected T98G glioblastoma cells modified to express a mutant Jun that acts as a dominant-negative inhibitor of wild-type c-Jun downstream targets. T98G cells express only mutant p53 (22) and, unlike many other cell types, including normal lung epithelial cells (23) , they express elevated, easily detectable levels of JNK activity, which can be activated an additional 5–10-fold by treatment with the DNA cross-linking agent cisplatin (16) . The Jun mutant construct used to modify T98G cells was originally derived by Binétruy et al. (19) and Smeal et al. (20) and has alanine substitutions at serine positions 63 and 73. Mutant Jun, therefore, cannot be phosphorylated by JNK. Expression of mutant Jun does not alter the basal or induced levels of JNK activity in these cells, indicating that mutant Jun has no direct effect on the JNK enzyme.4 However, it does strongly inhibit transactivation of AP-1 reporter plasmids in rodent fibroblasts (19 , 20) and T98G cells,4 indicating that mutant Jun acts as a competitive inhibitor in the formation of an active AP-1 complex and, therefore, greatly impedes phosphorylation-dependent transactivation functions of c-Jun (19 , 20) . Furthermore, in A549 human lung carcinoma cells, in which the JNK pathway is known to be required for the EGF-stimulated cell growth (23) , inhibition of the JNK pathway by the application of high affinity JNK oligonucleotides leads to inhibition of EGF-dependent growth in a manner indistinguishable from that caused by stable expression of mutant Jun (23) . Thus, stable expression of mutant Jun seems to be a potent and specific inhibitor of phosphorylation-dependent effects of endogenous c-Jun that are usually promoted by the action of JNK.

T98G cells that express mutant Jun have a marked increase in sensitivity to the DNA damaging drug cisplatin and to UV radiation, and this increased sensitivity to DNA damage correlates with an inability to repair DNA (16) . This suggests that phosphorylation of the wild-type c-Jun subunit of transcription factors, such as AP-1 and the c-Jun/ATF2 heterodimer, may contribute to DNA repair and survival after DNA damage through induction of DNA synthesis and repair genes such as topoisomerase I and DNA polymerase ß, both of which have functional AP-1 and ATF2/cAMP-responsive element binding protein sites (which bind to c-Jun/ATF2) in their promoters (24, 25, 26, 27) . Phosphorylation of c-Jun may also contribute to cell survival during the crisis phase of tumorigenic transformation by promoting repair of DNA strand breaks generated by the mechanisms that destabilize the genome during tumor progression.

Restoration of p53 function in T98G glioblastoma cells by exposure to p53-adenovirus promotes low levels of apoptosis at gene transfer efficiencies of 50–80% (28) . We have found that levels of apoptosis can be significantly increased in these cells when they are treated with p53 adenovirus in combination with DNA damaging agents, such as cisplatin and radiation. This is consistent with a model in which the level of DNA damage sustained by the cell is a strong determinant of p53-mediated apoptosis, as suggested by Chen et al. (29) . In this study, we hypothesized that inhibition of DNA repair by expression of mutant Jun, would also enhance p53-mediated apoptosis. It is known that various forms of genetic instability characteristic of cancer cells, including gene amplification, gene deletion, and broken chromosomes are related in origin through the involvement of strand breaks (see Ref. 30 , review). By blocking DNA repair, mutant Jun is predicted to promote elevated levels of strand breaks, which then serve as signals for p53-mediated apoptosis. The elevated level of strand breaks could also stimulate further gene amplification. In the studies reported here, we extend and confirm our earlier observations that mutant Jun expression leads to inhibition of DNA repair. Moreover, we show that mutant Jun expression predisposes cells to gene amplification as judged by the amplification of the DHFR gene. We further show that expression of mutant Jun greatly enhances p53-mediated apoptosis. These observations provide support for the hypothesis that inhibition of DNA repair in cancer cells with unstable genomes enhances sensitivity to DNA damaging chemotherapy and p53-dependent apoptosis.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
T98G Mutant Jun-expressing Cells Resemble Parental T98G Cells with Respect to Growth Rate and Plating Efficiency.
We have previously shown that mutant Jun-expressing T98G clone I-10-10 has decreased viability after treatment with cisplatin and other DNA-damaging agents, likely due to a defect in DNA repair (16) . Here, we examine this and a second mutant Jun-expressing clone, I-10-6, as well as a control c-Jun-expressing clone (T98GcJun) and parental T98G cells for the expression levels of total immunoreactive Jun, for proliferation rates, and for plating efficiencies. Fig. 1Citation shows a Western blot of lysates from each of these four cell lines using an antibody that recognizes both mutant Jun and c-Jun. Equivalent loading was confirmed by stripping the blots and reprobing them with an anti-ß-actin antibody (data not shown). As shown in the figure, mutant Jun-modified clones I-10-10 and I-10-6, as well as control c-Jun-modified clone T98GcJun, overexpress total Jun, consistent with expression of the exogenous constructs. The mutant Jun-expressing clones, as well as the control c-Jun-modified clone, show little difference from parental T98G cells with respect to proliferation rate or plating efficiency (Table 1)Citation . Only slight growth alterations were observed, but these did not correlate with expression of mutant Jun because clone I-10-10 proliferates about 20% faster than parental cells or c-Jun-modified cells and clone I-10-6 proliferates about 20% slower. Therefore, expression of mutant Jun in T98G glioblastoma cells inhibits their ability to repair DNA damage (see below), but it is not growth suppressive in itself.



View larger version (29K):
[in this window]
[in a new window]
  		Fig. 1. Increased expression of immunoreactive Jun in stable transfectants. Western blot analysis of total c-Jun plus mutant Jun in lysates of control c-Jun-modified clone T98GcJun (Lane 1), T98G mutant Jun-expressing clones I-10-6 (Lane 2) and I-10-10 (Lane 3), and parental T98G cells (Lane 4). Each lane represents the electrophoresis of 40 µg of total protein.

 

View this table:
[in this window]
[in a new window]
  		Table 1 Culture characteristics of T98G clones

 
T98G Mutant Jun-expressing Cells Are Defective in Repair of Cisplatin Adducts.
Cisplatin adduct formation and repair was analyzed by a PCR-based assay (PCR-stop assay), as described in "Materials and Methods." Because cisplatin adducts block PCR amplification by Taq polymerase, the intensity of the PCR signal derived from a given amplified region (in our case, the HPRT gene) is inversely proportional to the platination level and can be used as a quantitative measure of the number of cisplatin-DNA adducts on that region (31) . Platination levels determined by PCR amplification of a given housekeeping gene have been shown to correspond to determinations of platination levels on genomic DNA by atomic absorption, demonstrating that the PCR method reflects global DNA platination levels (31) . We have also observed in a variety of tumor cell lines that cells shown to be DNA repair deficient by the PCR assay were also defective in repairing a cisplatin-damaged reporter plasmid, as assayed by expression of the reporter gene 2 days after transfection.5 The PCR assay was chosen for these studies because it measures repair of an endogenous genomic sequence. Fig. 2Citation shows the results of a PCR-stop assay performed on genomic DNA isolated from parental T98G cells, mutant Jun-expressing I-10-10 and I-10-6 cells, and control c-Jun-expressing T98GcJun cells after treatment with cisplatin, with and without a subsequent 16-h recovery period. The bars represent the relative amounts of PCR product resulting from PCR amplification of a 2.7-kb region of the HPRT gene of genomic DNA from cisplatin-treated versus untreated cells. In all cases, the results have been corrected for sample to sample fluctuations in PCR efficiency by normalizing the results to a 170-base PCR product of the same gene (i.e., a fragment too small to register significant levels of platination and where fluctuation from sample to sample varied by <5%). The data show that treatment with 100 µM cisplatin results in an immediate decrease in the PCR signal intensity to about 85% of control signals obtained from DNA from untreated cells. On the basis of a Poisson relationship, this corresponds to an adduct density of about 0.16 adducts/2.7 kb. By 16 h after treatment, both T98G cells and c-Jun-expressing control cells (T98GcJun) have efficiently repaired the adducts, and the PCR signal strengths are equal to controls (i.e., no detectable adducts at 16 h). In contrast, mutant Jun-expressing clones I-10-10 and I-10-6 failed to repair the adducts, and PCR signal strengths remain unchanged after the 16-h recovery period (Fig. 2)Citation . These observations confirm and extend our earlier results (16) and strongly indicate that inhibition of the JNK pathway effectively blocks DNA repair.



View larger version (49K):
[in this window]
[in a new window]
  		Fig. 2. PCR-stop assays of cisplatin adduct formation and repair. A 2.7-kb region of the HPRT gene was PCR amplified from genomic DNA from untreated cells or from cells treated 1 h,15 min with 100 µM cisplatin and harvested either immediately or 16 h after treatment. Bars, the relative amounts of PCR product obtained from damaged versus undamaged templates. The results represent the averages of triplicate PCR reactions performed on two independent occasions.

 
T98G Mutant Jun-expressing Cells Give Rise to Methotrexate-resistant Clones with Higher Frequency Than Do Parental T98G Cells or c-Jun-expressing Cells.
Certain types of DNA repair defects contribute to tumorigenesis (32 , 33) and increased genome instability (34 , 35) . We examined the T98G clones described above for DHFR gene amplification, one measure of genome instability known to correlate with increased tumorigenicity after in vivo implantation of cells (36, 37, 38) . T98G cells were plated in the presence of concentrations of methotrexate five times the LD50 and nine times the LD50 determined for these cells (i.e., concentrations at which gene amplification of DHFR is known to be the predominant mechanism of resistance to the cytocidal effects of methotrexate; Refs. 39 and 40 ). Thus, the frequencies of appearance of methotrexate-resistant clones is a measure of genome instability. As shown in Table 2Citation , T98G I-10-10 cells produce methotrexate-resistant colonies at about 20 times the frequency of the parental T98G cells, and T98G I-10-6 cells produce methotrexate-resistant colonies at about 80 times the frequency of parental T98G cells (P = 0.006). In contrast, stable expression of wild-type c-Jun had no effect on the frequency of resistance, indicating strongly that interference with a phosphorylation-dependent function of JNK predisposed cells to form resistant colonies.


View this table:
[in this window]
[in a new window]
  		Table 2 Frequency of methotrexate-resistant colonies arising from T98G, T98GcJun, and T98G mutantJun clones I-10-10 and I-10-6

 
To verify the occurrence of gene amplification in methotrexate-resistant colonies, several colonies were picked from each selection condition and expanded. Genomic DNA was prepared and subjected to quantitative PCR analysis using 32P-labeled primers that define a 270-base fragment of the DHFR gene, which includes parts of exon 1 and intron A. PCR products were analyzed by agarose gel electrophoresis and quantitated by radioanalytic imaging, as described in "Material and Methods." The relative increase in PCR product from cellular DNA of methotrexate-resistant cells compared with unselected parental T98G cells was taken as a measure of the increased copy number of the DHFR gene and is indicated in Table 2Citation . The methotrexate-resistant clones derived from T98G mutant Jun-expressing clones have from two to four times the gene dosage of the DHFR gene, relative to parental T98G cells, indicating that the observed methotrexate resistance reflected an increased DHFR gene copy number. We conclude, therefore, that inhibition of DNA repair by mutant Jun in T98G glioblastoma cells leads to accumulated DNA damage that can promote gene amplification.

T98G Mutant Jun Cells Are More Susceptible Than Are Parental Cells to p53-mediated Growth Suppression.
T98G cells lack wild-type p53 function as a result of a methionine to isoleucine replacement in p53 at codon 237 (41) . Restoration of wild-type p53 in T98G cells through gene transfer results in partial G1 arrest (41) or apoptosis (28) . Furthermore, agents that promote DNA strand breaks and other forms of DNA damage enhance p53-mediated apoptosis (28) . On the basis of the observations above, indicating that mutant Jun-expressing cells are inhibited in DNA damage repair and predisposed to gene amplification, we predicted that strand breaks would accumulate in mutant Jun-expressing cells, thereby leading to increased p53-dependent growth inhibition and apoptosis. Fig. 3Citation compares the growth inhibition of Ad-p53-transduced cells relative to Ad-ßgal-transduced cells 6 days after infection. The results represent the average of two experiments performed on separate occasions, with each experiment being performed in triplicate. The infection efficiency, determined by X-gal staining of parallel cultures with Ad-ßgal, was about 50% in all cases, low enough to cause incomplete growth suppression of parental T98G cells and control cells modified to stably express wild-type c-Jun, as shown in Fig. 3Citation . Growth studies revealed that T98G I-10-10 and I-10-6 cells were considerably more growth suppressed upon expression of p53 under these conditions. Western blot analysis (Fig. 4)Citation of the p53-responsive gene product p21waf1/cip1 in cell lysates 48 h after infection shows induction of p21waf1/cip1 in all cases. The data, thus, show that p21waf1/cip1 is not a crucial player in this setting. Equivalent loading was confirmed by stripping the blots and reprobing them with an anti-ß-actin antibody (data not shown).



View larger version (31K):
[in this window]
[in a new window]
  		Fig. 3. Six-day viability assay of T98G subclones after treatment with Ad-p53, 100 pfu/cell for 3 h. Viability of Ad-p53-treated cultures is represented as a percentage of the same culture treated under identical conditions with Ad-ßgal.

 


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 4. Western blot analysis of p21waf1 protein in lysates from T98G parental cells, mutant Jun-expressing clones I-10-10 and I-10-6, and control c-Jun-expressing clone T98GcJun 48 h after treatment with Ad-ßgal or Ad-p53, 100 pfu/cell for 3 h. Each lane represents 40 µg of protein: Lane 1, T98G parental-Adßgal; Lane 2, T98G parental-Adp53; Lane 3, mutant Jun clone I-10-10-Adßgal; Lane 4, mutant Jun clone I-10-10-Adp53; Lane 5, mutant Jun clone I-10-6-Adßgal; Lane 6, mutant Jun clone I-10-6-Adp53; Lane 7, control T98GcJun-Ad-ßgal; Lane 8, control T98GcJun-Adp53.

 
T98G Mutant Jun-expressing Cells Are More Susceptible to p53-mediated Apoptosis.
To determine whether the p53-mediated growth inhibition of T98G mutant Jun-expressing cells observed in Fig. 3Citation could be accounted for by the induction of apoptosis, we assayed the cytoplasmic fractions of Ad-p53 or Ad-ßgal-infected cells 48 h after infection for the presence of oligonucleosomal fragments (Fig. 5)Citation . These fragments are released from the nuclei of cells undergoing apoptosis and can be detected by an ELISA assay using antihistone antibodies and anti-DNA peroxidase antibodies. We assayed for apoptosis 48 h after exposure to p53 adenovirus or ß-gal adenovirus because this is the point at which we have observed maximal transgene expression in Ad-ß-gal-infected cells.6 Fig. 5ACitation shows the results of the ELISA assay on the various T98G cell clones. Low levels of oligonucleosomal fragment release similar to levels observed in uninfected cells were observed in Ad-ß-gal-infected cells. Treatment of parental T98G cells and control wild-type c-Jun-expressing T98GcJun cells with Ad-p53 (100 pfu/cell, 3 h) resulted in virtually no induction of apoptosis under our conditions, consistent with growth assays showing no suppression of overall growth after treatment of these cell lines with Ad-p53. However, readily detectable and significantly increased levels of apoptosis were observed in mutant Jun-expressing clones I-10-10 and I-10-6. These results are consistent with the appearance of Ad-p53-treated cultures as shown in Fig. 5BCitation . Ad-p53-treated I-10-10 and I-10-6 cells lose contact with neighbors, become large, and contain cytoplasmic vacuoles. Thus, p53-mediated apoptosis is markedly enhanced in mutant Jun-expressing cells, possibly as a consequence of being triggered by endogenous strand breaks that fail to be repaired.



View larger version (84K):
[in this window]
[in a new window]
  		Fig. 5. A, ELISA apoptosis assay of cytoplasmic nucleosomes in untreated cells or in cells 48 h after being treated with 100 pfu/cell of Ad-ßgal or Ad-p53 for 3 h. B, light microscopy (x40) of untreated cells (top row), or cells 72 h after treatment with Ad-ßgal (middle row) or Ad-p53 (bottom row). a, T98G parental cells; b, c-Jun-modified clone T98GcJun; c, mutant Jun-modified clone I-10-6; d, mutant Jun-modified clone I-10-10.

 
To confirm a p53-dependent mechanism of apoptosis, we carried out a Western blot analysis of the apoptosis regulatory proteins bax and bcl2 in cells treated with Ad-p53 or Ad-ßgal (Fig. 6)Citation . The levels of the proapoptotic effector bax, the gene of which is induced by p53 (42) , increase after treatment with Ad-p53, as expected, whereas levels of the antiapoptotic protein bcl2 remain largely unchanged. A comparison of the bax to bcl2 protein is indicated by the ratios under the lanes in Fig. 6Citation . The bax:bcl2 ratio after treatment with Adp53 is significantly higher in mutant Jun-expressing cells I-10-10 and I-10-6 (ratios of 10 and 2.5, respectively) than in parental cells (ratio of 1.7) and c-Jun control cells (ratio of 0.8). Furthermore, a comparison of these ratios in uninduced versus induced cells (Adßgal-treated versus Adp53-treated) reveals a 3–4-fold increase for the Adp53-treated parental and cJun-expressing control cells compared with the same cells treated with Adßgal, whereas Adp53-treated mutant Jun-expressing cells I-10-10 and I-10-6 show an increase of some 8–25-fold, respectively, compared with the same cells treated with Adßgal. In accordance with other data suggesting that the bax:bcl2 ratio is a critical determinant of apoptosis (see Ref. 43 , review), these data support a role for bax in the increased apoptosis observed after Adp53 treatment of mutant Jun-expressing cells.



View larger version (41K):
[in this window]
[in a new window]
  		Fig. 6. Western blot analysis of bax and bcl2 protein in lysates from T98G parental cells, mutant Jun-expressing clones I-10-10 and I-10-6, and control c-Jun-expressing clone T98GcJun 48 h after treatment with Ad-ßgal or Adp53, 100 pfu/cell for 3 h. Each lane represents 15 µg of protein for the bax analysis and 30 µg of protein for the bcl2 analysis. After immunostaining and band detection with enhanced chemiluminescence Western reagent, bands were quantitated using Kodak digital software. Ratios of bax to bcl2 are indicated below the lanes. Experiment was carried out twice with similar results.

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
In this study, we have examined how a dominant-negative inhibitor of phosphorylated wild-type c-Jun downstream targets affects cell proliferation, DNA repair, susceptibility to p53-mediated apoptosis, and DHFR gene amplification in T98G glioblastoma cells. The JNK/stress-activated PK pathway is a cellular DNA damage and stress-response pathway that is activated by a variety of signals, including mitogens such as EGF (23) , oncogenes (19) , and numerous DNA-damaging agents such as UV radiation and cisplatin (15, 16, 17) . Phosphorylation of c-Jun by JNK activates the transcriptional potential of AP-1 and related transcription factors such as c-Jun/ATF2, which use c-Jun as a heterodimeric partner in the transcription complex. The nonphosphorylatable mutant Jun construct used in these and earlier studies resembles normal cellular c-Jun, except for two alanine replacements at positions 63 and 73. This change has been shown to abrogate the cotransformation properties of c-Jun with H-ras in rat embryo fibroblasts (19 , 20) , and to block EGF-induced proliferation of lung carcinoma cells (23) . T98G glioblastoma cells expressing this mutant Jun have reduced viability after treatment with the DNA-damaging drug cisplatin (16) , and this result is due to an inability to repair cisplatin adducts, as we show here and in an earlier study (16) . Thus, the JNK pathway may promote cell survival during transformation and in response to DNA damage.

In this study, we extend the analysis of the T98G mutant Jun clones analyzed previously. We observe that they grow with similar doubling times and have similar plating efficiencies as parental T98G cells or c-Jun-modified control cells, indicating that stable expression of mutant Jun does not substantially alter DNA synthesis. However, methotrexate-resistant clones arising in the presence of >= 5 x LD50 are generated at a 20–80-fold higher frequency in mutant Jun-expressing clones compared with parental T98G cells and the wild-type c-Jun-expressing clone T98GcJun. Under these conditions, resistance to methotrexate is known to be primarily due to amplification of the DHFR gene (39 , 40) . We confirmed a low, but detectable increase in DHFR gene copy number of about 2–4-fold compared with parental T98G cells by quantitative PCR analysis of genomic DNA isolated from several representative clones of methotrexate-selected T98G I-10-10 and I-10-6 cells. Although low, such an increase in copy number could explain the increase in methotrexate resistance observed in these cells and is supported by previous studies that showed that a low level of DHFR gene amplification was sufficient to confer resistance to methotrexate (44) . Furthermore, mutant Jun-expressing T98G cells, which do not express endogenous wild-type p53, exhibited increased growth suppression and apoptosis after exposure to p53 adenovirus and restoration of wild-type p53 function. Therefore, mutant Jun alone had little effect on the growth properties of T98G cells but manifested a negative effect on growth in the presence of wild-type p53.

Our results demonstrate that expression of a nonphosphorylatable mutant Jun, but not c-Jun, leads to a defect in DNA repair and contributes to increased gene amplification, one manifestation of genomic instability in mammalian cells. These observations are consistent with other examples in which DNA repair defects are seen to underlie a genome instability phenotype (34 , 35) . The results suggest that the DNA repair defect associated with expression of mutant Jun may generate elevated levels of strand breaks in T98G cells compared with T98G parental cells and c-Jun-modified cells, both of which have an intact JNK pathway. The elevated level of breaks may, in turn, serve as initiation events for increased gene amplification (45) , as well as triggers for DNA damage-induced stabilization of transduced wild-type p53, leading to apoptosis. Our results directly demonstrate both gene amplification and significantly increased p53-dependent apoptosis in mutant Jun-expressing cells in support of this hypothesis.

One possible explanation for our observations is that one or more downstream targets of wild-type c-Jun promotes repair of endogenous strand breaks. Candidate targets include DNA polymerase ß, PCNA, topoisomerase I, topoisomerase II, and GADD153, all of which have potential AP-1 or c-Jun/ATF2 binding sequences in their promoter regions (see Refs. 24, 25, 26, 27 and Ref. 46 , review). In the cases of DNA polymerase ß and topoisomerase I, these c-Jun/ATF2 binding sites are known to be functional and stress activated (46) . Moreover, all of these gene products have been implicated in the repair of cisplatin-DNA adducts (47) . Thus, although an intact JNK pathway in T98G parental cells and in c-Jun-modified control cells would not directly prevent DNA damage-induced p53 stabilization, the pathway would act indirectly to attenuate p53-mediated apoptosis by efficiently promoting repair of endogenous strand breaks that would trigger p53 stabilization.

An additional mechanism also may play a role in cells expressing endogenous wild-type p53. Shreiber et al. (21) have recently shown that c-Jun directly down-regulates p53 expression through binding to a variant AP-1 site in the endogenous cellular p53 promoter. In their study, negative regulation of p53 by c-Jun seemed to be crucial to cellular transformation in that transgenic mouse embryo fibroblasts lacking c-Jun displayed proliferation defects, elevated p53 expression, and prolonged transit through crisis before spontaneous immortalization. Thus, c-Jun may attenuate p53-mediated apoptosis both by down-regulating expression of p53 and by promoting repair of endogenous DNA damage that could trigger p53 stabilization and apoptosis.

Two independently derived mutant Jun-expressing clones show similar properties, whereas a third clone expressing wild-type c-Jun and maintained in culture for a similar period did not share any of these properties. These observations strengthen the argument that down-regulation of DNA repair as a consequence of mutant Jun expression underlies the elevation in DHFR gene amplification and enhanced predisposition to p53-mediated apoptosis. Our results suggest, in addition, that increased expression of the p53-regulated proapoptotic effector bax leads to an increased bax:bcl2 ratio that contributes to enhanced apoptosis in mutant Jun-expressing cells after exposure to p53 adenovirus. This is consistent with a variety of observations in other systems showing the importance of the bax:bcl2 ratio in determining apoptosis (see Ref. 43 , review). Thus, an elevated level of endogenous DNA strand breaks in mutant Jun-expressing cells may result in increased stabilization and activation of p53 and increased induction of bax.

The recent identification of p53 as a physiological substrate for JNK (48) indicates that the JNK response extends to other targets besides c-Jun, and these could mediate the various aspects of the stress response. Although inhibited in c-Jun phosphorylation, T98G cells modified with mutant Jun express constitutively active JNK at levels similar to the parental T98G cells.7 They would, therefore, be expected to carry out phosphorylation of other JNK substrates similarly to parental cells. The ability of T98G mutant Jun cells to carry out apoptosis after restoration of p53 activity suggests that any JNK-related apoptotic functions are not disrupted by the mutant Jun modification.

Consistent with our observations that the mutant Jun modification has no significant effect on cell growth or plating efficiency of T98G cells is a study demonstrating that ES cells lacking c-Jun had similar viability and growth rate as parental ES cells and were able to efficiently transactivate AP-1 reporter constructs (49) . Thus, most of the functions of c-Jun in ES cells seemed to be complemented by other Jun proteins. In our case, mutant Jun itself may be able to carry out the c-Jun functions required for basal growth. However, phosphorylation of c-Jun seems to be critical in the cellular response to DNA damage.

Our results can be understood in light of a growing body of evidence supporting a role for p53 in modulating apoptosis in response to DNA damage (see review, Ref. 50 ) and in proportion to the extent of damage (29) . p53 is a DNA damage recognition protein known to bind to a variety of types of DNA damage, including single-strand ends (2) , and insertion-deletion loops (3) . These types of damage, which could serve as triggers for p53-mediated apoptosis, are likely to be generated in tumor cells by the mechanisms that promote spontaneous gene rearrangements, deletions, and amplifications. As such, a failure of DNA repair in mutant Jun-expressing cells would promote the accumulation of strand breaks, which would, on the one hand, favor gene amplification and other manifestations of genome instability and, on the other hand, promote DNA damage-induced stabilization of p53 and apoptosis.

As depicted in the scheme in Fig. 7Citation , we hypothesize that activation of JNK and loss of p53 represent independent mechanisms by which tumor cells undergoing progression accommodate increased levels of genomic instability and insure survival while sustaining potentially lethal genome destabilizing events. By promoting DNA repair, the JNK pathway may limit damage to levels compatible with survival. Loss of p53 would further enhance survival owing to a down-regulated apoptotic response to unrepaired damage.



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 7. Model explaining how, through inhibition of potential c-Jun downstream targets leading to DNA repair (e.g., DNA polymerase ß, topoisomerase I, II, PCNA), mutant Jun promotes the accumulation of endogenous DNA strand breaks in genomically unstable tumor cells and, thus, collaborates with p53 to promote p53-mediated induction of bax and apoptosis.

 

   Materials and Methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Cell Lines.
T98G glioblastoma cells were obtained from Dr. Hoi U (University of California, San Diego, CA) and cultured at 37°C in 10% CO2 in DMEM supplemented with 10% newborn calf serum. The T98G clones that had been modified to express mutant Jun, termed T98G-dnJun-I-10-10 and I-10-6 (see Ref. 16 ), or simply T98G I-10-10 and T98G I-10-6, were cultured in the same way as were T98G cells, except that 100 µg/ml hygromycin was added to the culture medium. The pLHCmjun vector encodes a dominant negative mutant of c-Jun and was prepared, as described previously (51) , by insertion of DNA encoding mutant Jun [obtained by site-directed mutagenesis by Smeal et al. (20) ] into the retroviral vector pLHCX. Mutant Jun has ser -> ala substitutions at positions 63 and 73, two sites of DNA damage-induced phosphorylation in wild-type c-Jun, and cannot be phosphorylated at these sites. As a control, T98G cells modified to overexpress wild-type c-Jun (T98GcJun) were obtained by cotransfection with a c-jun expression vector, pSV2cjun and with pSV2neo, and were cultured similarly, with the addition of 100 µg/ml G418.

Western Blot Analysis.
Levels of total cellular Jun protein (c-Jun + mutant Jun), as well as levels of the gene products of the p53-regulated genes p21waf1, bax, and bcl2 were determined by Western blot analysis. Cell lysates (20–40 µg) were electrophoresed on a 12% acrylamide gel and blotted onto nylon membranes. Membranes were then treated with rabbit polyclonal anti c-Jun (1:200), or with mouse monoclonal anti p21waf1 (1:200), or with rabbit polyclonal anti-bax (1:200), or with mouse monoclonal anti-bcl2 (1:100), followed by an appropriate antirabbit or antimouse secondary antibody conjugated with horseradish peroxidase. All antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and used according to the protocol recommended by the manufacturer. Antibody reactive bands were revealed using the enhanced chemiluminescence Western detection system (Amersham Life Sciences, United Kingdom). For quantitation of bands, we used Kodak digital camera and analysis software.

Analysis of Repair of Cisplatin-DNA adducts.
Cisplatin (cis-diamminedichloroplatinum) adduct formation and repair was analyzed by a PCR-based DNA damage assay (PCR-stop assay; Ref. 31 ). The assay is based on observations that Taq polymerase is blocked at cisplatin adducts, was used to analyze cisplatin adduct formation and repair. Because DNA fragments are platinated randomly, the distribution of damage fits a Poisson distribution, where a mean level of one adduct/fragment (i.e., the portion of the genome defined by the forward and reverse PCR primers) will leave 37% of the fragments undamaged and these will be amplified to produce a PCR signal 37% of that from control DNA. For cisplatin treatments, cells were plated at 50% confluency in three wells of a 6-well plates in standard medium described above. After attachment, duplicate wells were treated with 100 µM cisplatin (Platinol, aqueous solution at 1 mg/ml, purchased from local pharmacies) for one h, 15 min, and one well was left untreated. After treatment, the untreated cells and one well of 100 µM cisplatin-treated cells were harvested, and genomic DNA was prepared. The remaining treated well was incubated an additional 16 h in the absence of cisplatin before harvesting. DNA was prepared using the QIAmp blood kit essentially following the manufacturer’s protocol, except that cells were lysed directly on the plate in the presence of PBS, Qiagen protease, and lysis buffer supplied in the kit. After purification, DNA was adjusted to 0.5 mg/ml in sterile water and stored at -20°C. Quantitative PCR was used to compare cisplatin adduct formation on a 2.7-kb region of the HPRT gene. As an internal control for PCR efficiency, we PCR-amplified from the same templates a 170-base nonoverlapping region of the same gene. The smaller region represents a target too small to register significant levels of damage under our conditions. We found that both the 2.7-kb and 170-base products increased linearly with input template over the range 0.1–0.5 µg DNA/25 µl reaction and we, therefore, routinely used 0.125–0.25 µg template/reaction. Reactions were performed in 25 µl using 0.125–0.25 µg DNA, 25 pmol each of forward and reverse primer, 250 µM dNTPs (Pharmacia), 1.25 units of Taq polymerase (Qiagen), 1 x buffer (Qiagen), and solution Q (Qiagen). Bands were quantitated using a Kodak digital camera and analysis software. The amplification program was as follows: 1 cycle (94°C, 1 min, 30 s); 25 cycles (94°C, 1 min; 57°C, 1 min; 70°C, 2 min, 30 s); 1 cycle (94°C, 1 min; 57°C, 1 min; 70°C, 7 min). All assays were performed in triplicate on two separate occasions.

Virus.
Replication-defective adenoviruses (Ad-p53 and Ad-ßgal), in which the human p53 coding sequence or the bacterial ß-galactosidase gene, respectively, replaced the viral early region E1A and E1B genes, were provided by Introgen Therapeutics, Inc. (Houston, TX).

Virus Treatments.
Cells at 80% confluence were placed in DMEM supplemented with 2% heat-inactivated fetal bovine serum and infected for 3 h at a multiplicity of 100 pfu/cell. The efficiency of infection was determined by X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) staining a sample of the ß-gal virus-infected cells (see Ref. 28 ) and was usually >=50%.

Viability and Growth Assays.
After infection, triplicate aliquots of cells were replated in 96-well plates at a density of 1000 cells/well. Plates were incubated for 5–7 days, and surviving cells were determined by adding a solution containing MTS [3-(4,5'-dimethylthiazol-2-yl)-5-(3-carboxymethoxylphenyl-2-(4-sulfophenyl)-2H-tetrazolium inner salt] and PMS (phenazine methosulfate; both purchased from Promega, Madison, WI.) for 1 h and determining A590 nm of the resulting formazan product, following procedures provided by the manufacturer. For growth assays, cells were plated at 1000/per well in 96-well plates. On successive days from day 1 through day 8, triplicate samples were stained with MTS, as described above.

Generation of Methotrexate-resistant Clones.
LD50 values for methotrexate were determined for the cell lines to be tested. Cells were seeded at a starting density of 103 cells/cm2 and allowed to attach for 16 h. Methotrexate (Sigma Chemical Co., St. Louis, MO) was then added to a concentration of 5 x LD50 or 9 x LD50, concentrations known to select for DHFR gene amplification (39 , 40) . Medium with fresh methotrexate was replaced weekly. When colonies developed and reached a size of about 100–200 cells (about 5 weeks), plates were washed in PBS and stained with 1% methylene blue in 70% methanol.

Analysis of DHFR Gene Copy Number.
To verify DHFR gene amplification after selection in methotrexate, as described above, several clones were picked and expanded. Genomic DNA from these clones, as well as from parental unselected cells was prepared from about 106 cells in each case using the QIAamp Blood KitÔ (Qiagen, Inc., Chatsworth, CA) and resuspended at 0.5 mg/ml in sterile H2O. Quantitative PCR was performed in 50-µl aliquots containing 0.2 µg of DNA, 50 pmol each of forward and reverse primers defining a 270-bp region of exon 1 and intron A of the DHFR gene (see below), 50 mm of KCL, 10 mm of Tris (pH 8.3), 1.5 mm of MgCl2, 250 mM dNTPs, 0.5 µl of Tac polymerase (Qiagen, Inc.), 10 µl of Q buffer (Qiagen, Inc.), and 1 pmol of radioactively end-labeled reverse primer (labeled with {gamma}-32P-dATP). PCR conditions were as follows: 1 cycle, 94°C (1 min, 30 s); 25 cycles, 94°C (1 min), 57°C (1 min), and 70°C (2 min, 30 s); 1 cycle, 94°C (1 min), 57°C (1 min), and 70°C (7 min). After PCR, 10 µl aliquots were electrophoresed on a 1% agarose gel. The gel was vacuum-dried for 2 h onto filter paper, and the PCR-amplified 270-bp band was quantitated using an Ambis4000 Radioanalytic Imaging system (Ambis, Inc., San Diego, CA). Quantitative conditions were established by demonstrating in control reactions with known amounts of DNA in 2-fold dilutions that product formation was directly proportional to input template. Primer sequences for the DHFR gene were: forward primer, 5'-GGTTCGCTAAACTGCATCGTCGC-3', and reverse primer, 5'-CAGAAATCAGCAACTGGGCCTCC-3'. An increase in DHFR gene copy number was then equal to the fold increase in the PCR product from cellular DNA of methotrexate-resistant clones compared with that of unselected parental cells.

Apoptosis Assay.
Apoptosis was assayed using the Cell Death Detection ELISA (Boehringer Mannheim, Indianopolis, IN), a quantitative photometric peroxidase immunoassay that detects cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) that are released from the nuclei of cells undergoing apoptosis. Cells (2 x 105) were plated in 24-well plates and infected the next day (when the cells were about 80% confluent) with Ad-p53 or Ad-ßgal, as described above. Forty-eight h after infection, cells were collected and cytoplasmic fractions were prepared and assayed for the presence of mono- and oligonucleosomes by following the manufacturer’s protocol.


   Acknowledgments
 
We thank M. Karin for providing the mutant Jun expression vectors and Dr. Hoi U for providing the T98G cells.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by National Cancer Institute Grants CA69546 (to R. A. G.), CA63783-05 (to D. A. M.), and CA76173-01 (to D. A. M.); Grant DAMD17-96-1-6038 from the Department of Defense (to R. A. G.); a grant from Introgen Therapeutics, Inc. (Houston, TX; to R. A. G.); and Grant 3CB-0246 from the Breast Cancer Research Program of the University of California (to D. A. M.). Back

2 To whom requests for reprints should be addressed, at Sidney Kimmel Cancer Center, 10835 Altman Row, San Diego, CA 92121. Phone: (619) 450-5990; Fax: (619) 450-3251; E-mail: rgjerset{at}skcc.org Back

3 The abbreviations used are: PK, protein kinase; JNK, Jun kinase; AP, activator protein; ATF, activating transcription factor; DHFR, dihydrofolate reductase; EGF, epidermal growth factor; ES, embryonal stem; HPRT, hypoxanthine phosphoribosyltransferase. Back

4 O. Potopova and D. Mercola, unpublished observations. Back

5 R. A. Gjerset and A. Haghighi, unpublished results. Back

6 Unpublished observations. Back

7 O. Potopova, unpublished observations. Back

Received for publication 12/29/98. Revision received 4/30/99. Accepted for publication 6/24/99.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

  1. Nowell P. C. The clonal evolution of tumor cell populations. Science (Washington DC), 194: 23-28, 1976.[Abstract/Free Full Text]
  2. Bakalkin G., Selivanova G., Yakovleva T., Kiseleva E., Kashuba E., Magnusson K. P., Szekely L., Klein G., Terenus L., Wiman K. G. p53 binds single stranded DNA ends through the C-terminal domain and internal DNA segments via the middle domain. Nucleic Acids Res., 23: 362-369, 1995.[Abstract/Free Full Text]
  3. Lee S., Elenbas B., Levine A., Griffith J. p53 and its 14-kDa C-terminal domain recognize primary DNA damage in the form of insertion/deletion mismatches. Cell, 81: 1013-1021, 1995.[Medline]
  4. Shieh S-Y., Ikeda M., Taya Y., Prives C. DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell, 91: 325-334, 1997.[Medline]
  5. Woo R. A., McLure K. G., Lees-Miller S. P., Rancourt D. E., Lee P. W. DNA-dependent protein kinase acts upstream of p53 in response to DNA damage. Nature (Lond.), 394: 700-704,
  6. Tainsky M. A., Bischoff F. Z., Strong L. C. Genomic instability due to germline p53 mutations drives preneoplastic progression toward cancer in human cells. Cancer Metastasis Rev., 14: 43-48, 1995.[Medline]
  7. Agapova L. S., Ilyinskaya G. V., Turovets N. A., Ivanov A. V., Chumakov P. M., Kopnin B. P. Chromosome changes caused by alterations of p53 expression. Mutat. Res., 354: 129-138, 1996.[Medline]
  8. Bertrand P., Rouillard D., Boulet A., Levalois C., Soussi T., Lopez B. S. Increase of spontaneous intrachromosomal homologous recombination in mammalian cells expressing a mutant p53 protein. Oncogene, 14: 1117-1122, 1997.[Medline]
  9. Bouffler S. D., Kemp C. J., Balmain A., Cox R. Spontaneous and ionizing radiation-induced chromosomal abnormalities in p53-deficient mice. Cancer Res., 55: 3883-3889, 1995.[Abstract/Free Full Text]
  10. Donehower L. A, Godley L. A., Aldaz C. M., Pyle R., Shi Y-P., Pinkel D., Gray J., Bradley A., Medina D., Varmus H. E. Deficiency of p53 accelerates mammary tumorigenesis in wnt-1 transgenic mice and promotes chromosomal instability. Genes Dev., 9: 882-895, 1995.[Abstract/Free Full Text]
  11. Lanza G., Jr., Maestri I., Dubini A., Gafa R., Santini A., Ferretti S., Cavazzini L. p53 expression in colorectal cancer: relation to tumor type, DNA ploidy pattern and short-term survival. Am. J. Clin. Pathol., 105: 604-612, 1996.[Medline]
  12. Gryfe R., Swallow C., Bapat B., Redston M., Gallinger S., Couture J. Molecular biology of colorectal cancer. Curr. Probl. Cancer, 21: 233-300, 1997.[Medline]
  13. Levine A. J. p53, the cellular gatekeeper for growth and division. Cell, 88: 323-331, 1997.[Medline]
  14. Derijard B., Hibi M., Wu I. H., Barrett T., Su B., Deng T., Karin M., Davis R. J. JNK1: a protein kinase stimulated by UV light and Ha-ras that binds and phosphorylates the c-Jun activation domain. Cell, 76: 1025-1037, 1994.[Medline]
  15. Adler V., Fuchs S. Y., Kim J., Kraft A., King M. P., Pelling J., Ronai Z. Jun-NH2-terminal kinase activation mediated by UV-induced DNA lesions in melanoma and fibroblast cells. Cell Growth Differ., 6: 1437-1446, 1995.[Abstract]
  16. Potapova O., Haghighi A., Bost F., Liu C., Birrer M. J., Gjerset R., Mercola D. The JNK/stress-activated protein kinase pathway functions to regulate DNA repair and inhibition of the pathway sensitizes tumor cells to cisplatin. J. Biol. Chem., 272: 14041-14044, 1997.[Abstract/Free Full Text]
  17. Saleem A., Datta R., Yuan Z. M., Kharbanda S., Kufe D. Involvement of stress-activated protein kinase in the cellular response to 1-ß-D-arabinofunanosylcytosine and other DNA damaging agents. Cell Growth Differ., 6: 1651-1658, 1995.[Abstract]
  18. Osborne M. T., Chambers T. C. Role of stress-activated/c-Jun NH2-terminal protein kinase pathway in the cellular response to adriamycin and other chemotherapeutic drugs. J. Biol. Chem., 271: 30950-30955, 1996.[Abstract/Free Full Text]
  19. Binétruy B., Smeal T., Karin M. Ha-ras augments c-Jun activity and stimulates phosphorylation of its activation domain. Nature (Lond.), 351: 122-127, 1991.[Medline]
  20. Smeal T., Binétruy B., Mercola D. A., Birrer M., Karin M. Oncogenic and transcriptional cooperation with Ha-ras requires phosphorylation of c-Jun on serines 63 and 73. Nature (Lond.), 354: 494-496, 1991.[Medline]
  21. Schreiber M., Kolbus A., Piu F., Szabowski A., Mohle-Steinlein U., Tian J., Karin M., Angel P., Wagner E. F. Control of cell cycle progression by c-Jun is p53 dependent. Genes Dev., 13: 607-619, 1999.[Abstract/Free Full Text]
  22. Takahashi J. A., Fukumoto M., Kozai Y., Ito N., Oda Y., Kikuchi H., Hatanaka M. Inhibition of cell growth and tumorigenesis of human glioblastoma cells by neutralizing antibody against human basic fibroblast growth factor. FEBS Lett., 288: 65-71, 1991.[Medline]
  23. Bost F., McKay R., Dean N., Mercola D. The Jun kinase/stress-activated protein kinase pathway is required for epidermal growth factor stimulation of growth of human A549 lung carcinoma cells. J. Biol. Chem., 272: 33422-33429, 1997.[Abstract/Free Full Text]
  24. Srivastava D. K., Rawson T. Y., Showalter S. D., Wilson S. H. Phorbol ester abrogates up-regulation of DNA polymerase ß by DNA-alkylating agents in Chinese hamster ovary cell. J. Biol. Chem., 270: 16402-16408, 1995.[Abstract/Free Full Text]
  25. Kedar P. S., Widen S. G., Englander E. W., Fornace A. J., Jr., Wilson S. H. The ATF/CREB transcription factor-binding site in the polymerase ß promoter mediates the positive effect of N-methyl-N-nitro-N-nitrosoguanidine on transcription. Proc. Natl. Acad. Sci. USA, 88: 3729-3733, 1991.[Abstract/Free Full Text]
  26. Baumgartner B., Heiland S., Kunze N., Richter A., Knippers R. Conserved regulatory elements in the type I DNA topoisomerase gene promoters of mouse and man. Biochim. Biophys. Acta, 1218: 123-127, 1994.[Medline]
  27. Heiland S., Knippers R., Kunze N. The promoter region of the human type-I-DNA-topoisomerase gene. Protein-binding sites and sequences involved in transcriptional regulation. Eur. J. Biochem., 217: 813-822, 1993.[Medline]
  28. Gjerset R. A., Turla S. T., Sobol R. E., Scalise J. J., Mercola D., Collins H., Hopkins P. J. Use of wild-type p53 to achieve complete treatment sensitization of tumor cells expressing endogenous mutant p53. Mol. Carcinog., 14: 275-285, 1997.
  29. Chen X., Ko L. J., Jayaraman L., Prives C. p53 levels, functional domains, and DNA damage determine the extent of the apoptotic response of tumor cells. Genes Dev., 10: 2438-2451, 1996.[Abstract/Free Full Text]
  30. Stark G. R. Regulation and mechanisms of mammalian gene amplification. Adv. Cancer Res., 61: 87-113, 1993.[Medline]
  31. Jennerwein M. M., Eastman A. A polymerase chain reaction-based method to detect cisplatin adducts in specific genes. Nucleic Acids Res., 19: 6209-6214, 1991.[Abstract/Free Full Text]
  32. Robbins J. H., Burk P. G. Relationship of DNA repair to carcinogenesis in xeroderma pigmentosa. Cancer Res., 33: 929-935, 1973.[Abstract/Free Full Text]
  33. Nacht M., Strasser A., Chan Y. R., Harris A. W., Schlissel M., Bronson R. T., Jacks T. Mutations in the p53 and SCID genes cooperate in tumorigenesis. Genes Dev., 10: 2055-2066, 1996.[Abstract/Free Full Text]
  34. Melton D., Ketchen A. M., Nuñez F., Bonatti-Abbondandolo S., Abbondandolo A., Squires S., Johnson R. Cells from ERCC1-deficient mice show increased genome instability and a reduced frequency of homologous recombination. J. Cell. Sci., 111: 395-404, 1998.[Abstract]
  35. Lengauer C., Kinzler K. W., Vogelstein B. Genetic instability in colorectal cancers. Nature (Lond.), 386: 623-627, 1997.[Medline]
  36. Sager R., Gadi I. K., Stephens L., Grabowy C. T. Gene amplification: an example of accelerated evolution in tumorigenic cells. Proc. Natl. Acad. Sci. USA, 82: 7015-7019, 1985.[Abstract/Free Full Text]
  37. Otto E., McCord S., Tlsty T. D. Increased incidence of CAD gene amplification in tumorigenic rat lines as an indicator of genomic instability of neoplastic cells. J. Biol. Chem., 264: 3390-3396, 1989.[Abstract/Free Full Text]
  38. Tlsty T. D. Normal diploid human and rodent cells lack a detectable frequency of gene amplification. Proc. Natl. Acad. Sci. USA, 87: 3132-3136, 1990.[Abstract/Free Full Text]
  39. Brown P. C., Tlsty T. D., Schimke R. T. Enhancement of methotrexate resistance and dihydrofolate reductase gene amplification by treatment of mouse 3T6 cells with hydroxyurea. Mol. Cell. Biol., 3: 1097-1107, 1983.[Abstract/Free Full Text]
  40. Tlsty T. D., Brown P. C., Schimke R. T. UV-radiation facilitates methotrexate resistance and amplification of the dihydrofolate reductase gene in cultured 3T6 mouse cells. Mol. Cell. Biol., 4: 1050-1056, 1984.[Abstract/Free Full Text]
  41. Mercer W. E., Shields M. T., Amin M., Sauve G. J., Appela E., Roman J. W., Ullrich S. J. Negative growth regulation in a glioblastoma tumor line that conditionally expresses human wild-type p53. Proc. Natl. Acad. Sci. USA, 87: 6166-6170, 1990.[Abstract/Free Full Text]
  42. Miyashita T., Reed J. C. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell, 80: 293-299, 1995.[Medline]
  43. Basu A., Haldar S. The relationship between BcI2, Bax and p53: consequences for cell cycle progression and cell death. Mol. Hum. Reprod., 4: 1099-1109, 1998.[Abstract/Free Full Text]
  44. Windle B., Draper B. W., Yin Y. X., O’Gorman S., Wahl G. M. A central role for chromosome breakage in gene amplification, deletion formation, and amplicon integration. Genes Dev., 5: 160-174, 1991.[Abstract/Free Full Text]
  45. Smith K. A., Agarwal M. L., Chernov M. V., Chernova O. B., Deguchi Y., Ishizaka Y., Patterson T. E., Poupon M. F., Stark G. R. Regulation and mechanisms of gene amplification. Philos. Trans. R. Soc. Lond. B. Biol. Sci., 347: 49-56, 1995.[Abstract/Free Full Text]
  46. Gjerset, R. A., and Mercola, D. Sensitization of tumors to chemotherapy through gene therapy. In: N. Nagy (ed.), Cancer Gene Therapy: Past Achievements and Future Challenges. New York, London, and Moscow: Plenum Publishing Corporation, in press, 1999.
  47. Zamble D. B., Lippard S. J. Cisplatin, and DNA repair in cancer chemotherapy. Trends Biochem. Sci., 20: 435-439, 1995.[Medline]
  48. Milne D. M., Campbell L. E., Campbell D. G., Meek D. W. P53 is phosphorylated in vitro and in vivo by an ultraviolet radiation-induced protein kinase characteristic of the c-Jun kinase, JNK1. J. Biol. Chem., 270: 5511-5518, 1995.[Abstract/Free Full Text]
  49. Hilberg F., Wagner E. F. Embryonic stem (ES) cells lacking functional c-Jun: consequences for growth and differentiation, AP-1 activity and tumorigenicity. Oncogene, 7: 2371-2380, 1992.[Medline]
  50. Harris C. C. Structure and function of the p53 tumor suppressor gene: clues for rational cancer therapeutic strategies. J. Natl. Cancer Inst., 88: 1442-1445, 1996.[Abstract/Free Full Text]
  51. Potapova O., Fahkrai H., Baird S., Mercola D. Platelet-derived growth factor-B/v-sis confers a tumorigenic and metastatic phenotype to human T98G glioblastoma cells. Cancer Res., 56: 280-286, 1996.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
K. J. Hughes, G. P. Meares, K. T. Chambers, and J. A. Corbett
Repair of Nitric Oxide-damaged DNA in {beta}-Cells Requires JNK-dependent GADD45{alpha} Expression
J. Biol. Chem., October 2, 2009; 284(40): 27402 - 27408.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. Rouget, Y. Auclair, M. Loignon, E. B. Affar, and E. A. Drobetsky
A Sensitive Flow Cytometry-based Nucleotide Excision Repair Assay Unexpectedly Reveals That Mitogen-activated Protein Kinase Signaling Does Not Regulate the Removal of UV-induced DNA Damage in Human Cells
J. Biol. Chem., February 29, 2008; 283(9): 5533 - 5541.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
G. Fritz and B. Kaina
Late Activation of Stress Kinases (SAPK/JNK) by Genotoxins Requires the DNA Repair Proteins DNA-PKcs and CSB
Mol. Biol. Cell, February 1, 2006; 17(2): 851 - 861.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
B. W. Ennis, K. E. Fultz, K. A. Smith, J. K. Westwick, D. Zhu, M. Boluro-Ajayi, G. K. Bilter, and B. Stein
Inhibition of Tumor Growth, Angiogenesis, and Tumor Cell Proliferation by a Small Molecule Inhibitor of c-Jun N-terminal Kinase
J. Pharmacol. Exp. Ther., April 1, 2005; 313(1): 325 - 332.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
A. MacLaren, E. J. Black, W. Clark, and D. A. F. Gillespie
c-Jun-Deficient Cells Undergo Premature Senescence as a Result of Spontaneous DNA Damage Accumulation
Mol. Cell. Biol., October 15, 2004; 24(20): 9006 - 9018.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
C.-K. Youn, M.-H. Kim, H.-J. Cho, H.-B. Kim, I.-Y. Chang, M.-H. Chung, and H. J. You
Oncogenic H-Ras Up-Regulates Expression of ERCC1 to Protect Cells from Platinum-Based Anticancer Agents
Cancer Res., July 15, 2004; 64(14): 4849 - 4857.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
Y. Huang, T. Tyler, N. Saadatmandi, C. Lee, P. Borgstrom, and R. A. Gjerset
Enhanced Tumor Suppression by a p14ARF/p53 Bicistronic Adenovirus through Increased p53 Protein Translation and Stability
Cancer Res., July 1, 2003; 63(13): 3646 - 3653.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
I. A. Vasilevskaya, T. V. Rakitina, and P. J. O'Dwyer
Geldanamycin and its 17-Allylamino-17-Demethoxy Analogue Antagonize the Action of Cisplatin in Human Colon Adenocarcinoma Cells: Differential Caspase Activation as a Basis for Interaction
Cancer Res., June 15, 2003; 63(12): 3241 - 3246.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
G. Mathonnet, C. Leger, J. Desnoyers, R. Drouin, J.-P. Therrien, and E. A. Drobetsky
UV wavelength-dependent regulation of transcription-coupled nucleotide excision repair in p53-deficient human cells
PNAS, June 10, 2003; 100(12): 7219 - 7224.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Hayakawa, C. Depatie, M. Ohmichi, and D. Mercola
The Activation of c-Jun NH2-terminal Kinase (JNK) by DNA-damaging Agents Serves to Promote Drug Resistance via Activating Transcription Factor 2 (ATF2)-dependent Enhanced DNA Repair
J. Biol. Chem., May 30, 2003; 278(23): 20582 - 20592.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gjerset, R. A.
Right arrow Articles by Mercola, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gjerset, R. A.
Right arrow Articles by Mercola, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cell Growth & Differentiation