![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Cancer Research Center and Department of Medicine, Boston University School of Medicine, Boston, Massachusetts,1 Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama2
Received 23 August 2001/ Returned for modification 22 October 2001/ Accepted 29 January 2002
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Tumor formation is a multistep process involving positive, growth-stimulatory signals which permit uncontrolled cell proliferation and negative, protective regulation that eliminates unlimited growth through the induction of an apoptotic crisis (21, 43, 49, 51, 53). These two conflicting signals often occur simultaneously during the transformation process, in which p53 has been implicated as a crucial regulator (3, 28). A functional p53, together with other cell cycle regulators, elicits the apoptotic crisis during the establishment of myc-induced immortalization or the transformation process mediated by Abelson murine leukemia virus (3, 28, 48). p53, as a transcriptional factor, also regulates the induction and function of several other genes, including Bax and p21, which participate in either p53-dependent apoptosis or growth arrest (28, 36, 37). It has been observed that mutations in both p53 and ras genes often occur in the same cancer cell (5, 8, 30, 48). In colon cancer cells, Ras mutations have been demonstrated to precede p53 mutations (30). The possible mechanism has been proposed to be one in which the Ras signaling pathway suppresses p53 during an early stage of tumor development by targeting MDM2 activity (42).
Bax is a cell death agonist with homology to the antiapoptotic Bcl-2 protein (36, 41). Mice deficient in Bax have selective hyperplasia and show resistance to certain apoptotic stimuli (36, 37). Overexpression of p53 increases Bax expression in various cell lines, and this increase correlates with the induction of apoptosis in various cell types (36, 37). Overexpression of Bcl-2 protects cells from certain types of apoptosis, including Ras-mediated cell death, and also counteracts the proapoptotic function of Bax elicited by various apoptotic stimuli (11, 36, 37, 39, 41). However, the involvement of Bax in Ras-induced cell death has not yet been fully explored.
The complex relationship between cell proliferation or differentiation and apoptosis mediated by oncogenic ras has begun to be elucidated (11, 17, 35, 46). We previously reported that mouse or human lymphocytes and fibroblasts containing oncogenic ras die after downregulation of PKC activity (11, 12, 13). The integration of Ras signaling has also been observed in Fas-induced apoptosis (12, 24). Furthermore, ras-transformed cells are susceptible to apoptosis upon suppression of NF-
B activation in a p53-independent fashion (35). These data provide evidence that, although oncogenic ras promotes unlimited cell proliferation under normal growth conditions, cells expressing activated ras retain the ability to undergo apoptosis. Other studies have indicated that abnormalities of tumor suppressors are important in the regulation of the susceptibility of tumor cells to apoptosis. It is not completely understood how changes in these tumor suppressors may regulate the threshold of the sensitivity of cells to apoptotic stimulations, and subsequently affect their fate. Since Ras activity has been directly linked to the inactivation of p53 and to the regulation of the susceptibility of cells to apoptosis (11, 17, 35, 46), it led us to investigate the role of p53 in Ras-mediated apoptosis. The expression of wild-type (wt) p53 in BALB or Swiss mouse fibroblasts did not change after transient expression of v-ras or acute increase of endogenous Ras activity by GAP antisense oligonucleotide. However, in stable ras transfectants, p53 was mutated. We also demonstrated that cells transiently transfected with ras or treated with GAP antisense oligonucleotides became susceptible to apoptosis, and this apoptotic process was dramatically augmented after PKC suppression, accompanied with Bax induction, in a p53-dependent fashion. In contrast, stable ras transfectants were less sensitive to PKC inhibition, and Bax was not induced. The results suggest that PKC is crucial for cells containing high Ras activity to survive and to be further transformed. Apoptosis mediated by transiently increased Ras activity is p53 dependent. After escaping from the Ras-elicited, p53-dependent apoptotic crisis, the stable ras transfectants still retain the ability to die, under circumstances in which endogenous PKC activity is eliminated, and this apoptosis is p53 independent.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
For an acute increase of Ras activity or suppression of Bax expression, a 15 or 20 µM concentration of GAP or Bax antisense oligonucleotides was added into a cell culture for 48 h, and subsequently the cells were refed with medium containing a half dose of the oligonucleotides every 2 days. For transient transfection, cells were infected with ras or E6 and exposed to various treatments 48 h after the transfection.
GDP-GTP exchange assay. BALB and Swiss cells without or with transiently or stably expressed ras were washed with phosphate-free RPMI medium containing 10% dialyzed fetal bovine serum and cultured in the same medium with [32P]orthophosphate (0.5 mCi/ml) for 4 h. Subsequently, cells were lysed in lysis buffer (24). Equal amounts of cell lysates were immunoprecipitated with an anti-Ras antibody (anti-Ras Ab) (Oncogene Science, Uniondale, N.Y.), and this was followed by the addition of goat anti-rat immunoglobulin-conjugated protein A-Sepharose beads. Precipitates were washed with lysis buffer, and bound nucleotides were eluted with the elution buffer containing 20 mM EDTA and a 25 µM concentration (each) of cold GDP and GTP at 65°C. The eluted products were separated on polyethyleneimine-cellulose plates (24). Quantitation was performed by densitometric scanning of autoradiograms using a laser densitometer.
Cell viability assays. Cells (0.5 x 106 cells/ml) were cultured in the medium containing various concentrations of 1-O-hexadecyl-2-O-methyl-rac-glycerol (HMG; Calbiochem) for the times indicated in the figures or in the figure legends. Viable cells were determined and enumerated using trypan blue exclusion (11).
Flow cytometric determination of nuclear DNA fragmentation and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. Cytofluorometric analysis was performed with a FACScan (Becton Dickinson, Mountain View, Calif.). The data analysis and display were performed using the Cell-Fit software program (Becton Dickinson). Cell-Fit provides data from the flow cytometer and real-time statistical analysis of the data, computed at 1-s intervals, and also discriminates doublets or adjacent particles. Briefly, following various treatments with HMG or other stimuli, cells (0.5 x 106/ml) were washed with 1x phosphate-buffered saline, fixed with 70% ethanol, and treated with RNase (10 ng/ml). Subsequently, cells were stained with propidium iodide (50 µg/ml). The stained samples were kept in the dark at 4°C overnight before flow cytometric analysis.
For the TUNEL assay, an in situ cell death detection kit (Roche Diagnostics Corp., Indianapolis, Ind.) was used. After the treatment, cells (0.5 x 106) were washed with 1x phosphate-buffered saline and incubated in 2% paraformaldehyde solution at room temperature. Following centrifugation, cells were resuspended in the permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice and then stained with TUNEL reaction mixture. The samples were then analyzed with a FACScan and the Lysis II software program (Becton Dickinson).
RNA isolation and Northern blot analysis. Following various treatments, total RNAs were isolated using Trizol reagent (Life Technologies, Inc.), quantified, separated by electrophoresis on formaldehyde-agarose gels, and transferred to nitrocellulose. 32P-labeled DNA probes were made by the random oligonucleotide primer method.
Protein analysis. Cells were labeled with 100 µCi of [35S]methionine (New England Nuclear) for 4 h and lysed in ice-cold lysis buffer containing 150 mM NaCl, 50 mM Tris (pH 8.0), 2 mM EDTA, 5 mM EGTA, 1% Nonidet P-40, and 1 mM phenylmethylsulfonyl fluoride (47). The protein concentrations of cell lysates were normalized. Equal amounts of total proteins from different cell lysates were immunoprecipitated with 1 µg of Abs recognizing wt p53, mutant (mu) p53, or the common region of p53 (generous gift from C. Prives, Columbia University). The immune complexes were recovered by using Sepharose CL-4B (Pharmacia) and were subsequently separated on a sodium dodecyl sulfate (SDS)-polyacrylamide gel.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
To further demonstrate that the loss of viability in the cells with high Ras activity is the result of apoptosis, a TUNEL assay was performed (Fig. 1c). With or without HMG treatment, BALB, BALB/GAP, or Ki-BALB cells were permeabilized and labeled with tagged dUTP by terminal deoxynucleotidyl transferase to detect DNA strand breaks. The percentages of labeled BALB/GAP and Ki-BALB cells were 42.3 and 22.6%, respectively. In contrast, there was no significant increase in the terminal deoxynucleotidyl transferase labeling in BALB cells after treatment with 150 nM HMG. Again, under normal growth conditions, 11.4% of BALB/GAP cells contained broken DNA strands. Overall, the results from the TUNEL assay are comparable with those obtained from the DNA fragmentation analysis.
p53 is mutated in stable ras transfectants but not in cells with transiently increased Ras activity. Tumor suppressor p53 possesses multiple functions, including regulation of apoptosis (50, 52). The deregulation of p53 mediated by various oncogenes frequently occurs in tumorigenesis (28). During the early stage of transformation, activated ras suppresses p53 through targeting MDM2 (42). Because cells transiently expressing ras were more susceptible to apoptosis after HMG treatment than the stable transfectants, the expression of p53 was examined in BALB, BALB/GAP, and Ki-BALB cells. After labeling the cells with [35S]methionine, whole-cell lysates were prepared, and subsequently p53 was immunoprecipitated with anti-wt, anti-mu, or anti-pan (recognizing both wt and mu proteins) p53 Ab (Fig. 2a). BALB and BALB/GAP cells expressed wt p53 only. Ki-BALB cells had a p53 protein that was recognized by the anti-pan p53 Ab, in which the expressed p53 mainly reacted with an anti-mu p53 Ab and only a detectable amount of the protein could be recognized by an anti-wt-p53 Ab. The reduction of wt p53 and the increase in mutated p53 were also observed in SH1 cells but not in Swiss/ras cells (data not shown). The same experiment was performed in several other stable ras clones, and similar results were obtained (data not shown). Thus, the data indicated that the accumulation of p53 mutations appears to be one event mediated by activated ras in the process of the establishment of the transformation.
|
Bax is induced in cells with transient increase of Ras activity.
Bax, a p53-regulated gene, is involved in p53-dependent apoptosis in certain cell types (36, 37). To test whether Bax is induced in cells with increased Ras activity, before the onset of Ras-mediated apoptosis, Northern blot analysis was conducted (Fig. 3a, upper panel). The different doses of HMG (100 nM for the cells with a transient increase of Ras activity or 150 nM for the ras transfectants) were chosen based on the different sensitivities of these cells to the inhibitor for the induction of apoptosis (Fig. 1). Other apoptotic stimuli (doxorubicin and 
-irradiation) were used as positive controls. Bax was induced in BALB/GAP cells following treatment with doxorubicin (0.5 µg/ml, 15 h), -irradiation (400 rads), or HMG (100 nM, 15 h). The stronger induction of Bax by doxorubicin than other reagents (about 1.5-fold) may reflect the different usage of the promoter(s) and/or enhancer(s). Bax was moderately expressed in untreated BALB/GAP cells. This is consistent with the data shown in Fig. 1, in which a moderate magnitude of apoptosis was elicited in these cells. In contrast, Bax was slightly expressed in Ki-BALB cells after HMG, -irradiation, or doxorubicin treatment. However, in parental BALB cells, Bax was not induced by HMG, because PKC inhibition was not apoptotic in BALB cells. Perhaps, under such conditions, a functional transcription complex for the induction of Bax is unable to assemble in BALB cells. Equal loading of total RNA was monitored by the expression of 18S on the Northern blot (Fig. 3a, lower panel).
|
E6 or Bax antisense oligonucleotides suppress apoptosis mediated by transient increases in Ras activity. To further determine the p53 dependency of Bax expression and the induction of apoptosis mediated by transient increases in Ras activity, a retroviral vector containing the papillomavirus E6 gene (which targets and causes the rapid degradation of p53) was employed. Following the coinfection of Swiss cells with v-ras and E6, the induction of Bax mediated by two different treatments was examined by Northern blot analysis (Fig. 4a, upper panel). A low level of Bax was detected in Swiss/ras cells under normal growth conditions, and the expression was further induced by HMG (1.8-fold) and doxorubicin (about 4-fold). However, the introduction of E6 blocked Bax expression in untreated Swiss/ras cells and also inhibited the induction of the gene mediated by HMG or doxorubicin in these cells. The same results were obtained from BALB/GAP cells (data not shown). The equal loading of the samples was also monitored by 18S expression on the Northern blot (Fig. 4a, lower panel).
|
We next tested whether Bax antisense oligonucleotides also have a negative effect on the apoptotic process. The concentration of the antisense oligonucleotide required to suppress Bax expression was determined by a dose titration experiment (Fig. 5, inserted panel). Swiss/ras cells were exposed to a 15 or 20 µM concentration of Bax antisense oligonucleotide or 20 µM Bax sense oligonucleotide for 48 h, and Northern blotting of Bax expression was then conducted with or without HMG treatment. A 15 µM concentration of the antisense oligonucleotide moderately inhibited Bax induction, and a 20 µM concentration of the antisense oligonucleotide significantly suppressed Bax expression. The addition of the sense oligonucleotide did not affect the induction of the gene. Subsequently, Swiss, Swiss/ras, or SH1 cells were cultured in the medium containing a 20 µM concentration of either the Bax sense or antisense oligonucleotide for 48 h, and then the DNA fragmentation assay was performed, under normal growth conditions or after PKC suppression (Fig. 5). The Bax antisense oligonucleotide partially inhibited the generation of DNA fragmentation mediated by HMG (100 nM) in Swiss/ras. It is notable that the inhibitory effect of the antisense oligonucleotide is similar to that of E6. The addition of the sense oligonucleotide did not affect Ras-mediated apoptosis in the same cells. A moderate number of SH1 cells had fragmented DNA after the addition of 150 nM HMG, and Bax antisense oligonucleotide had no effect on the formation of DNA fragmentation. Apoptosis did not occur in parental Swiss cells in the presence of either the sense or antisense oligonucleotide. The data again provide evidence that Bax is involved only in the regulation of apoptosis mediated by transient expression of Ras.
|
|
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
During the early stage of transformation, cells undergo a period of crisis in which the majority of cells die before permanent establishment of malignancy (21, 43, 49, 51, 53). This phenomenon has also been noticed in the establishment of immortal cell lines. For instance, myc overexpression rapidly activates p53 and triggers an apoptotic crisis in primary mouse embryo fibroblasts (53). Some mouse embryo fibroblasts survive the crisis by inactivating wt p53 or other cell cycle checkpoint regulators during the process and become immortal. Our study, which compares the effects of transient and stable v-ras expressions, provides a new model for further understanding potential interactions among activated Ras, p53, and PKC. Studies have shown that PKC is involved in a variety of diverse signaling pathways, including its interaction with Ras. Moreover, PKC activity is important for tumor promotion (6, 29, 38). Our study demonstrates that a transient but persistent increase of Ras activity in cells transmits a signal to downstream effectors; however, the decision to grow or to undergo apoptosis depends upon p53 and PKC. In general, p53, as a guardian of the genome, eliminates those cells containing abnormally high Ras activity (pretransformed) via apoptosis during the early stages of transformation. PKC, as a survival-promoting factor, suppresses the activities of various proapoptotic factors, thereby helping cells to escape from p53 surveillance. The elimination of PKC activity releases the apoptotic factors from this suppression and dramatically enhances Ras-mediated apoptosis. Alternatively, under such conditions, suppression of endogenous PKC activity further provokes the collision among internal, unstable signals mediated by increases of Ras activity and in turn activates massive cell death signaling pathways. In the stable ras transfectants, PKC appears to be required for adapting gene mutations and genetic changes mediated by oncogenic ras. At the same time, this kinase may, together with oncogenic ras, block programmed cell death, especially p53-mediated apoptotic signaling. However, transformed cells are not absolutely immortal and, under certain circumstances, have the propensity to commit suicide. Our study demonstrates that the stable ras transfectants undergo programmed cell death once PKC activity is inhibited, in a p53-independent fashion. It is possible that the blockade of PKC activity causes clashes of hyperproliferative signals in the cells with unstable genome mediated by loss of wt p53 and in turn lifts the ban for p53-independent apoptosis.
Mutation of the p53 gene is a common event in tumorigenesis and often occurs in the late stage of malignancy (5, 8, 30, 48). In various types of cells transformed by oncogenic ras, p53 appears to be mutated. For example, the introduction of Ha-ras into mouse prostate cells mediates the mutation of endogenous p53 and induces hyperplasia in the reconstituted mouse prostate organ (33). In colon cancer cells, ras mutation precedes p53 mutations (30, 44, 45). Recently, it has been reported that, in the early stage of transformation, Ras activity negatively regulates p53 through modulating MDM2 (42). The BALB and Swiss cell lines employed in this study possess wt p53. Therefore, it is possible that, during this process, the autoregulatory loop for maintaining normal function of p53 is disrupted by persistent increase of Ras activity, which facilitates error-prone mutations, including p53 mutation. The loss of wt p53 expression or function mediated by oncogenic ras in cells also cripples apoptotic crisis-related signals and leads to the increased tolerance to abnormally high Ras activity. However, MCF7 breast cancer cells are tumor cells that express wt p53 and contain nonmutated Ras. In the process of tumorigenesis, the loss of the expression or function of other tumor suppressors, for example BRCA1 and -2, may play an important role in the regulation of the susceptibility of the cells to apoptotic crisis.
Bax, a transcriptional target of p53, has been suggested to be involved in certain types of apoptotic processes (9, 20). Bax is induced by a transient increase of Ras activity. The introduction of E6 into the cells with a transient increase of Ras activity to degrade p53 inhibits the Bax induction, and, at the same time, dramatically reduces Ras-mediated apoptosis. The apoptotic process was also greatly diminished by treating the same cells with Bax antisense oligonucleotide, with a magnitude similar to that achieved by E6, which presents a linear relationship between p53 and Bax in the regulation of Ras-mediated apoptosis. Therefore, we conclude that a transient increase of Ras activity activates p53-mediated apoptotic machinery in which Bax activity is required. In addition, the study shows that HMG does not induce Bax expression in parental BALB or Swiss cells that contain wt p53, and only mediates G1 arrest in these cells (data not shown). p53 is able to sense or distinguish growth arrest and apoptotic signals (22, 34, 50, 54). Because PKC suppression is not apoptotic to these cells, it is reasonable to believe that the p53-dependent transcriptional factors which regulate the apoptotic process do not converge under this condition in the cells. It is also conceivable that other cis elements exist within the promoter-enhancer region of Bax which discriminates such differences. Overall, wt p53 and Bax appear to be the key players in the induction of Ras-mediated apoptosis initiated by transient increase of Ras activity. The incomplete blockades of the apoptotic process by either E6 or Bax antisense oligonucleotide indicate that multiple pathways are involved in Ras-induced apoptosis.
The present study also demonstrates that the pattern of the induction of p21 in the cells containing a transient increase of Ras activity (BALB/GAP), in response to PKC suppression, is very different from Bax expression. Under normal growth conditions, p21 could not be detected in these cells, and the addition of HMG only moderately induced the gene, in comparison with that mediated by serum starvation in the same cells or by the same drug in BALB cells. In BALB/GAP cells, the apoptotic signaling elicited by HMG may increase the recruitment of p53 and further allow for the specific binding of p53 to a death-related element(s) (such as Bax), whereas less p53 may transactivate p21 expression. This model was suggested by the experiments with irradiated cells, in which Bax is maximally induced and GADD45 is only slightly expressed, despite the apparently lower affinity of wt p53 for the Bax site (22, 52). Under apoptotic conditions, the activation of apoptosis-related promoters, such as the Bax promoter, by p53 may be favored. However, the mechanism of the preferential activation of p53 is still unclear.
There are considerable data indicating that inappropriately increasing Ras activity interferes with the normal function(s) of cell cycle checkpoints (4, 16, 18, 19, 23, 31). Selective induction of activated Ha-ras stimulates serum-depleted cells to progress from G1 arrest into S phase. Other studies demonstrated that oncogenic ras shortens the G1 phase of the cell cycle. It is possible that, in order to tolerate the environment or to survive, activated ras, together with prosurvival factors such as PKC and NF-B, cripples cell cycle surveillance by targeting tumor suppressors and promotes cell proliferation. The results generated from 3-4 cells indicate that p53 is a target for oncogenic ras to regulate the apoptotic process. After restoration of the wt conformation of p53, the cells regain the susceptibility to Ras-induced cell death in a p53/Bax-dependent fashion. The fact that MCF7 cells become susceptible to Ras-mediated cell death once Ras activity is transiently increased provides further evidence for the connection between Ras and wt p53 in the regulation of apoptosis. In the future, the identification of the factors which are involved in the regulation of the p53-independent, apoptotic process in stable ras transfectants will help us to further understand the mechanism(s) of Ras-mediated apoptosis and may offer a potential cancer therapy for targeting elements at different stages of oncogenic ras-mediated malignancy.
In the process of signal transduction, PKC transmits the proper message for cell activation or cell cycle progression. Suppression of PKC by various methods causes normal cells to arrest in the G1 phase of the cell cycle; however, it augments or elicits the apoptotic process in cells containing high Ras activity. This indicates that PKC is a crucial factor for the survival and further transformation of cells with abnormally high Ras activity. The present investigation also suggests that the cells at various stages of ras-mediated transformation possess different susceptibilities to apoptosis. The discrepancy of the susceptibilities to apoptosis is regulated, in part, by Ras, PKC, and p53.
|
ACKNOWLEDGMENTS |
|---|
This work was supported by a grant from the American Cancer Society (RPG-00-111-01-MGO awarded to C.-Y.C.).
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Ameyar, M., V. Shatrov, C. Bouquet, C. Capoulade, Z. Cai, R. Stancou, C. Badie, H. Haddada, and S. Chouaib. 1999. Adenovirus-mediated transfer of wild-type p53 gene sensitizes TNF resistant MCF7 derivatives to the cytotoxic effect of this cytokine: relationship with c-myc and Rb. Oncogene 18:5464-5472.[CrossRef][Medline]
3. Amundson, S. A., T. G. Myers, and A. J. Fornace, Jr. 1998. Roles for p53 in growth arrest and apoptosis: putting on the brakes after genotoxic stress. Oncogene 17:3287-3299.[CrossRef][Medline]
4. Arber, N., T. Sutter, M. Miyake, S. M. Kahn, V. S. Venkatraj, A. Sobrino, D. Warburton, P. R. Holt, and I. B. Weinstein. 1996. Increased expression of cyclin D1 and the Rb tumor suppressor gene in c-K-ras transformed rat enterocytes. Oncogene 12:1903-1908.[Medline]
5. Azzoli, C. G., M. Sagar, A. Wu, D. Lowry, H. Hennings, D. L. Morgan, and W. C. Weinberg. 1998. Cooperation of p53 loss of function and v-Ha-ras in transformation of mouse keratinocyte cell lines. Mol. Carcinogenesis 21:50-61.[CrossRef][Medline]
6. Baier-Bitterlich, G., F. Ulberall, B. Bauer, F. Fresser, G. Wachter, H. Grunicke, G. Utermann, A. Altman, and G. Baier. 1996. Protein kinase C-theta isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes. Mol. Cell. Biol. 16:1842-1850.[Abstract]
7.
Bos, J. L. 1989. ras oncogenes in human cancer: a review. Cancer Res. 49:4682-4689.
8. Boukamp, P., W. Peter, U. Pascheberg, S. Altmeier, C. Fasching, E. J. Stanbridge, and N. E. Fusenig. 1995. Step-wise progression in human skin carcinogenesis in vitro involves mutational inactivation of p53, ras oncogene activation and additional chromosome loss. Oncogene 11:961-969.[Medline]
9. Caelles, C., A. Heimberg, and M. Karin. 1994. p53-dependent apoptosis in the absence of transcriptional activation of p53-target genes. Nature 370:220-224.[CrossRef][Medline]
10. Cai, Z., C. Capoulade, C. Moyret-Lalle, M. Amor-Gueret, J. Feunteun, A. K. Larsen, B. B. Paillerets, and S. Chouaib. 1997. Resistance of MCF7 human breast carcinoma cells to TNF-induced cell death is associated with loss of p53 function. Oncogene 15:2817-2826.[CrossRef][Medline]
11. Chen, C.-Y., and D. V. Faller. 1995. Direction of p21Ras-generated signals cell growth or apoptosis is determined by protein kinase C and Bcl-2. Oncogene 11:1487-1498.[Medline]
12.
Chen, C.-Y., J. Liou, L. W. Forman, and D. V. Faller. 1998. Differential regulation of discrete apoptotic pathways by Ras. J. Biol. Chem. 273:16700-16709.
13. Chen, C-Y., J. Liou, L. W. Forman, and D. V. Faller. 1998. Correlation of genetic instability and apoptosis in the presence of oncogenic Ki-Ras. Cell Death Diff. 5:984-995.[CrossRef][Medline]
14.
Chen, X., J. Bargonetti, and C. Prives. 1995. p53, through p21(WAF1/CIP1), induces cyclin D1 synthesis. Cancer Res. 55:4257-4263.
15.
Clark, G. J., J. K. Westwick, and C. J. Der. 1997. p120GAP modulates Ras activation of Jun kinase and transformation. J. Biol. Chem. 272:1677-1681.
16. Downward, J. 1997. Cell cycle: routine role for Ras. Curr. Biol. 7:258-260.
17. Downward, J. 1998. Ras signalling and apoptosis. Curr. Opin. Genet. Dev. 8:49-54.[CrossRef][Medline]
18. Downward, J., J. D. Graves, P. H. Warne, S. Rayter, and D. A. Cantrell. 1990. Stimulation of p21ras upon T-cell activation. Nature 346:719-723.[CrossRef][Medline]
19. Filmus, J., A. I. Robles, W. Shi, M. J. Wong, L. L. Colombo, and C. J. Conti. 1994. Induction of cyclin D1 overexpression by activated ras. Oncogene 9:3627-3633.[Medline]
20. Fisher, D. E. 1994. Apoptosis in cancer therapy: crossing the threshold. Cell 78:539-542.[CrossRef][Medline]
21. Franza, B. R., K. Maruyama, J. I. Garrels, and H. E. Ruley. 1986. In vitro establishment is not a sufficient prerequisite for transformation by activated Ras oncogenes. Cell 44:409-418.[CrossRef][Medline]
22. Friedlander, P., Y. Haupt, C. Prives, and M. Oren. 1996. A mutant p53 that discriminates between p53-responsive gene cannot induce apoptosis. Mol. Cell. Biol. 16:4961-4971.[Abstract]
23.
Gille, H., and J. Downward. 1999. Multiple Ras effector pathways contribute to G1 cell cycle progression. J. Biol. Chem. 274:22033-22042.
24. Gulbins, E., R. Bissonnette, A. Mahoubi, S. Martin, W. Nishioka, T. Brunner, G. Baier, G. Baier-Bitterlich, C. Byrd, F. Lang, R. Kolesnick, and D. Green. 1995. Fas-induced apoptosis is mediated via a ceramide-initiated Ras signaling pathway. Immunity 2:341-351.[CrossRef][Medline]
25. Johnson, L., K. Mercer, D. Greenbaum, R. T. Bronson, D. Crowley, D. A. Tuveson, and T. Jacks. 2001. Somatic activation of the K-ras oncogene causes early onset lung cancer in mice. Nature 410:1111-1116.[CrossRef][Medline]
26. Joneson, T., M. A. White, M. H. Wigler, and D. Bar-Sagi. 1996. Stimulation of membrane ruffling and MAP kinase activation by distinct effectors of Ras. Science 271:810-812.[Abstract]
27. Khosravi-Far, R., and C. J. Der. 1994. The Ras signal transduction pathway. Cancer Metastasis Rev. 13:67-89.[CrossRef][Medline]
28.
Ko, L. J., and C. Prives. 1996. p53: puzzle and paradigm. Genes Dev. 10:1054-1072.
29.
Lee, J. Y., Y. A. Hannun, and L. M. Obeid. 1996. Ceramide inactivates cellular protein kinase C alpha. J. Biol. Chem. 271:13169-13174.
30.
Lengauer, C., W. Kenneth, and B. Vogelstein. 1997. DNA methylation and genetic instability in colorectal cancer cells. Proc. Natl. Acad. Sci. USA 94:2545-2550.
31. Liu, J., J. Chao, M. Jiang, S. Ng, J. J. Yen, and H. Yang-Yen. 1995. Ras transformation results in an elevated level of cyclin D1 and acceleration of G1 progression in NIH 3T3 cells. Mol. Cell. Biol. 15:3654-3663.[Abstract]
32. Lowy, D. R. 1993. Function and regulation of Ras. Annu. Rev. Biochem. 62:851-891.[CrossRef][Medline]
33. Lu, X., S. H. Park, T. C. Thompson, and D. P. Lane. 1992. ras-induced hyperplasia occurs with mutation of p53, but activated ras and myc together can induce carcinoma without p53 mutation. Cell 70:153-161.[CrossRef][Medline]
34. Ludwig, R. L., S. Bates, and K. H. Vousden. 1996. Differential activation of target cellular promoters by p53 mutants with impaired apoptotic function. Mol. Cell. Biol. 16:4952-4960.[Abstract]
35.
Mayo, M. W., C.-Y. Wang, P. C. Cogswell, K. S. Rogers-Graham, S. W. Lowe, C. J. Der, and A. S. Baldwin. 1997. Requirement of NFB activation to suppress p53-independent apoptosis induced by oncogenic Ras. Science 278:1812-1815.
36. Miyashita, T., S. Krajewski, M. Krajewska, H. G. Wang, H. K. Lin, D. A. Liebermann, B. Hoffman, and J. C. Reed. 1994. Tumor suppressor p53 is a regulator of bcl-2 and Bax gene expression in vitro and in vivo. Oncogene 9:1799-1805.[Medline]
37. Miyashita, T., and J. C. Reed. 1995. Tumor suppressor p53 is a direct transcriptional activator of the human Bax gene. Cell 80:293-299.[CrossRef][Medline]
38. Monks, C. R. F., H. Kupfer, I. Tamir, A. Barlow, and A. Kupfer. 1997. Selective modulation of protein kinase C-theta during T-cell activation. Nature 385:83-86.[CrossRef][Medline]
39. Oltvai, Z., C. Milliman, and S. J. Korsmeyer. 1993. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74:609-619.[CrossRef][Medline]
40. Quin, R.-G., F. McCormick, and M. Symons. 1996. An essential role for Rac in Ras transformation. Nature 374:457-459.
41.
Reed, J. C. 1994. Bcl-2 and the regulation of programmed cell death. J. Cell Biol. 124:1-6.
42. Ries, S., C. Biederer, D. Woods, O. Shifman, S. Shirasawa, T. Sasazuki, M. McMahon, M. Oren, and F. McCormick. 2000. Opposing effects of Ras and p53: transcriptional activation of mdm2 and induction of p19ARF. Cell 103:321-330.[CrossRef][Medline]
43.
Rosenberg, N., and D. Baltimore. 1976. A quantitative assay for transformation of bone marrow cells by Abelson murine leukemia. J. Exp. Med. 143:1453-1463.
44.
Shaw, P., R. Bovey, S. Tardy, R. Sahli, B. Sordat, and J. Costa. 1992. Induction of apoptosis by wild-type p53 in a human colon tumor-derived cell line. Proc. Natl. Acad. Sci. USA 89:4495-4499.
45.
Shirasawa, S., M. Furuse, N. Yokoyama, and T. Sasazuki. 1993. Altered growth of human colon cancer lines disrupted at activated Ki-ras. Science 260:85-88.
46. Tanaka, N., M. Ishihara, M. Kitagawa, H. Harada, T. Kimura, T. Matsuyama, M. S. Lamphier, S. Aizawa, T. W. Mak, and T. Taniguchi. 1994. Cellular commitment to oncogene-induced transformation or apoptosis is dependent on the transcription factor IRF-1. Cell 77:829-839.[CrossRef][Medline]
47. Thome, K. C., A. Radfar, and N. Rosenberg. 1997. Mutation of Tp53 contributes to the malignant phenotype of Abelson virus-transformed lymphoid cells. J. Virol. 71:8149-8156.[Abstract]
48. Tuveson, D. A., and T. Jacks. 1999. Modeling human lung cancer in mice: similarities and shortcomings. Oncogene 18:5318-5324.[CrossRef][Medline]
49.
Unnikrishnan, I., A. Radfar, J. Jenab-Wolocott, and N. Rosenberg. 1999. p53 mediates apoptotic crisis in primary Abelson virus-transformed pre-B cells. Mol. Cell. Biol. 19:4825-4831.
50. Vogelstein, B., and K. W. Kinzler. 1992. p53 function and dysfunction. Cell 70:523-526.[CrossRef][Medline]
51. Vousden, K. H. 2000. p53: death star. Cell 103:691-694.[CrossRef][Medline]
52. Vousden, K. H., and G. F. Vande Woude. 2000. The ins and outs of p53. Nat. Cell Biol. 2:E178-E180.[CrossRef][Medline]
53.
Weinber, R. A. 1989. Oncogenes, antioncogenes, and the molecular bases of multistep carcinogenesis. Cancer Res. 49:3713-3736.
54.
Whitlock, C. A., S. F. Ziegler, and O. N. Witte. 1983. Progression of the transformed phenotype in clonal lines of Abelson virus-infected lymphocytes. Mol. Cell. Biol. 3:596-604.
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| J. Bacteriol. | J. Virol. | Eukaryot. Cell |
|---|
| Microbiol. Mol. Biol. Rev. | Clin. Vaccine Immunol. | All ASM Journals |
|---|
