![[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}
Previous Article | Next Article 
Molecular and Cellular Biology, October 2003, p. 6836-6848, Vol. 23, No. 19
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.19.6836-6848.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
Received 19 March 2003/ Returned for modification 9 May 2003/ Accepted 17 June 2003
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The adenovirus early region 1A protein (E1A) induces chemosensitization among different categories of anticancer drugs, including cisplatin, adriamycin, etopside, staurosporine, 5-fluorouracil, and paclitaxel (Taxol) (5, 14, 16, 32, 45, 53), suggesting that a general cellular mechanism may exist to regulate E1A-mediated chemosensitization. However, the molecular mechanisms underlying E1A-mediated chemosensitization are still not completely defined. Earlier studies on normal fibroblast cells revealed that E1A-mediated sensitization to cytotoxic anticancer drugs depends on the expression of functional p53 and p19ARF, an alternative splicing form of p16INK4a (12, 31, 32). E1A was also shown to downregulate Her-2/neu overexpression and facilitate E1A-mediated sensitization to the cytotoxicity of anticancer drugs in human breast and ovarian cancer cells (53, 55, 56). In another study, E1A was reported to mediate sensitization to anticancer drugs in human osteosarcoma cells (16) in a p53- and Her-2/neu-independent manner. Similarly, there is no correlation between p53 protein level and sensitivity of DNA-damaging agents in keratinocytes carrying adenovirus E1A (45). A few other critical molecules were also proposed to be involved in E1A-induced chemosensitization, such as the proapoptotic protein Bax, caspase 9, or a yet unidentified inhibitor that ordinarily provides protection against cell death (14, 15, 34, 43, 49, 52). However, none of the above molecules or pathways can really serve as a general cellular mechanism for E1A-mediated sensitization to apoptosis in a diverse cellular context. Recently, transcriptional upregulation of procaspases (such as pro-caspase 3, 7, 8, and 9) through E1A-mediated disruption of pRB function and subsequent release of free E2F-1 was reported to contribute to both p53-dependent and p53-independent drug sensitization by E1A in diploid normal fibroblast cells (37). In the present study, we found that E1A can activate p38 and inactivate Akt and showed that this pathway may provide a general cellular mechanism for E1A to mediate sensitization to different categories of anticancer drugs.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
For the analysis of basal Akt and p38 expression and activity, cells were serum starved overnight before harvesting. Cells were then washed twice with cold phosphate-buffered saline (PBS) and lysed in a lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate (NaVO3), and 1.5% aprotinin. The cell extracts were clarified by centrifugation, and protein concentrations were determined by using a Bio-Rad (Hercules, Calif.) protein assay reagent and analyzed in a spectrophotometer with bovine serum album (Sigma, St. Louis, Mo.) as the protein standard. Aliquots of protein were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Millipore Corp., Bedford, Mass.) by using standard procedures. The membranes were then subjected to Western blotting, and the blots were developed with the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, N.J.).
Primary antibodies. For Western blot analysis, rabbit polyclonal antibodies against phospho-Akt (Ser 473, catalog no. 9271, 1:1,000 dilution) and phospho-p38 (Thr 180/Tyr 182, catalog no. 9211, 1:1,000 dilution) were purchased from Cell Signaling Technology (Beverly, Mass.). Rabbit polyclonal antibodies against nonphosphorylated total Akt (catalog no. 9272, 1:500 dilution), p38 (catalog no. 9212, 1:500 dilution), and cleaved poly(ADP-ribose) polymerase (PARP) (Asp214, catalog no. 9541, 1:1,000 dilution) were also from Cell Signaling Technology. A rabbit polyclonal antibody against beta-actin was used as a loading control for Western blotting and was purchased from Sigma. For immunohistochemical (IHC) study, we used an IHC-specific rabbit polyclonal antibody against phospho-Akt (Ser 473, catalog no. 9277) or an IHC-specific monoclonal antibody against phospho-p38 (Thr180/Tyr 182, catalog no. 9216) at a 1:75 dilution for both antibodies. The monoclonal antibody used against the E1A proteins was M58 (PharMingen, San Diego, Calif.). Rabbit polyclonal anti-Bax antibodies, a hamster anti-human Bcl-2 monoclonal antibody, and rabbit polyclonal anti-ASK1 (H-300) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.). Mouse monoclonal antibodies against human caspase 3 and 7 were from Transduction Laboratories (1:1,000 dilution, C31720; Lexington, Ky.) and BD PharMingen (1:1,000 dilution, 66871A), respectively. Rabbit polyclonal antibodies against human caspase 8 and 9 were from Santa Cruz Biotechnology (1:500 dilution, SC-7890/H-134) and Cell Signaling Technology (1:500 dilution, no. 9502), respectively. To detect hemagglutinin (HA)-tagged proteins, a monoclonal anti-HA antibody was used (1:1,000 dilution, catalog no. 1,583,816; Boehringer Mannheim, Indianapolis, Ind.). A monoclonal anti-FLAG antibody (M2) was purchased from Sigma (1:1,000 dilution).
Transient transfection, MTT assay, luciferase assay, and FACS analysis. The standard MTT assay was performed to measure the viable cells after treatment with anticancer drugs as described previously (52). Expression vectors for HA-p38, constitutively active Akt (CA-Akt), dominant-negative Akt (DN-Akt), and cytomegalovirus driving luciferase (pcDNA3-Luc) were used in this study. First, 105 cells in a 60-mm dish were transfected with 2.2 µg of total DNA by using the DC-Chol cationic liposome as described previously (52). After 48 h, the cells were split into three sets: one used for a luciferase assay after exposure with or without paclitaxel for 24 h, one used to analyze Akt and p38 protein expression, and one fixed in 75% ethanol, stained with propidium iodide (25 µg/ml), and sent for fluorescence-activated cell sorter (FACS) analysis. The percentage of paclitaxel-treated cells that exhibited luciferase activity was normalized by using the luciferase activity of the untreated cells as the baseline (100%). Standard deviations from three independent experiments were calculated.
Establishment of IPTG-inducible DN-p38 stable cell lines.
One E1A-expressing MDA-MB-231 clone was cotransfected with an isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible DN-p38
construct (a gift from Philipp E. Schere, Albert Einstein College of Medicine, Bronx, N.Y.) and the plasmid pCMVLacI (Stratagene, La Jolla, Calif.). Stable clones were selected in the presence of 200 µg of hygromycin/ml.
Immunoprecipitation. After transient transfection with HA-tagged p38 or CA-Akt, cells were stimulated with 10 µM insulin for 15 min. Cells were then lysed, and cell lysates were centrifuged at 16,000 g for 30 min. The supernatants were then transferred to a fresh tube. Proteins were cleared via addition of a normal mouse or rabbit immunoglobulin G and immunoprecipitated with anti-p38, anti-Akt, or anti-HA antibodies. Immunoprecipitates were resolved by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Akt, p38, and ASK1 were detected by Western blotting.
Kinase assay. Nonradioactive kinase assay kits for p38 and Akt were purchased from Cell Signaling (New England BioLabs, Beverly, Mass.). The p38 and Akt kinase activities were measured according to the manufacturer's protocol with glutathione S-transferase (GST)-ATF-2 as the substrate for p38 and GST-GSK-3-ß as the substrate for Akt.
Tissue microarray and immunohistochemistry. Tissue microarray slides (HistoArray no. IMH-343/BA2 and IMH-304/CB2) were purchased from IMGENEX (San Diego, Calif.). Detailed information about each slide is available online. Slide processing and immunohistochemical staining were performed according to the manufacturer's protocol. Briefly, tissue slides were heated at 60°C, deparaffinized in xylene, hydrated in graded ethanol, and then immersed in tap water. Antigen retrieval was performed with 0.01 M citrate buffer at pH 6.0 for 20 min in a 95°C water bath. Endogenous peroxidase activity was quenched in 3% hydrogen peroxide solution followed by three sequential PBS washes (5 min each). Slides were then blocked by the respective normal serum for each primary antibody, incubated with primary antibody diluted in TBS-T (50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.1% Tween 20) containing 1% ovalbumin and 1 mg of sodium azide/ml, incubated with biotinylated secondary antibody for 30 min at room temperature, washed with PBS again, and incubated with avidin-biotin complex (Vestastain Elite ABC kit; Vector Laboratories, Inc., Burlingame, Calif.). Slides were washed with PBS again, incubated with the AEC (3-amino-9-ethylcarbazole) substrate kit (catalog no. Sk-4200; Vector Laboratories, Inc.), and then counterstained in Meyer's xylene.
One representative slide per case was evaluated with the antibodies mentioned above. The intensities of staining seen in different areas of the same slide were analyzed according to criteria described previously in the literature (1). The intensity was designated 0 when no cells stained, 1+ when 10 to 20% of cells stained (weak), 2+ when 20 to 50% of cells stained (moderate), and 3+ when more than 50% of cells stained (strong).
Statistics.
For statistical analysis, groups scored as 0 and 1+ were combined as weak staining while groups scored as 2+ and 3+ were combined as strong staining. Similarly, to simplify the statistical analysis, breast tumors with stages 1 and 2 were combined as early stages of tumors (n = 25 cases) while tumors with stages 3 and 4 were combined as late stages of tumors (n = 25 cases). Statistical analysis was performed by using 
2 analysis.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Upregulation of p38 activity and downregulation of Akt activity correlate with E1A-mediated sensitization to paclitaxel-induced apoptosis. To test whether alteration of the kinase activity of Akt or p38 played a role in E1A-mediated sensitization to apoptosis, we compared the kinetics of phosphorylation of Akt or p38 with paclitaxel-induced apoptosis in 231-E1A cells by using PARP cleavage and Bcl-2 phosphorylation as apoptotic cell death markers. PARP cleavage and Bcl-2 phosphorylation occurred after decreased Akt phosphorylation and increased p38 phosphorylation in 231-E1A cells after exposure to 0.01 µM paclitaxel (Fig. 2A). However, no significant change was detected in the protein levels of p53 and Bax (Fig. 2A). The same concentration of paclitaxel did not trigger PARP cleavage, induce Bcl-2 phosphorylation, or modulate the levels of phosphorylated p38 and Akt in the parental MDA-MB-231 cells (data not shown). To trigger a similar response in parental MDA-MB-231 cells, a much higher dosage was required (Fig. 2B). The results suggest that downregulation of Akt and upregulation of p38 activities may be involved in the E1A-mediated sensitization to paclitaxel-induced apoptosis.
|
|
Activation of p38 and inactivation of Akt represent a general cellular mechanism in response to different apoptotic stimuli. To determine whether the same mechanism of E1A-mediated sensitization to paclitaxel applies to other anticancer drugs, we tested the effects of four additional drugs used in the clinic for treatment of human cancer. These four anticancer drugs induce antitumor activities through different modes of action: doxorubicin-adriamycin (topoisomerase II inhibitor), cisplatin (DNA-damaging agent), methotrexate (antimetabolite drug), and gemcitabine (antimetabolite drug). The expression of E1A significantly enhanced each drug's cytotoxicity in MDA-MB-231 cells, determined by using the MTT assay (Fig. 4A). In addition, downregulation of Akt phosphorylation and upregulation of p38 phosphorylation and PARP cleavage were observed in 231-E1A cells but not in 231-Vect cells treated with each of the drugs at the same dosage (Fig. 4B). These results suggest that activation of p38 and inactivation of Akt may contribute to E1A-mediated sensitization to apoptosis induced by these different drugs.
|
To determine the physiological relevance of inactivation of Akt and activation of p38 in the execution of apoptosis, we extended our investigation to apoptosis induced by serum starvation, tumor necrosis factor alpha (TNF-), and UV irradiation. We observed that phosphorylation of p38 and dephosphorylation of Akt were correlated with serum starvation-, TNF--, and UV-induced PARP cleavage in 231-E1A cells, especially in detached apoptotic cells (Fig. 4D, lanes 1 to 8). However, a dose of 10 times higher is required for inducing a response in parental MDA-MB-231 cells similar to that in 231-E1A cells (Fig. 4D, lanes 9 to 11). Taken together, downregulation of Akt activation and upregulation of p38 activation may also represent a general cellular mechanism in response to different apoptotic stimuli.
The physiological regulation of p38 activity by Akt is through ASK1 and MEKK3, the upstream kinases of p38. The above results suggest that both downregulation of Akt and upregulation p38 activities are involved in E1A-mediated sensitization to apoptosis. We noticed that reduced Akt phosphorylation occurs before enhanced p38 phosphorylation in the kinetic study of E1A-mediated sensitization to paclitaxel-induced PARP cleavage (Fig. 2A). We therefore asked whether Akt may act upstream of p38. To this end, Akt activity was blocked by either a specific PI3K inhibitor, wortmannin, or a genetic method to knock out Akt expression. Blocking Akt activation with wortmannin in MDA-MB-231 cells resulted in decreased Akt phosphorylation and increased p38 phosphorylation (Fig. 5A). And when Akt phosphorylation was recovered, the p38 phosphorylation was reduced again (8- to 24-h time points). These results indicate that Akt phosphorylation was required for repressing p38 activation, suggesting that the former is upstream from the latter. This conclusion was further supported by the study with Akt1-knockout MEFs and myr-Akt1-transfected stable cells. We observed that the level of phosphorylated p38 was increased in Akt1-/- MEFs compared with that in Akt+/+ and Akt+/- MEFs. Furthermore, the phospho-p38 protein was undetectable in the Akt constitutively activated myr-Akt1 stable cells (Fig. 5B). These results indicate that Akt is able to inhibit p38 activity.
|
p38 inactivation is associated with Akt activation in human cancer. The above results suggest that Akt acts upstream of p38 and blocks p38 activation. Because activation of Akt is a common phenomenon in different types of human cancers, we asked whether p38 inactivation is also a common phenomenon in human cancer cells and correlates with Akt activation. To test whether p38 inactivation was accompanied by Akt activation in human tumor tissues in vivo, we utilized tissue array slides to screen phospho-p38 and phospho-Akt expression in tumor tissues of different origins and normal or parallel normal organ tissues. We found that the phospho-Akt level was dramatically higher while phospho-p38 was undetectable in most of the cancer tissues obtained from different types of solid tumors, such as breast, lung, liver, bile duct, gastric, colorectal, renal cell, ovarian, and uterine cancers; malignant lymphoma; and Schwannoma. In contrast, the intensity of phospho-p38 protein staining was relatively strong while that of phospho-Akt staining was very weak in normal organs and parallel healthy tissues. Representative data on the expression of phospho-p38 and phospho-Akt in healthy versus tumor tissues obtained from the breast, lung, liver, and biliary duct are shown in Fig. 6A. By screening a panel of breast, ovarian, prostate, pancreatic, and colorectal cancer cell lines with phosphospecific antibody against p38 or Akt, we also observed a correlation between enhanced Akt phosphorylation and reduced p38 phosphorylation in these human cancer cell lines (data not shown). These data support the hypothesis that p38 activity is repressed in different types of human cancer, which is associated with enhanced Akt activation.
|
The CR2 domain of E1A is required for downregulation of Akt phosphorylation and chemosensitization.
The above results suggested that Akt regulated p38 activation in both physiological and pathological conditions, which indicates that E1A-mediated downregulation of Akt activity and upregulation of p38 activity are accompanied events, i.e., by repression of Akt activation, E1A enhanced p38 activity and sensitized cells to drug-induced apoptosis. To lay further genetic support for these conclusions, we proposed to map the domain(s) of E1A that is responsible for downregulation of Akt activity and demonstrate that the same domain is also critical for E1A-mediated upregulation of p38 and sensitization to drug-induced apoptosis. It is known that among the three conserved domains (CR) of E1A, CR1 and CR2 are associated with E1A-mediated sensitization to apoptosis (46). Therefore, we established E1A functional domain deletion mutation stable cells in MDA-MB-231 cells, including wild-type E1A, and deletion mutations of CR1 (
CR1) and CR2 (CR2) (Fig. 7A). We found that deletion mutations of the CR2 domain dramatically disrupted E1A's ability to downregulate Akt kinase activity, eliminated E1A-mediated upregulation of p38 kinase activity, and remarkably repressed E1A-mediated sensitization to paclitaxel-induced apoptosis (Fig. 7B and C) while the CR1 domain mutant only slightly affected E1A-mediated chemosensitization and downregulation of Akt and upregulation of p38 activities (Fig. 7B and C). These results indicate that the same CR2 domain required for downregulation of Akt is also required for upregulation of p38 and sensitization to drug-induced apoptosis and thus supports the conclusions that Akt represses p38 activity and that E1A, by downregulation of Akt activity, enhances p38 activation and sensitizes cells to anticancer drug-induced apoptosis.
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We have shown that activation of p38 follows inactivation of Akt (Fig. 2 and 4B to D) when cells underwent apoptosis, suggesting that the pro- and antiapoptotic signals may integrate each other to prepare cells to commit suicide. The relative Akt and p38 activity may determine a cell's response to apoptotic stimuli, as they can be observed in E1A-mediated sensitization to apoptosis induced by serum starvation, TNF-, UV irradiation, and different categories of chemotherapeutic drugs (Fig. 4B to D). Expression of E1A may shift the balance of the pro- and antiapoptotic signals by repressing Akt activity and enhancing p38 activity, thereby favoring the proapoptotic signal. Additional approaches could also be used by E1A to shift the intracellular signal integration to favor a proapoptotic signal, such as activation of p53 and caspase proenzymes, but these pathways may not contribute to the present study. For example, p53 and p14ARF were deleted in MDA-MB-231 cells and we did not detect any change in the expression level of p53 or p14ARF in E1A versus parental control cells (data not shown), i.e., the p53-dependent mechanisms may not contribute to E1A-mediated sensitization to apoptosis in the present study. Although expression of E1A by infection of cells with either retroviral or adenoviral vector resulted in the accumulation of caspase proenzymes, such as caspase 3, 7, 8, and 9, by a direct transcriptional mechanism through enforced E2F-1 release in normal diploid human fibroblasts (IMR90) (37). We did not observe a consistent increase of these caspase proenzymes in the E1A stable cells established in human cancer cells with epithelial origin (Fig. 1D). In addition, sensitization to the DNA damage agent adriamycin-induced apoptosis by E1A is dependent on p53 status in normal fibroblasts (37) while E1A dramatically sensitized the adriamycin therapeutic effect in ovarian cancer SKOV3.ip1 (5) and breast cancer MDA-MB-231 cells (Fig. 4A), which do not express functional p53. The discrepancy between normal diploid fibroblasts and epithelial carcinoma cells in E1A-mediated sensitization to apoptosis may reflect the nature of the intrinsic difference between normal fibroblasts and carcinoma cells. However, downregulation of Akt activity by E1A was also observed in E1A-mediated sensitization to cisplatin in human normal IMR90 fibroblasts (54). Thus, targeting the key oncogenic survival factor Akt may represent a critical mechanism for E1A-mediated sensitization to anticancer drug-induced apoptosis in human cancer cells and normal fibroblasts as well.
E1A has been shown to facilitate cytochrome c release from the mitochondria, which also contributes to E1A-mediated sensitization to anticancer drugs. However, the mechanism by which E1A facilitates cytochrome c release is unclear (14). Although we did not test whether E1A expression affected Bax translocation, which may also facilitate cytochrome c release, E1A expression or treatment with paclitaxel did not affect the levels of Bax protein in our system (Fig. 2A). Akt is known to play an important role in maintaining mitochondrial integrity and inhibiting the release of cytochrome c (11, 27). Overexpression of Akt confers resistance to paclitaxel by inhibiting paclitaxel-induced cytochrome c release (41). However, p38 is also involved in regulation of cytochrome c release (2). Therefore, it is possible that E1A may alter mitochondrial potential by downregulating Akt and upregulating p38, thereby facilitating the release of cytochrome c upon treatment with chemotherapeutic drugs, such as paclitaxel. Thus, downregulation of key survival factor Akt activity and subsequent upregulation of a proapoptotic factor p38 activity by E1A may constitute a fundamental approach for E1A-mediated sensitization to apoptosis.
The mechanisms underlying E1A-mediated downregulation of Akt activity are not yet clear. Obviously, E1A-mediated downregulation of Her-2/neu and/or Axl may contribute to reduced Akt activation, as activation of either Her-2/neu or Axl leads to PI3K-Akt kinase activation and downregulation of Her-2/neu and/or Axl also contributes to E1A-mediated sensitization to apoptosis (21, 29, 30, 58). However, downregulation of Akt activity by E1A may not necessarily depend on E1A-mediated downregulation of Her-2/neu and/or Axl, because both MDA-MB-231 and MCF-7 cells are low Her-2/neu-expressing cells and MCF-7 cells have an undetectable expression level of Axl (35). In addition, deletion mutation of the CR2 domain affects E1A-mediated downregulation of Akt (Fig. 7B), but it has no effect on E1A-mediated transcriptional repression of Her-2/neu (9). Thus, in addition to the Her-2/neu-dependent pathway, a Her-2/neu-independent pathway must exist for E1A to mediate downregulation of Akt activity leading to sensitization to apoptosis. In addition, overexpression of Her-2/neu or activation of Akt also leads to p53 destabilization (50, 59), suggesting that downregulation of Akt activity by E1A may constitute an alternative pathway for stabilization of p53. Like Her-2/neu and p53, Akt also plays a critical role in the regulation of apoptotic cell death and the development of human cancer. Therefore, E1A-mediated downregulation of Akt and upregulation of p38 activities may have general implications for E1A-mediated tumor suppression and sensitization to apoptosis.
|
ACKNOWLEDGMENTS |
|---|
This work was supported by grant RO1-CA58880 and the SPORE grant for ovarian cancer research from the National Institutes of Health (to M.-C.H.) and by grant DAMD17-01-1-0300 from the U.S. Department of Defense Army Breast Cancer Research Program (to Y.L.).
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Assefa, Z., A. Vantieghem, M. Garmyn, W. Declercq, P. Vandenabeele, J. R. Vandenheede, R. Bouillon, W. Merlevede, and P. Agostinis. 2000. p38 mitogen-activated protein kinase regulates a novel, caspase-independent pathway for the mitochondrial cytochrome c release in ultraviolet B radiation-induced apoptosis. J. Biol. Chem. 275:21416-21421.
3. Berra, E., M. T. Diaz-Meco, and J. Moscat. 1998. The activation of p38 and apoptosis by the inhibition of Erk is antagonized by the phosphoinositide 3-kinase/Akt pathway. J. Biol. Chem. 273:10792-10797.
4. Blain, S. W., and J. Massague. 2002. Breast cancer banishes p27 from nucleus. Nat. Med. 8:1076-1078.[CrossRef][Medline]
5. Brader, K. R., J. K. Wolf, M. C. Hung, D. Yu, M. A. Crispens, K. L. van Golen, and J. E. Price. 1997. Adenovirus E1A expression enhances the sensitivity of an ovarian cancer cell line to multiple cytotoxic agents through an apoptotic mechanism. Clin. Cancer Res. 3:2017-2024.[Abstract]
6. Bulavin, D. V., S. A. Amundson, and A. J. Fornace. 2002. p38 and Chk1 kinases: different conductors for the G(2)/M checkpoint symphony. Curr. Opin. Genet. Dev. 12:92-97.[CrossRef][Medline]
7. Bulavin, D. V., O. N. Demidov, S. Saito, P. Kauraniemi, C. Phillips, S. A. Amundson, C. Ambrosino, G. Sauter, A. R. Nebreda, C. W. Anderson, A. Kallioniemi, A. J. J. Fornace, and E. Appella. 2002. Amplification of PPM1D in human tumors abrogates p53 tumor-suppressor activity. Nat. Genet. 31:210-215.[CrossRef][Medline]
8. Chen, D., R. V. Fucini, A. L. Olson, B. A. Hemmings, and J. E. Pessin. 1999. Osmotic shock inhibits insulin signaling by maintaining Akt/protein kinase B in an inactive dephosphorylated state. Mol. Cell. Biol. 19:4684-4694.
9. Chen, H., D. Yu, G. Chinnadurai, D. Karunagaran, and M. C. Hung. 1997. Mapping of adenovirus 5 E1A domains responsible for suppression of neu-mediated transformation via transcriptional repression of neu. Oncogene 14:1965-1971.[CrossRef][Medline]
10. Chen, W. S., P. Z. Xu, K. Gottlob, M. L. Chen, K. Sokol, T. Shiyanova, I. Roninson, W. Weng, R. Suzuki, K. Tobe, T. Kadowaki, and N. Hay. 2001. Growth retardation and increased apoptosis in mice with homozygous disruption of the Akt1 gene. Genes Dev. 15:2203-2208.
11. Cross, T. G., D. S. Toellner, N. V. Henriquez, E. Deacon, M. Salmon, and J. Lod. 2000. Serine/threonine protein kinases and apoptosis. Exp. Cell Res. 256:34-41.[CrossRef][Medline]
12. de Stanchina, E., M. E. McCurrach, F. Zindy, S. Y. Shieh, G. Ferbeyre, A. V. Samuelson, C. Prives, M. F. Roussel, C. J. Sherr, and S. W. Lowe. 1998. E1A signaling to p53 involves the p19(ARF) tumor suppressor. Genes Dev. 12:2434-2442.
13. De Zutter, G. S., and R. J. Davis. 2001. Pro-apoptotic gene expression mediated by the p38 mitogen-activated protein kinase final transduction pathway. Proc. Natl. Acad. Sci. USA 98:6168-6173.
14. Duelli, D. M., and Y. A. Lazebnik. 2000. Primary cells suppress oncogene-dependent apoptosis. Nat. Cell Biol. 2:859-862.[CrossRef][Medline]
15. Fearnhead, H. O., J. Rodriguez, E. E. Govek, W. Gou, R. Kobayashi, G. Hannon, and Y. A. Lazebnik. 1998. Oncogene-dependent apoptosis is mediated by caspase-9. Proc. Natl. Acad. Sci. USA 95:13664-13669.
16. Frisch, S. M., and K. E. Dolter. 1995. Adenovirus E1a-mediated tumor suppression by a c-erbB-2/neu-independent mechanism. Cancer Res. 55:5551-5555.
17. Gratton, J. P., M. Morales-Ruiz, Y. Kureishi, D. Fulton, K. Walsh, and W. C. Sessa. 2001. Akt down-regulation of p38 signaling provides a novel mechanism of vascular endothelial growth factor-mediated cytoprotection in endothelial cells. J. Biol. Chem. 276:30359-30365.
18. Hanahan, D., and R. A. Weinberg. 2000. The hallmarks of cancer. Cell 100:57-70.[CrossRef][Medline]
19. Heidenreich, K. A., and J. L. Kummer. 1996. Inhibition of p38 mitogen-activated protein kinase by insulin in cultured fetal neurons. J. Biol. Chem. 271:9891-9894.
20. Hsu, S. C., M. A. Gavrilin, M. H. Tsai, J. Han, and M. Z. Lai. 1999. p38 mitogen-activated protein kinase is involved in Fas ligand expression. J. Biol. Chem. 274:25769-25776.
21. Hung, M. C., G. N. Hortobagyi, and N. T. Ueno. 2000. Development of clinical trial of E1A gene therapy targeting HER-2/neu-overexpressing breast and ovarian cancer. Adv. Exp. Med. Biol. 465:171-180.[Medline]
22. Hunter, T. 2000. Signalin—2000 and beyond. Cell 100:113-127.[CrossRef][Medline]
23. Ichijo, H., E. Nishida, K. Irie, P. ten Dijke, M. Saitoh, T. Moriguchi, M. Takagi, K. Matsumoto, K. Miyazono, and Y. Gotoh. 1997. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 275:90-94.
24. Igney, F. I., and P. H. Krammer. 2002. Death and anti-death: tumor resistance to apoptosis. Nat. Rev. Cancer 2:277-288.[CrossRef][Medline]
25. Inoki, K., Y. Li, T. Zhu, J. Wu, and K. L. Guan. 2002. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat. Cell Biol. 4:648-657.[CrossRef][Medline]
26. Kandel, E. S., J. Skeen, N. Majewski, A. Di Cristofano, P. P. Pandolfi, C. S. Feliciano, A. Gartel, and N. Hay. 2002. Activation of Akt/protein kinase B overcomes a G(2)/m cell cycle checkpoint induced by DNA damage. Mol. Cell. Biol. 22:7831-7841.
27. Kennedy, S. G., E. S. Kandel, T. K. Cross, and N. Hay. 1999. Akt/protein kinase B inhibits cell death by preventing the release of cytochrome c from mitochondria. Mol. Cell. Biol. 19:5800-5810.
28. Kim, A. H., G. Khursigara, X. Sun, T. F. Franke, and M. V. Chao. 2001. Akt phosphorylates and negatively regulates apoptosis signal-regulating kinase 1. Mol. Cell. Biol. 21:893-901.
29. Lee, W. P., Y. Liao, D. Robinson, H. J. Kung, E. T. Liu, and M. C. Hung. 1999. Axl-gas6 interaction counteracts E1A-mediated cell growth suppression and proapoptotic activity. Mol. Cell. Biol. 19:8075-8082.
30. Lee, W. P., Y. Wen, B. Varnum, and M. C. Hung. 2002. Akt is required for Axl-Gas6 signaling to protect cells from E1A-mediated apoptosis. Oncogene 21:329-336.[CrossRef][Medline]
31. Lowe, S. W. 1999. Activation of p53 by oncogenes. Endocr. Relat. Cancer 6:45-48.[Abstract]
32. Lowe, S. W., H. E. Ruley, T. Jacks, and D. E. Housman. 1993. p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 74:957-967.[CrossRef][Medline]
33. Mayo, L. D., and D. B. Donner. 2001. A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus. Proc. Natl. Acad. Sci. USA 98:11598-11603.
34. McCurrach, M. E., T. M. F. Connor, C. M. Knudson, S. J. Korsmeyer, and S. W. Lowe. 1997. bax-deficiency promotes drug resistance and oncogenic transformation by attenuating p53-dependent apoptosis. Proc. Natl. Acad. Sci. USA 94:2345-2349.
35. Meric, F., W. P. Lee, A. Sahin, H. Zhang, H. J. Kung, and M. Hung. 2002. Expression profile of tyrosine kinases in breast cancer. Clin. Cancer Res. 8:361-367.
36. Meric, F., Y. Liao, W. P. Lee, R. E. Pollock, and M. C. Hung. 2000. Adenovirus 5 early region 1A does not induce expression of the Ewing sarcoma fusion product EWS-FLI1 in breast and ovarian cancer cell lines. Clin. Cancer Res. 6:3832-3836.
37. Nahle, Z., J. Polakoff, R. V. Davuluri, M. E. McCurrach, M. D. Jacobson, M. Narita, M. Q. Zhang, Y. Lazebnik, D. Bar-Sagi, and S. W. Lowe. 2002. Direct coupling of the cell cycle and cell death machinery by E2F. Nat. Cell Biol. 4:859-864.[CrossRef][Medline]
38. Nebreda, A. R., and A. Porras. 2000. p38 MAP kinases: beyond the stress response. Trends Biochem. Sci. 25:257-260.[CrossRef][Medline]
39. Nicholson, K. M., and N. G. Anderson. 2002. The protein kinase B/Akt signalling pathway in human malignancy. Cell. Signal. 14:381-395.[CrossRef][Medline]
40. Okumura, E., T. Fukuhara, H. Yoshida, S. Hanada, R. Kozutsumi, M. Mori, K. Tachibana, and T. Kishimoto. 2002. Akt inhibits Myt1 in the signalling pathway that leads to meiotic G2-M-phase transition. Nat. Cell Biol. 4:111-116.[CrossRef][Medline]
41. Page, C., H. J. Lin, Y. Jin, V. P. Castle, G. Nunez, M. Huang, and J. Lin. 2000. Overexpression of Akt/AKT can modulate chemotherapy-induced apoptosis. Anticancer Res. 20:407-416.[Medline]
42. Pearce, A. K., and T. C. Humphrey. 2001. Integrating stress-response and cell-cycle checkpoint pathways. Trends Cell Biol. 11:426-433.[CrossRef][Medline]
43. Putzer, B. M., T. Stiewe, K. Parssanedjad, S. Rega, and H. Esche. 2000. E1A is sufficient by itself to induce apoptosis independent of p53 and other adenoviral gene products. Cell Death Differ. 7:177-188.[CrossRef][Medline]
44. Rane, M. J., P. Y. Coxon, D. W. Powell, R. Webster, J. B. Klein, W. Pierce, P. Ping, and K. R. McLeish. 2001. p38 kinase-dependent MAPKAPK-2 activation functions as 3-phopshoinositide-dependent kinase-2 for AKt in human neutrophils. J. Biol. Chem. 276:3517-3523.
45. Sanchez-Prieto, R., M. Lleonart, and S. Ramon y Cajal 1995. Lack of correlation between p53 protein level and sensitivity of DNA-damaging agents in keratinocytes carrying adenovirus E1a mutants. Oncogene 11:675-682.[Medline]
46. Shisler, J., P. Duerksen-Hughes, T. M. Hermiston, W. M. Wold, and L. R. Gooding. 1996. Induction of susceptibility to tumor necrosis factor by E1A is dependent on binding to either p300 or p105-Rb and induction of DNA synthesis. J. Virol. 70:68-77.[Abstract]
47. Shtivelman, E., J. Sussman, and D. Stokoe. 2002. A role of PI 3-kinase and PKA activity in the G2/M phase of the cell cycle. Curr. Biol. 12:919-924.[CrossRef][Medline]
48. Skorski, T. 2002. Oncogenic tyrosine kinases and the DNA-damage repsonse. Nat. Rev. Cancer 2:351-360.[CrossRef][Medline]
49. Teodoro, J. G., G. C. Shore, and P. E. Branton. 1995. Adenovirus E1A proteins induce apoptosis by both p53-dependent and p53-independent mechanisms. Oncogene 11:467-474.[Medline]
50. Testa, J. R., and A. Bellacosa. 2001. AKT plays a central role in tumorigenesis. Proc. Natl. Acad. Sci. USA 98:10983-10985.
51. Tobiume, K., A. Matsuzawa, T. Takahashi, H. Nishitoh, K. Morita, K. Takeda, O. Minowa, K. Miyazono, T. Noda, and H. Ichijo. 2001. ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis. EMBO Rep. 2:222-228.[CrossRef][Medline]
52. Ueno, N. T., C. Bartholomeusz, J. L. Herrmann, Z. Estrov, R. Saho, M. Andreeff, J. Price, R. W. Paul, P. Anklesaria, D. Yu, and M. C. Hung. 2000. E1A-mediated paclitaxel sensitization in Her-2/neu-overexpressing ovarian cancer SKOV3. ip1 through apoptosis involving the caspase-3 pathway. Clin. Cancer Res. 6:250-259.
53. Ueno, N. T., D. Yu, and M. C. Hung. 1997. Chemosensitization of Her-2/neu-overexpressing human breast cancer cells to paclitaxel (Taxol) by adenovirus type 5 E1A. Oncogene 15:953-960.[CrossRef][Medline]
54. Viniegra, J. G., J. H. Losa, V. J. Sanchez-Arevalo, C. P. Cobo, V. M. Soria, S. Ramon y Cajal, and R. Sanchez-Prieto 2002. Modulation of PI3K/Akt pathway by E1a mediates sensitivity to cisplatin. Oncogene 21:7131-7136.[CrossRef][Medline]
55. Yu, D., and M. C. Hung. 2000. Overexpression of ErbB2 in cancer and ErbB2-targeting strategies. Oncogene 19:6115-6121.[CrossRef][Medline]
56. Yu, D., and M. C. Hung. 2000. Role of erbB2 in breast cancer chemosensitivity. Bioessays 22:673-680.[CrossRef][Medline]
57. Yuan, Z. Q., R. I. Feldman, G. E. Sussman, D. Coppola, S. V. Nicosia, and J. Q. Cheng. 15 April 2003, posting date. AKT2 inhibition of cisplatin-induced JNK/p38 and Bax activation by phosphorylation of ASK1: implication of AKT2 in chemoresistance. J. Biol. Chem. 278:23432-23440. [Online.] http://www.jbc.org.
58. Zhou, B. P., Y. Liao, W. Xia, B. Spohn, M. H. Lee, and M. C. Hung. 2001. Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells. Nat. Cell Biol. 3:245-252.[CrossRef][Medline]
59. Zhou, B. P., Y. Liao, W. Xia, Y. Zou, B. Spohn, and M. C. Hung. 2001. HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation. Nat. Cell Biol. 3:973-982.[CrossRef][Medline]
This article has been cited by other articles:
| ||||||||||||||||||||||||||||||||||||||||
