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• W1967975669 abstract "Many cell lines derived from neuroblastoma (NB) carry the wild-type p53 gene with a p53-dependent apoptotic pathway that is responsive to DNA damaging agents. A recent study has demonstrated that retinoic acid (RA) pretreatment of NB cells promotes chemoresistance to apoptosis induced by chemotherapeutic agents. We examine here the possible contribution of the p53 pathway to the chemoresistance response associated with the RA treatment in NB cells. Upon treatment with RA (1–10 μm) for 4 days, the human NB cells, SH-SY5Y, developed resistance selectively to p53-dependent apoptotic stimuli including γ-irradiation, etoposide, and 1-(5-isoquinolinyl sulfonyl)-2-methylpiperazine (H-7). Interestingly, RA affected the ability of H-7 to induce nuclear accumulation of the p53 protein without altering its effect on elevating the steady-state level of p53, suggesting that drug-induced up-regulation and nuclear accumulation of the wild-type p53 protein are separable processes. The modulation of nuclear import of p53 protein by RA may thus represent a potential mechanism by which certain tumor cells with the wild-type p53 gene develop resistance to chemotherapeutic agents. Many cell lines derived from neuroblastoma (NB) carry the wild-type p53 gene with a p53-dependent apoptotic pathway that is responsive to DNA damaging agents. A recent study has demonstrated that retinoic acid (RA) pretreatment of NB cells promotes chemoresistance to apoptosis induced by chemotherapeutic agents. We examine here the possible contribution of the p53 pathway to the chemoresistance response associated with the RA treatment in NB cells. Upon treatment with RA (1–10 μm) for 4 days, the human NB cells, SH-SY5Y, developed resistance selectively to p53-dependent apoptotic stimuli including γ-irradiation, etoposide, and 1-(5-isoquinolinyl sulfonyl)-2-methylpiperazine (H-7). Interestingly, RA affected the ability of H-7 to induce nuclear accumulation of the p53 protein without altering its effect on elevating the steady-state level of p53, suggesting that drug-induced up-regulation and nuclear accumulation of the wild-type p53 protein are separable processes. The modulation of nuclear import of p53 protein by RA may thus represent a potential mechanism by which certain tumor cells with the wild-type p53 gene develop resistance to chemotherapeutic agents. It is well established that tumor cells are able to acquire resistance to chemotherapeutic agents through a variety of mechanisms including enhanced drug metabolism, altered drug accumulation, drug-target amplification, and repair of damaged drug target (1Biedler J.L. Cancer Res. 1994; 54: 666-678PubMed Google Scholar). More recently, induction of apoptosis has been discovered as a novel mechanism by which many anti-neoplastic therapies achieve their therapeutic effects (2Dive C. Hickman J.A. Br. J. Cancer. 1991; 64: 192-196Crossref PubMed Scopus (413) Google Scholar, 3Barry M.A. Behnke C.A. Eastman A. Biochem. Pharmacol. 1990; 40: 2353-2362Crossref PubMed Scopus (914) Google Scholar, 4Sen S. D'Incalci M. FEBS Lett. 1992; 307: 122-127Crossref PubMed Scopus (265) Google Scholar). Therefore, the inability of a tumor cell to activate the apoptotic response has been proposed as a pathway of resistance to antineoplastic therapy (5Lowe S.W. Ruley H.E. Jacks T. Housman D.E. Cell. 1993; 74: 957-967Abstract Full Text PDF PubMed Scopus (2963) Google Scholar). Neuroblastoma (NB) 1The abbreviations used are: NB, neuroblastoma; H-7, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine; RA, all-trans-retinoic acid; RB, retinoblastoma; MTT, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; TPA, 12-O-tetradecanoyl-13-acetate; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; TBST, TBS with Tween 20; RAR, retinoic acid receptor; RXR, retinoid X receptor. is one of the most common malignancies in childhood. Unlike other tumor types, nearly all human NBs were found to carry the wild-type p53gene (6Vogan K. Bernstein M. Leclerc J.M. Brisson L. Brossard J. Brodeur G.M. Pelletier J. Gros P. Cancer Res. 1993; 53 (1993): 5269-5273PubMed Google Scholar, 7Komuro H. Hayashi Y. Kawamura M. Hayashi K. Kaneko Y. Kamoshita S. Hanada R. Yamamoto K. Hongo T. Yamada M. Cancer Res. 1993; 53: 5284-5288PubMed Google Scholar, 8Imamura J. Bartram C.R. Berthold F. Harms D. Nakamura H. Koeffler H.P. Cancer Res. 1993; 53: 4053-4058PubMed Google Scholar, 9Hosoi G. Hara J. Okamura T. Osugi Y. Ishihara S. Fukuzawa M. Okada A. Okada S. Tawa A. Cancer. 1994; 73: 3087-3093Crossref PubMed Scopus (74) Google Scholar). Wild-type p53 protein is expressed in many of the cell lines derived from NB (10Davidoff A.M. Pence J.C. Shorter N.A. Iglehart J.D. Marks J.R. Oncogene. 1992; 7: 127-133PubMed Google Scholar). However, the p53 protein is found to be restricted primarily to the cytoplasmic compartment of the NB cells (11Moll U.M. LaQuaglia M. Benard J. Riou G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4407-4411Crossref PubMed Scopus (391) Google Scholar). This finding has led to the proposal that the p53-mediated response in NB cells is impaired because of an unknown cytoplasmic sequestration mechanism that prevents its translocation into the nucleus (11Moll U.M. LaQuaglia M. Benard J. Riou G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4407-4411Crossref PubMed Scopus (391) Google Scholar, 12Moll U.M. Ostermeyer A.G. Haladay R. Winkfield B. Frazier M. Zambetti G. Mol. Cell. Biol. 1996; 16: 1126-1137Crossref PubMed Scopus (253) Google Scholar). Our laboratory has recently reported a protein kinase inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine (H-7) that possesses novel activities of inducing nuclear accumulation of the p53 protein and triggering p53-dependent apoptosis in several NB cell lines that carry the wild-type p53 gene (13Ronca F. Chan S.L. Yu V.C. J. Biol. Chem. 1997; 272: 4252-4260Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). H-7, similar to DNA damaging agents, increased the steady-state level of p53 in NB cells, which at least, in part accounted for the dramatic nuclear accumulation of p53 in these cells (12Moll U.M. Ostermeyer A.G. Haladay R. Winkfield B. Frazier M. Zambetti G. Mol. Cell. Biol. 1996; 16: 1126-1137Crossref PubMed Scopus (253) Google Scholar, 13Ronca F. Chan S.L. Yu V.C. J. Biol. Chem. 1997; 272: 4252-4260Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). All-trans-retinoic acid (RA) and many of its natural and synthetic derivatives, collectively known as retinoids, have been investigated extensively as potential therapeutics for human cancer because of their effects in inhibiting cell proliferation, inducing cell differentiation, and promoting apoptosis in many types of cancer, including NBs (14Aboseif S.R. Dahiya R. Narayan P. Cunha G.R Prostate. 1997; 31: 161-167Crossref PubMed Scopus (36) Google Scholar, 15Kaba S.E. Kyritsis A.P. Conrad C. Gleason M.J. Newman R. Levin V.A. Yung W.K. J. Neurooncol. 1997; 34: 145-151Crossref PubMed Scopus (48) Google Scholar, 16Szondy Z. Reichert U. Bernardon J.M. Michel S. Toth R. Ancian P. Ajzner E. Fesus L. Mol. Pharmacol. 1997; 51: 972-982Crossref PubMed Scopus (83) Google Scholar, 17Melino G. Thiele C.J. Knight R.A. Piacentini M. J. Neurooncol. 1997; 31: 65-83Crossref PubMed Scopus (76) Google Scholar). The activities of retinoids are mediated by a network of RA receptors (RARs) and retinoid X receptors (RXRs) (18Yu V.C. Delsert C. Andersen B. Holloway J.M. Devary O.V. Naar A.M. Kim S.Y. Boutin J.M. Glass C.K. Rosenfeld M.G. Cell. 1991; 67: 1251-1266Abstract Full Text PDF PubMed Scopus (1062) Google Scholar, 19Yu V.C. Naar A.M. Rosenfeld M.G. Curr. Opin. Biotechnol. 1992; 3: 597-602Crossref PubMed Scopus (35) Google Scholar, 20Kastner P. Mark M. Chambon P. Cell. 1995; 83: 859-869Abstract Full Text PDF PubMed Scopus (940) Google Scholar, 21Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2843) Google Scholar). RA at low concentration activates RARs in vivo(18Yu V.C. Delsert C. Andersen B. Holloway J.M. Devary O.V. Naar A.M. Kim S.Y. Boutin J.M. Glass C.K. Rosenfeld M.G. Cell. 1991; 67: 1251-1266Abstract Full Text PDF PubMed Scopus (1062) Google Scholar, 19Yu V.C. Naar A.M. Rosenfeld M.G. Curr. Opin. Biotechnol. 1992; 3: 597-602Crossref PubMed Scopus (35) Google Scholar, 20Kastner P. Mark M. Chambon P. Cell. 1995; 83: 859-869Abstract Full Text PDF PubMed Scopus (940) Google Scholar, 21Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2843) Google Scholar). RA can also be metabolized to several active metabolitesin vivo, including 9-cis-RA, the high-affinity ligand for RXR (22Heyman R.A. Mangelsdorf D.J. Dyck J.A. Stein R.B. Eichele G. Evans R.M. Thaller C. Cell. 1992; 68: 397-406Abstract Full Text PDF PubMed Scopus (1570) Google Scholar). In addition, RXRs are also known to heterodimerize with RARs and a subset of other nuclear receptors and co-regulator molecules to participate in a complex array of signaling events (18Yu V.C. Delsert C. Andersen B. Holloway J.M. Devary O.V. Naar A.M. Kim S.Y. Boutin J.M. Glass C.K. Rosenfeld M.G. Cell. 1991; 67: 1251-1266Abstract Full Text PDF PubMed Scopus (1062) Google Scholar, 19Yu V.C. Naar A.M. Rosenfeld M.G. Curr. Opin. Biotechnol. 1992; 3: 597-602Crossref PubMed Scopus (35) Google Scholar, 20Kastner P. Mark M. Chambon P. Cell. 1995; 83: 859-869Abstract Full Text PDF PubMed Scopus (940) Google Scholar, 21Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2843) Google Scholar). Besides having a role in differentiation, RA has also been implicated in mediating apoptosis in NB cells (23Piacentini M. Annicchiarico-Petruzzelli M. Oliviero S. Piredda L. Biedler J.L. Melino G. Int. J. Cancer. 1992; 52: 271-278Crossref PubMed Scopus (110) Google Scholar). Paradoxically, a recent study has shown that treatment of NB cells with RA resulted in the development of resistance to apoptosis induced by chemotherapeutic agents (24Lasorella A. Iavarone A. Israel M.A. Cancer Res. 1995; 55: 4711-4716PubMed Google Scholar). In this report, we examine the possible contribution of the p53 pathway in the chemoresistance response rendered by RA treatment in the NB cell line, SH-SY5Y. RA treatment was found to confer resistance selectively to p53-dependent apoptotic stimuli in these cells. The effect was associated with the suppression of H-7-induced nuclear accumulation rather than the enhancement of protein stability of wild-type p53 protein in SH-SY5Y cells, suggesting that drug-induced up-regulation and nuclear accumulation of the p53 protein are separable processes. Isoquinoline (1-(5-isoquinolinesulfonyl)-2-methylpiperazine; H-7) and staurosporine were purchased from RBI.N-Acetyl-d-erythro-sphingosine was from Calbiochem. All other chemicals were purchased from Sigma unless otherwise indicated. The human neuroblastoma cell lines SH-SY5Y, LA-N-5, and IMR-32 have been described previously (13Ronca F. Chan S.L. Yu V.C. J. Biol. Chem. 1997; 272: 4252-4260Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). RPMI 1640 medium was supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.) and antibiotics (100 mg/ml streptomycin and 100 IU/ml penicillin, Life Technologies, Inc.). H-7 was dissolved in water; all the other compounds were dissolved in Me2SO-ethanol (1:1). The cells were grown to subconfluence and then treated with RA for different durations and with different concentrations of RA. The medium was replaced every 48 h. When reagents dissolved in Me2SO-ethanol were used, equal volume of the solvent was added to the control cells. The following antibodies were purchased from Transduction Laboratories (Lexington, KY): anti-p53 monoclonal antibody P21020 that recognizes the C terminus of the human p53 protein, anti-RB monoclonal antibody, and anti-ICH-1L monoclonal antibody. Anti-p53 (DO-1) monoclonal antibody, anti-Bcl-2 (100) monoclonal antibody, rabbit anti-Bcl-xL/S (S-19), anti-Bax (N-20), anti-c-Jun/AP-1 (N), and anti-c-Myc (A-14) polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-p53 (Ab-2) monoclonal antibody was purchased from Oncogene Science (Uniondale, NY). Horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG were from Amersham Pharmacia Biotech (Buckinghamshire, UK). Drug-induced cytotoxicity was assessed by (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay carried out in 96-well microtiter plates. The cells were treated with RA for different durations and concentrations, as described in the figure legends, and then exposed to the apoptotic drugs for additional 24 h. 3 h before the end of drug treatment, MTT was added to each well to a final concentration of 1 mg/ml and incubated at 37 °C. The reaction was terminated by removing the supernatant and the dye was dissolved by adding 100 μl Me2SO and 10 μl of Sorensen glycine buffer (0.1 m glycine, 0.1m NaCl, pH 10.5), followed by thorough mixing. In a parallel set of experiments, the cells were exposed to the indicated amounts of ionizing radiation at a dose rate of 1 Gy/min in a137Cs γ-irradiator and then incubated at 37 °C for 6 h before the MTT assay was performed. The plates were read at 562 nm on a Ceres 900 microtiter plate reader (Bio-tek Instruments Inc.). The means and standard deviations were derived from six replicates. Cells were grown on 60-mm tissue culture dishes (Nunc). After drug treatment, the cells were harvested, washed once with ice-cold PBS, resuspended in 250 μl of lysis buffer (10 mm Tris-HCl, pH 7.6, 20 mm EDTA, pH 8.0, 0.5% v/v Triton X-100) in a microcentrifuge tube, and the samples were incubated at room temperature for 15 min. After centrifugation at 16,000 × g for 5 min, the supernatant was transferred to another microcentrifuge tube and sequential extractions were carried out. The samples were extracted once with equal volume of phenol, once with phenol-chloroform (1:1), and once with chloroform-isoamyl alcohol (24:1). DNA was precipitated with 0.1 volume of 3 m sodium acetate, pH 4.8, and 2.5 volumes of ethanol at −20 °C overnight. The DNA was then pelleted at 16,000 × g for 30 min the next day. After washing once with 70% ethanol, the samples were digested with 0.1 μg of RNase A at 37 °C for 30 min before loading onto 1.5% agarose gels and the DNA was visualized by ethidium bromide staining. Cells were grown in 90-mm dishes, cultured, and treated as described. To prepare the whole cell lysates, the medium was removed and cells were washed twice with ice-cold Tris buffered saline (TBS) (150 mm NaCl, 10 mm Tris, pH 7.6) and lysed with 500 μl of lysis buffer (10 mm Tris, 150 mm NaCl, 1% Triton X-100, 5 mm EDTA, 25 mm β-glycerophosphate, 1 mm PMSF, 2 mm benzamidine, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mm sodium orthovanadate) for 15 min. The lysed cells were transferred into 1.5-ml microcentrifuge tubes, centrifuged at 15,000 × g for 10 min at 4 °C, and the supernatants were collected. To prepare the nuclear extracts, the cells were washed with cold PBS twice, and detached from plates by adding 1 ml of detachment buffer (150 mm NaCl, 1 mm EDTA, pH 8.0, 40 mm Tris, pH 7.6) for 5 min at room temperature. The cells were then transferred to microcentrifuge tubes and centrifuged at 300 × g for 4 min at 4 °C. The supernatant was discarded, and the pellet was resuspended in 400 μl of cold buffer A (10 mm Hepes, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 1 mm DTT, 0.5 mm PMSF, 2 mm benzamidine, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mm sodium orthovanadate) and incubated on ice for 15 min. 10 μl of 10% Nonidet P-40 was added, and the mixture was vortexed briefly. Nuclei were pelleted by centrifuging at 2800 × g for 4 min at 4 °C and then resuspended in 50 μl of ice-cold buffer B (20 mm Hepes, pH 7.9, 0.4 m NaCl, 1 mm EDTA, 1 mmDTT, 1 mm PMSF, 2 mm benzamidine, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mm sodium orthovanadate). The mixture was shaken vigorously for 15 min at 4 °C, centrifuged at 15,000 × g for 5 min, and the supernatant was collected. The protein concentration was determined by the Bradford assay (25Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217507) Google Scholar). Total cell lysates or nuclear extracts, volume adjusted according to the protein concentration, were resolved by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and the proteins were transferred to 0.2-μm pore size nitrocellulose membrane (Amersham Pharmacia Biotech). Filters were blocked with TBS containing 3% nonfat dry milk and 0.1% Tween 20 (TBST) for 3 h at room temperature and probed with primary antibodies diluted according to the supplier's instructions for 1 h in the above buffer. The filters were then washed three times with TBST containing 0.1% Tween 20, probed with a 1:10,000 dilution of horseradish peroxidase-conjugated anti-mouse IgG (Amersham Pharmacia Biotech), washed again in TBST, and developed using the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech). To ensure identical amounts of protein extracts from different conditions were loaded, a duplicate set of 10% SDS-PAGE gels was routinely prepared and the gel was subjected to Coomassie staining to allow a visual inspection of the overall protein staining patterns. Cells were seeded onto glass coverslips and treated with various reagents after which monolayers were washed twice with ice-cold PBS, pH 7.4. The immunofluorescence staining procedure has been described previously (26Yee K.S. Yu V.C. J. Biol. Chem. 1998; 273: 5366-5374Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Briefly, cells were fixed for 3 min at 4 °C with absolute methanol (−20 °C). The washing step with PBS was then repeated once. The cells were blocked at 37 °C for 30 min with 2% bovine serum albumin, 5% fetal bovine serum, 5% normal goat serum in PBS. The cells were incubated at room temperature for 45 min with the monoclonal anti-p53 (Ab-2) antibody in blocking buffer and washed with PBS. The cells were then incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Molecular Probes Inc., Eugene, OR). After washing, the coverslips were mounted in 80% glycerol in PBS containing 1 mg/mlp-phenylenediamine, and examined with a Zeiss Axioplan microscope. Cells were grown in 90-mm plates, treated with solvent or RA for 4 days and then subjected to 50 μm H-7. The cells were labeled by incubation for 3 h in methionine-free medium (Life Technologies, Inc.) supplemented with dialyzed fetal bovine serum and 25 μCi/ml [35S]methionine (NEN Life Science Products). After the labeling, the cells were washed three times with PBS before refeeding with fresh medium and then harvested 2, 4, 6, and 8 h later. The cells were lysed in lysis buffer (10 mm phosphate buffer, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.1% SDS, 1 mm PMSF, and 2 mg/ml aprotinin) and subjected to centrifugation at 140,000 ×g at 4 °C for 20 min. Radioactivities of the supernatants collected were determined by scintillation counting, and protein concentrations were determined. Immunoprecipitation of p53 protein was performed from samples containing equivalent amount of total cellular protein. The lysates were incubated with 1 μg of p53 monoclonal antibody, DO-1, at 4 °C for 1 h. The immune complexes were bound with protein A (4 °C, 1 h), followed by three washes with lysis buffer. Finally, samples were denatured by boiling in SDS-loading buffer (100 mm Tris, pH 6.8, 2% SDS, 0.1% bromphenol blue, 10% glycerol, 25 μm β-mercaptoethanol) and loaded onto a 10% SDS-polyacrylamide gel. Autoradiography was then performed on the dried gel. The intensities of the bands were quantitated with a phosphorimager using ImageQuantTM(Molecular Dynamics). To facilitate our investigation into the mechanism underlying the chemoresistance response in the neuroblastoma cells, SH-SY5Y, associated with RA treatment (24Lasorella A. Iavarone A. Israel M.A. Cancer Res. 1995; 55: 4711-4716PubMed Google Scholar), we evaluated the effect of RA on cellular levels of a panel of proteins known to have a role in various apoptosis pathways, with and without H-7 treatment. Many of these proteins are known activators or suppressors (e.g.Bcl-xL/S, Bcl-2, Bax, c-Myc, RB) of apoptosis, and their expressions in SH-SY5Y cells have been documented (13Ronca F. Chan S.L. Yu V.C. J. Biol. Chem. 1997; 272: 4252-4260Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 27Hanada M. Krajewski S. Tanaka S. Cazals-Hatem D. Spengler B.A. Ross R.A. Biedler J.L. Reed J.C. Cancer Res. 1993; 53: 4978-4986PubMed Google Scholar, 28Hammerling U. Bjelfman C. Pahlman S. Oncogene. 1987; 2: 73-77PubMed Google Scholar). Protein levels were evaluated by Western analyses. With the exception of Bcl-2 and p53, no changes were noted in levels of all the proteins tested in mock- or RA-treated cells, with or without H7 (Fig.1 A). Similar to the results obtained in the earlier study, the level of Bcl-2 protein was found to be up-regulated in RA-treated SH-SY5Y cells (24Lasorella A. Iavarone A. Israel M.A. Cancer Res. 1995; 55: 4711-4716PubMed Google Scholar) (Fig. 1 A). However, H-7 treatment did not affect the Bcl-2 level in either mock- or RA-treated cells (Fig. 1 A). Interestingly, while RA treatment did not seem to affect the level of nuclear p53 in these cells, it suppressed the H-7-mediated nuclear accumulation of p53 that was observed in solvent-treated (Mock) SH-SY5Y cells (Fig. 1 A). The effect of RA in preventing nuclear accumulation of p53 mediated by H-7 in SH-SY5Y cells was further documented by immunofluorescence staining of endogenous p53 protein in these cells subjected to various treatments. Diffuse staining of p53 in the cytosol of SH-SY5Y cells was apparent in untreated cells (Fig. 1 B). Upon stimulation by H-7, nuclear p53 signal was dramatically enhanced in the cells treated with solvent (Mock). However, in RA-treated cells, the strong nuclear staining was no longer observed upon H-7 treatment (Fig.1 B), supporting the conclusion drawn from Western analysis that RA treatment blocked H-7-mediated accumulation of nuclear p53 protein. The ability of RA to block nuclear p53 induction in SH-SY5Y cells was not restricted to the H-7 paradigm, as essentially identical results were obtained with etoposide treatment (Fig. 1 C), which is also an established paradigm for the induction of nuclear p53 (29Fritsche M. Haessler C. Brandner G. Oncogene. 1993; 8: 307-318PubMed Google Scholar). The proteins that we have examined in Western analysis represent only a fraction of all the possible proteins that might have a role in the apoptotic pathway affected by RA. In order to assess whether the up-regulation of Bcl-2 and the suppression of H-7-induced p53 nuclear accumulation by RA had any functional impact on the apoptotic response of SH-SY5Y cells, we compared the apoptotic responses of these cells upon induction by paradigms that are either p53-dependent or p53-independent. H-7, etoposide, and γ-irradiation have been regarded as p53-dependent apoptotic paradigms in many experimental systems (5Lowe S.W. Ruley H.E. Jacks T. Housman D.E. Cell. 1993; 74: 957-967Abstract Full Text PDF PubMed Scopus (2963) Google Scholar, 11Moll U.M. LaQuaglia M. Benard J. Riou G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4407-4411Crossref PubMed Scopus (391) Google Scholar, 12Moll U.M. Ostermeyer A.G. Haladay R. Winkfield B. Frazier M. Zambetti G. Mol. Cell. Biol. 1996; 16: 1126-1137Crossref PubMed Scopus (253) Google Scholar, 13Ronca F. Chan S.L. Yu V.C. J. Biol. Chem. 1997; 272: 4252-4260Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 30Lowe S.W. Schmitt E.M. Smith S.W. Osborne B.A. Jacks T. Nature. 1993; 362: 847-849Crossref PubMed Scopus (2767) Google Scholar), while staurosporine,N-acetyl-d-erythro-sphingosine, and menadione are believed to act through p53-independent pathways (31Bertrand R. Solary E. O'Connor P. Kohn K.W. Pommier Y. Exp. Cell. Res. 1994; 211: 314-321Crossref PubMed Scopus (471) Google Scholar, 32Santana P. Pena L.A. Haimovitz-Friedman A. Martin S. Green D. McLoughlin M. Cordon-Cardo C. Schuchman E.H. Fuks Z. Kolesnick R. Cell. 1996; 86: 189-199Abstract Full Text Full Text PDF PubMed Scopus (729) Google Scholar, 33Nishikawa Y. Carr B.I. Wang M. Kar S. Finn F. Dowd P. Zheng Z.B. Kerns J. Naganathan S. J. Biol. Chem. 1995; 270: 28304-28310Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Indeed, treatment of SH-SY5Y cells with staurosporine,N-acetyl-d-erythro-sphingosine, or menadione effectively resulted in apoptosis as demonstrated by DNA ladder assay (Fig. 2 A), and yet none of these paradigms induced nuclear accumulation of p53 under identical conditions (Fig. 2 B), confirming that their apoptotic effects are likely to involve p53-independent mechanisms. On the other hand, treatment of SH-SY5Y cells with H-7, etoposide, or γ-irradiation induced apoptosis as well as nuclear accumulation of p53 (Fig. 2, A and B). We next assessed the effect of RA pretreatment on the sensitivity of SH-SY5Y cells to these apoptotic stimuli. The cells were treated with either solvent or 10 μm RA for 4 days before exposure to the different apoptosis-inducing paradigms, and the cell viability was measured by MTT assay (Fig. 3). RA treatment did not alter the apoptotic response of the cells to staurosporine,N-acetyl-d-erythro-sphingosine, and menadione (Fig. 3, D–F). Interestingly, RA-treated cells were considerably more resistant to apoptosis induced by H-7, etoposide, or γ-irradiation (Fig. 3, A–C), as demonstrated by a shift of the dose-response curves to the right. These results support the idea that pretreatment of these cells with RA led to the development of resistance selectively to the p53-dependent apoptotic stimuli. In agreement with a previous study, the protein level of Bcl-2 was found to be up-regulated by 3–4-fold in SH-SY5Y cells upon RA treatment (24Lasorella A. Iavarone A. Israel M.A. Cancer Res. 1995; 55: 4711-4716PubMed Google Scholar). Since Bcl-2 has been reported to be an effective suppressor of apoptosis triggered by a variety of apoptotic stimuli (34Huang D.C. Cory S. Strasser A. Oncogene. 1997; 14: 405-414Crossref PubMed Scopus (231) Google Scholar, 35Cai J. Jones D.P. J. Biol. Chem. 1998; 273: 11401-11404Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar, 36Yoshimura S. Banno Y. Nakashima S. Takenaka K. Sakai H. Nishimura Y. Sakai N. Shimizu S. Eguchi Y. Tsujimoto Y. Nozawa Y. J. Biol. Chem. 1998; 273: 6921-6927Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 37Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar), it is therefore possible that the chemoresistance response observed in SH-SY5Y cells was a consequence of the elevated Bcl-2 level. The observation that RA-treated cells develops resistance selectively to only p53-dependent apoptosis argues against the elevated Bcl-2 levels to have a dominant role in conferring the RA-mediated resistance response in these cells. To clarify the issue further, we subjected SH-SY5Y cells to 12-O-tetradecanoyl-13-acetate (TPA) treatment, which is known to up-regulate Bcl-2 in SH-SY5Y cells (27Hanada M. Krajewski S. Tanaka S. Cazals-Hatem D. Spengler B.A. Ross R.A. Biedler J.L. Reed J.C. Cancer Res. 1993; 53: 4978-4986PubMed Google Scholar). Similar to RA, TPA treatment (16 nm × 5 days) resulted in 3–5-fold induction of the Bcl-2 protein (Fig.4 A). In contrast to RA-treated cells, TPA-treated cells remained sensitive to H-7 and exhibited nuclear accumulation of p53 (Fig. 4 B). Moreover, TPA was unable to confer resistance to the p53-dependent apoptotic response mediated by H-7. Despite a rise in Bcl-2 level, SH-SY5Y cells pre-exposed to TPA remained as sensitive as the solvent-treated cells (Mock) to H-7-mediated apoptosis, as evidenced by the DNA ladder assay (Fig. 4 C). The effect of RA in conferring resistance to p53-dependent apoptosis appeared to be time- and concentration-dependent. The effect was not detectable within the first 24 h of exposure (Fig.5 A). After 48 h of exposure to RA, resistance to p53-dependent apoptosis became apparent and the degree of resistance was enhanced in a time-dependent manner. The chemoresistance effect of RA was found to be dose-dependent as higher concentrations produced more pronounced effects (Fig. 5 A). RA at concentration above 10 μm was toxic to the cells and therefore was not used. In contrast to an earlier study, which showed that SH-SY5Y cells treated with RA at 10–100 nm for 4 days developed chemoresistance to adriamycin and cisplatin (24Lasorella A. Iavarone A. Israel M.A. Cancer Res. 1995; 55: 4711-4716PubMed Google Scholar), the resistance effect studied here appeared to require a relatively higher concentration of RA (1 μm). This raises the possibility that a metabolite of RA, rather than RA itself, was responsible for this effect. DNA fragmentation, a hallmark of late stage apoptosis, induced by H-7, was dramatically reduced only when the SH-SY5Y cells were pretreated with RA for more than 24 h (Fig. 5 B). These data provide evidence to support the hypothesis that the effect of RA on NB cells was to prevent death resulting from apoptosis. The loss of sensitivity of SH-SY5Y cells to p53-dependent apoptosis upon RA treatment may represent a gradual metabolic process, which may involve re-programming of complex signaling processes in the cells. We further investigated the effect of RA in preventing the induction of functional p53 and subsequent apoptosis mediated by H-7 in two additional NB cell lines. These two cell lines, LA-N-5 and IMR-32, have been previously demonstrated to be sensitive to the effect of H-7 in mediating nuclear accumulation of p53 and apoptosis (13Ronca F. Chan S.L. Yu V.C. J. Biol. Chem. 1997; 272: 4252-4260Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In our experiments, only LA-N-5 cells responded to RA in a similar manner as SH-SY5Y cells. LA-N-5 cells showed a reduction in sensitivity to H-7 in the induction of nuclear p53 as well as apoptosis upon treatment with 10 μm RA for 4 days (Fig.6 A). In contrast, RA pretreatment had no effect on H-7-induced nuclear p53 accumulation and also failed to confer resistance to apoptosis in IMR-32 cells (Fig.6 B). H-7 has been shown to elevate the steady-state level of p53 in NB cells by increasing the half-life of the protein (13Ronca F. Chan S.L. Yu V.C. J. Biol. Chem. 1997; 272: 4252-4260Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The effect may, in part, account for the nuclear accumulation of p53 in the cells upon exposure to this drug. It is conceivable that RA treatment in SH-SY5Y cells may affect the protein stabilizing effect of H-7 on p53 in these cells. We examined the amount of p53 protein in whole cell lysates and nuclear extracts of SH-SY5Y cells by immunoblotting before and after exposure to RA treatment. RA treatment did not significantly affect the low basal level of p53 detected in the whole cell lysate of SH-SY5Y cells (Fig. 7, A, lanes 1and 3, and B). Upon H-7 treatment, however, a marked increase in p53 level was detected in the whole cell extract of both mock and RA-treated cells (Fig. 7,A, lanes 2and 4, and B). Apparently, H-7 was still able to enhance the steady-state level of p53 in RA-treated cells to a similar extent as in the control cells (Fig. 7 B). The effect of H-7 on nuclear accumulation of p53, however, was essentially absent in RA-treated SH-SY5Y cells (Fig. 7, A,lane 8, and B). Indeed, pulse-chase experiments revealed that pre-exposure to 10 μm RA for 4 days had minimal effect on the H-7-induced stabilization of p53 protein in SH-SY5Y cells (Fig. 7 C), suggesting that the effect of RA on blocking the H-7-induced nuclear accumulation of p53 is independent from its effect on the stability of p53 in these cells. Therefore, RA may modulate the p53-dependent response in SH-SY5Y cells by inhibiting the nuclear localization process from taking place despite the high level of p53 in the cytoplasm induced by drug treatments. In this study, we investigated the role of the p53 pathway in the development of chemoresistance response to p53-dependent apoptosis in NB cells as a result of exposure to RA. The SH-SY5Y, LA-N-5, and IMR-32 NB cell lines are known to be sensitive to p53-dependent apoptosis pathway regulated by H-7 (13Ronca F. Chan S.L. Yu V.C. J. Biol. Chem. 1997; 272: 4252-4260Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). RA treatment effectively conferred resistance in SH-SY5Y and LA-N-5 but not IMR-32 cells to H-7-mediated apoptosis. The differential responses among NB cell lines to RA may be due to a variation of genetic backgrounds in NBs. RA-treated SH-SY5Y and LA-N-5 cells failed to accumulate nuclear p53 upon H-7 treatment, consistent with the suggestion that the p53 pathway might be involved in this effect. Interestingly, RA did not significantly affect the H-7-mediated up-regulation of the cytoplasmic pool of the wild-type p53 protein in SH-SY5Y cells, raising the possibility that the drug-induced nuclear accumulation of p53 is a separable process from enhanced protein stability. It is widely believed that restoring and/or enhancing wild-type p53 functions in tumor cells represent a promising treatment strategy for human cancers (38Lowe S.W. Curr. Opin. Oncol. 1995; 7: 547-553Crossref PubMed Scopus (199) Google Scholar). Unlike other tumors, NBs have an extremely low rate of mutation in their p53 gene (6Vogan K. Bernstein M. Leclerc J.M. Brisson L. Brossard J. Brodeur G.M. Pelletier J. Gros P. Cancer Res. 1993; 53 (1993): 5269-5273PubMed Google Scholar, 8Imamura J. Bartram C.R. Berthold F. Harms D. Nakamura H. Koeffler H.P. Cancer Res. 1993; 53: 4053-4058PubMed Google Scholar). Irradiation and DNA damaging agents are known to induce p53 expression in NB cells harboring the wild-type gene (12Moll U.M. Ostermeyer A.G. Haladay R. Winkfield B. Frazier M. Zambetti G. Mol. Cell. Biol. 1996; 16: 1126-1137Crossref PubMed Scopus (253) Google Scholar). Recently, we reported that the protein kinase inhibitor, H-7, has the ability to elevate the p53 level in the nucleus of NB cells, thus leading to apoptosis (13Ronca F. Chan S.L. Yu V.C. J. Biol. Chem. 1997; 272: 4252-4260Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). H-7 treatment is known to enhance the level of p53 in SH-SY5Y cells by increasing its stability (13Ronca F. Chan S.L. Yu V.C. J. Biol. Chem. 1997; 272: 4252-4260Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). As a result, a large increase in p53 protein level in both cytoplasmic and nuclear compartments was noted upon stimulation (12Moll U.M. Ostermeyer A.G. Haladay R. Winkfield B. Frazier M. Zambetti G. Mol. Cell. Biol. 1996; 16: 1126-1137Crossref PubMed Scopus (253) Google Scholar,13Ronca F. Chan S.L. Yu V.C. J. Biol. Chem. 1997; 272: 4252-4260Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). However, it is not clear whether the increase in p53 in the nucleus mediated by drug treatments is an active or passive process and whether it is separable from the effect of the increased stability of the protein. Indeed, Moll et al. (12Moll U.M. Ostermeyer A.G. Haladay R. Winkfield B. Frazier M. Zambetti G. Mol. Cell. Biol. 1996; 16: 1126-1137Crossref PubMed Scopus (253) Google Scholar) proposed that the cytoplasmic retention of p53 in NB cells is a saturable process and a sudden increase in p53 level can saturate the storage capacity of the cytoplasmic pool of p53 leading to nuclear accumulation of the protein. In this study, RA treatment did not substantially affect the H-7-induced enhancement of the p53 steady-state level but dramatically inhibited the nuclear accumulation of p53. This observation leads us to propose that the nuclear import of p53 is an independent and active process that can be selectively regulated by RA in a subset of NB cells that expresses wild-type p53 protein. While the loss of p53-dependent response has been associated with chemoresistance in certain tumor cells, the molecular basis of the resistance has primarily been associated with gene mutation and protein stability (5Lowe S.W. Ruley H.E. Jacks T. Housman D.E. Cell. 1993; 74: 957-967Abstract Full Text PDF PubMed Scopus (2963) Google Scholar, 39Shaulian E. Resnitzky D. Shifman O. Blandino G. Amsterdam A. Yayon A. Oren M. Oncogene. 1997; 15: 2717-2725Crossref PubMed Scopus (96) Google Scholar). Our current findings represent the first documentation that the process of nuclear import of wild-type p53 protein can be a target for modulating the chemoresistance response in some tumor cells. An earlier study has reported that RA treatment, at 10–100 nm, renders chemoresistance to adriamycin and cisplatin in SH-SY5Y cells (24Lasorella A. Iavarone A. Israel M.A. Cancer Res. 1995; 55: 4711-4716PubMed Google Scholar). Bcl-2 up-regulation upon RA treatment was proposed as a likely mechanism for the development of the chemoresistance (24Lasorella A. Iavarone A. Israel M.A. Cancer Res. 1995; 55: 4711-4716PubMed Google Scholar). In agreement with this report, we observed a 3–4-fold up-regulation of total Bcl-2 protein in SH-SY5Y cells upon RA (10 μm) treatment (Fig. 4). However, Bcl-2 has been shown to prevent apoptosis resulting from treatments with various apoptotic stimuli including sphingosine, staurosporine, and chemotherapeutic agents (34Huang D.C. Cory S. Strasser A. Oncogene. 1997; 14: 405-414Crossref PubMed Scopus (231) Google Scholar, 35Cai J. Jones D.P. J. Biol. Chem. 1998; 273: 11401-11404Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar, 36Yoshimura S. Banno Y. Nakashima S. Takenaka K. Sakai H. Nishimura Y. Sakai N. Shimizu S. Eguchi Y. Tsujimoto Y. Nozawa Y. J. Biol. Chem. 1998; 273: 6921-6927Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 37Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). Since RA treatment did not confer resistance to apoptotic stimuli rendered by sphingosine or staurosporine in our system, it is therefore questionable whether the mild up-regulation of Bcl-2 protein could have a major role in conferring selective resistance to the p53-dependent apoptosis. TPA is as effective as RA in up-regulating Bcl-2 in SH-SY5Y cells. However, only RA is able to confer resistance to p53-dependent apoptosis in SH-SY5Y cells. These data offer further evidence arguing against the up-regulation of Bcl-2 alone being responsible for the RA-mediated chemoresistance response studied here. However, it remains formally possible that Bcl-2 might have a role in collaborating with p53 or some other molecule in the apoptotic pathway to confer the resistance response seen in these cells. The network of signaling pathways influenced by RA is extremely complex and diverse (40Gudas L.J. Sporn M.B. Roberts A.B. Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry, and Medicine. 2nd Ed. Raven Press, New York1994: 443-520Google Scholar). Indeed, some synthetic retinoids, which have selective affinity to a particular RAR or RXR subtype, have been shown to promote different effects in NB cells in vitro (41Ponzoni M. Bocca P. Chiesa V. Decensi A. Pistoia V. Raffaghello L. Rozzo C. Montaldo P.G. Cancer Res. 1995; 55: 853-861PubMed Google Scholar, 42Hsu C.A. Rishi A.K. Su-Li X. Gerald T.M. Dawson M.I. Schiffer C. Reichert U. Shroot B. Poirer G.C. Fontana J.A. Blood. 1997; 89: 4470-4479Crossref PubMed Google Scholar). It is possible that the pleiotropic effect of RA on NB cells is mediated by a complex interplay of multiple retinoid signaling pathways. Additional studies involving receptor subtype-specific retinoid ligands are, therefore, necessary to further our understanding of the contribution of individual retinoid signaling pathways to the processes of differentiation, proliferation, apoptosis, and chemoresistance in NB cells. We are grateful to Drs. Wolfang Sádee and R. C. Seeger for human neuroblastoma cell lines. We thank Dr. Shing-Leng Chan and Karen M. L. Tan for critical reading of the manuscript." @default.
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