Molecular Cancer Therapeutics Funding AM No Date
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vaziri, S. A.J.
Right arrow Articles by Ganapathi, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vaziri, S. A.J.
Right arrow Articles by Ganapathi, R.
Mol Cancer Ther. 2005;4:1880-1890
© 2005 American Association for Cancer Research

Sensitization of DNA damage–induced apoptosis by the proteasome inhibitor PS-341 is p53 dependent and involves target proteins 14-3-3{sigma} and survivin

Susan A.J. Vaziri, Jason Hill, Kenichi Chikamori, Dale R. Grabowski, Nagio Takigawa, Mamta Chawla-Sarkar, Lisa R. Rybicki, Andrei V. Gudkov, Tarek Mekhail, Ronald M. Bukowski, Mahrukh K. Ganapathi and Ram Ganapathi

Experimental Therapeutics Program, Taussig Cancer Center, Cleveland Clinic Foundation, Cleveland, Ohio

Requests for reprints: Ram Ganapathi, Experimental Therapeutics Program, Taussig Cancer Center, Cleveland Clinic Foundation, R40, 9500 Euclid Avenue, Cleveland, OH 44195. Phone: 216-444-2085; Fax: 216-444-7115. E-mail: ganapar{at}ccf.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Proteasome inhibition following DNA damage results in the synergistic induction of apoptosis via a nuclear factor-{kappa}B–independent mechanism. In this study, we identify the role of p53 in mediating apoptosis by the sequence-specific treatment involving the DNA-damaging, topoisomerase I–targeting drug SN-38 followed by the proteasome inhibitor PS-341 (SN-38->PS-341). The p53-dependent sensitization of DNA damage–induced apoptosis by PS-341 is accompanied by persistent inhibition of proteasome activity and increased cytosolic accumulation of p53, including higher molecular weight forms likely representing ubiquitinated species. In contrast, pretreatment with PS-341 followed by treatment with SN-38 (PS-341->SN-38), which leads to an antagonistic interaction, results in transient inhibition of proteasome activity and accumulation of significantly lower levels of p53 localized primarily to the nucleus. Whereas cells treated with PS-341->SN-38 undergo G2 + M cell cycle arrest, cells treated with SN-38->PS-341 exhibit a decreased G2 + M block with a concomitant increase in the sub-G1 population. Decreased accumulation of cells in the G2 + M phase of the cell cycle in SN-38->PS-341–treated cells compared with PS-341->SN-38–treated cells correlates with enhanced apoptosis and reduced expression of two p53-modulated proteins, 14-3-3{sigma} and survivin, both of which play critical roles in regulating G2 + M progression and apoptosis. The functional role of 14-3-3{sigma} or survivin in regulating the divergent function of p53 in response to SN-38->PS-341 and PS-341->SN-38 treatment in inducing apoptosis versus G2 + M arrest/DNA repair, respectively, was confirmed by targeted down-regulation of these proteins. These results provide insights into the mechanisms by which inhibition of proteasome activity modulates DNA damage–induced apoptosis via a p53-dependent pathway. [Mol Cancer Ther 2005;4(12):1880–90]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cellular response mechanisms to stress or DNA damage are important for maintaining genomic stability and cellular integrity. Based on the type of injury, the cell either repairs the damaged DNA or initiates the cell death program. Thus, cancer therapy often relies on DNA-damaging strategies to initiate apoptosis. An important chemotherapeutic target is the nuclear enzyme topoisomerase, type I and II, which alters DNA topology for the efficient processing of genetic material, by creating a transient, cleavable, covalent DNA-topoisomerase linkage (1, 2). In the presence of DNA-damaging topoisomerase-targeting drugs, the transient cleavable DNA-topoisomerase linkage is trapped into a stable complex, which results in DNA damage and apoptosis (3, 4). However, development of resistance to these agents, which often limits their therapeutic potential, warrants alternate treatment strategies.

Among the different mechanisms involved in the physiologic response to DNA damage, the tumor suppressor protein p53 plays a pivotal role (5–7). The functional status of p53 or p53-mediated signaling pathways, which are frequently altered during tumorigenesis, often predicts success of chemotherapeutic strategies relying on DNA-damaging mechanisms. Under normal physiologic conditions, p53 is maintained at low steady-state levels by the p53-interacting protein MDM2, an E3 ubiquitin ligase, which ubiquitinates p53 and targets it for degradation by the 26S proteasome pathway (8–10). Following stress or DNA damage, p53 and MDM2 undergo different post-translational modifications, including phosphorylation, acetylation, ubiquitination, and sumoylation, which disrupt the interaction between these two proteins and lead to activation of p53 (8–12). Depending on the type of post-translational modification, p53 activates different signaling pathways that regulate transcription of distinct subsets of genes involved in cell cycle arrest, DNA repair, differentiation, or apoptosis (13–17).

Although induction of p21 has been shown to represent a key mechanism by which p53 arrests cells in the G1 and/or G2 phase of the cell cycle (18, 19), expression of yet another p53-modulated protein, 14-3-3{sigma}, is involved in G2 arrest and prevention of mitotic catastrophe (20, 21). However, p53-dependent regulation of apoptosis is poorly defined. p53 has been shown to target several genes involved in the mitochondrial apoptotic pathway (22, 23). These genes include the proapoptotic proteins Bax, PERP, NOXA, and PUMA (24–28). In addition to the proapoptotic genes, p53 represses transcription of survivin (29, 30), a member of the inhibitor of apoptosis family. Whereas overexpression of this gene has been shown to inhibit apoptosis, down-regulation of survivin in cells transfected with survivin mutant adenovirus or antisense oligonucleotides enhances apoptosis (31, 32). The antiapoptotic function of survivin has been shown to require phosphorylation of this protein at Thr34 by the mitotic kinase p34cdc2-cyclinB (33).

Because the 26S proteasome plays a major physiologic role in apoptosis by regulating the cellular level of p53 and other target proteins, including p21, p27, and nuclear factor-{kappa}B (NF-{kappa}B), manipulation of proteasomal activity has been employed to alter apoptosis (34–36). Inhibition of the proteasomal activity has been shown generally to promote apoptosis primarily due to prevention of proteasomal degradation of the NF-{kappa}B inhibitory protein, I{kappa}-B, which leads to attenuation of the transcriptional activity of NF-{kappa}B (35). In addition, proteasomal inhibitors sensitize cells to the apoptotic effect of topoisomerase I–targeting drugs by preventing degradation of ubiquitinated topoisomerase I (37, 38). Our previous studies evaluating the effect of the proteasome inhibitor, MG132, on apoptosis induced by the DNA-damaging topoisomerase-targeting agents, etoposide or SN-38, revealed that MG132 synergistically augments apoptosis via a NF-{kappa}B-independent signaling pathway (39). Enhancement of apoptosis was observed only when proteasomal inhibition followed DNA damage. Inhibition of proteasomal activity before DNA damage led to an antagonistic effect. In this study, we evaluated the mechanistic basis for the sequence-dependent effect of proteasomal inactivation on DNA damage–induced apoptosis. Our results show the requirement of p53 in induction of apoptosis by the proteasome inhibitor, PS-341, and more significantly in the potentiating effect on apoptosis following post-treatment with PS-341 after DNA damage induced by SN-38. The sensitization of DNA damage–induced apoptosis was correlated with prolonged inhibition of proteasomal activity, accumulation of p53 in the cytosol, reduced expression of 14-3-3{sigma}, and down-regulation of the antiapoptotic protein, survivin. The functional role of 14-3-3{sigma} and survivin in attenuating the apoptotic response was confirmed by studies showing enhanced apoptosis in cells deficient in these proteins or transfected with dominant-negative mutant survivin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and Antibodies
The topoisomerase I–targeting drug SN-38 (active metabolite of irinotecan) and the proteasome inhibitor bortezomib (PS-341) were obtained from Pharmacia & Upjohn Co. (Kalamazoo, MI) and Millenium Pharmaceuticals (Cambridge, MA), respectively. Cell culture medium and fetal bovine serum were obtained from BioWhittaker, Inc. (Gaithersburg, MD). Antibodies to p53 (DO-1), 14-3-3{sigma}, Bax, and survivin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The antibody to PUMA was a generous gift from Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD). The antibody to cleaved caspase-3 was purchased from Cell Signaling Technology (Beverly, MA).

Oligonucleotides and Expression Vectors
The small interfering RNA (siRNA) targeted to p53 and green fluorescent protein (GFP) was transcribed from a modified pBabe-puromycin retroviral vector described by Brummelkamp et al. (40). The 19-nucleotide p53 targeting sequence was 5'-GACTCCAGTGGTAATCTAC-3' and the GFP targeting sequence was 5'-GCAGCACGACTTCTTCAAG-3'. The siRNA sequence targeted to survivin (5'-GGCUGGGAGCCAGAUGACG-3') and the scrambled siRNA oligonucleotide (5'-GCGCGCTTTGTAGGATTCG-3') were synthesized by Dharmacon, Inc. (Lafayette, CO). The full-length wild-type survivin cDNA under the control of the cytomegalovirus promoter and cloned into the pcDNA3 expression vector was obtained from Science Reagents, Inc. (El Cajon, CA). This vector was used to prepare Thr34Ala (T34A) mutant survivin by site-directed mutagenesis with the QuikChange Site-Directed Mutagenesis kit (Stratagene, Inc., La Jolla, CA). The primers used for mutation were sense 5'-GCTGCGCCTGCGCCCCGGAGCGGATGG-3' and antisense 5'-CCATCCGCTCCGGGGCGCAGGCGCAGC-3'. The site-directed mutagenesis of T34A was confirmed by PCR and DNA sequence analysis.

Cell Culture and Transfections
HCT116 human colorectal carcinoma cell lines with different genetic backgrounds were obtained from Dr. Bert Vogelstein. The wild-type HCT116 cell line expresses wild-type p53 and 14-3-3{sigma} and is called HCT116 (p53+/+) or HCT116 (14-3-3{sigma}+/+), respectively. The two knockout cell lines used were HCT116 (p53–/–) and HCT116 (14-3-3{sigma}–/–), in which the p53 or 14-3-3{sigma} gene, respectively, was disrupted by homologous recombination (18, 21). These cells, which have a doubling time in vitro of ~22 hours, were maintained in McCoy's 5A medium supplemented with 10% fetal bovine serum and 2 mmol/L L-glutamine. Log-phase cultures of these cells were treated with PS-341 and/or SN-38. Treatment with drugs alone involved incubation with 1 µmol/L PS-341 for 30 minutes or 0.2 µmol/L SN-38 for 60 minutes. Combination treatment with the proteasome inhibitor and SN-38 involved either a pretreatment (PS-341->SN-38) or a post-treatment (SN-38->PS-341) with PS-341 for 30 minutes and 0.2 µmol/L SN-38 for 60 minutes. Following treatment, cells were washed and reincubated in drug-free medium for various periods before being harvested by trypsinization for analysis of apoptosis and/or expression of target proteins.

Stable integration of vectors containing target-specific sequences for transcription of p53 or GFP siRNAs in HCT116 (p53+/+) was carried out according to the procedure of Gurova et al. (41). Cells were subsequently selected for viral integration with 1 µg/mL puromycin for 1 week. Transfection of HCT116 (p53+/+) cells with mutant T34A survivin in pcDNA3 expression vector or the empty pcDNA3 expression vector was carried out using 4 µg DNA/5 x 105 cells and LipofectAMINE 2000. Stable transfectants were selected by culturing in 2 mg/mL G418. Transient transfection with survivin siRNA or scrambled siRNA (80 nmol/3 x 105 cells) was carried out for 4 hours in the presence of LipofectAMINE 2000.

Measurement of Apoptosis and Cell Cycle Traverse Perturbations
Wild-type or knockout HCT116 cells were treated with PS-341 and/or SN-38 as described above. Following 12 to 24 hours of incubation in drug-free medium, cells were washed, trypsinized, and stained with Hoechst 33342 and propidium iodide for quantifying apoptosis by fluorescent microscopy (39). Flow cytometry for cell cycle traverse perturbations was carried out following a 16-hour incubation of the cells in drug-free medium. Control and treated cells were stained with propidium iodide (39) and analyzed in a Coulter Epics XL-MCL cytometer (Beckman-Coulter, Miami, FL). Cell cycle phase compartment analysis was determined using Modfit LT3.0 software (Verity Software House, Topsham, ME).

Preparation of Cell Lysates and Western Blotting
Control or treated cells were trypsinized and lysed in the radioimmunoprecipitation assay buffer containing 20 mmol/L Tris-HCl (pH 8.0), 0.45 mol/L NaCl, 0.5% deoxycholate, 0.1% SDS, 1% NP40, 2 mmol/L EDTA, 0.5 mmol/L EGTA, and 10 mmol/L ß-mercaptoethanol plus protease and phosphatase inhibitors (39). Aliquots of cell lysates (10–40 µg) were electrophoresed on a 10% bis-Tris gel (Invitrogen Life Technologies, Carlsbad, CA) and subjected to Western blot analysis (39). The relative intensity of the signal was determined for the protein band of interest and normalized with the relative intensity of actin or tubulin.

Measurement of 20S Proteasome Activity
Extracts of control and treated cells were prepared in lysis buffer containing 10 mmol/L Tris, 150 mmol/L NaCl, 5 mmol/L EDTA, 2 mmol/L DTT, 1 mg/mL CHAPS, and 1 mmol/L phenylmethylsulfonyl fluoride (pH 8.0). Proteasome activity was determined using the 20S proteasome assay kit (BostonBiochem, Cambridge, MA), which employs the substrate succinyl-Leu-Leu-Val-Tyr-7 amino methyl coumarin. Fluorescence was determined in Wallac Victor 2 and the 20S proteasome activity was expressed as a percent of the control normalized for protein content.

Preparation of Cytosolic and Nuclear Fractions
Subcellular fractionation was done according to the procedure of Li et al. (42). Aliquots of the cytosolic and nuclear fractions corresponding to an equivalent amount of cells were electrophoresed and subjected to Western blot analysis as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Enhancement of SN-38-Induced Apoptosis by the Proteasome Inhibitor PS-341 Is p53 Dependent
We showed previously that potentiation of SN-38-induced apoptosis by post-treatment with the proteasome inhibitor, MG132, in NSCLC-3 or NSCLC-5 cells was NF-{kappa}B independent (39). To determine the molecular mechanisms involved in modulation of DNA damage–induced apoptosis by inhibition of proteasome activity, we examined the role of p53 in this process. For this study, we evaluated the effect of the combination of the proteasome inhibitor, PS-341, and SN-38 on apoptosis in p53-expressing HCT116 (p53+/+) and p53-null HCT116 (p53–/–) colon carcinoma cells. In these cells, PS-341 alone induced apoptosis in a dose-dependent (0.25–1 µmol/L) manner, whereas SN-38 (0.2–1 µmol/L) failed to induce apoptosis (data not shown). Induction of apoptosis by PS-341 alone was significantly higher in HCT116 (p53+/+) cells compared with HCT116 (p53–/–) cells (Fig. 1A ). Evaluation of the combination of PS-341 with SN-38 in HCT116 (p53+/+) cells revealed that, similar to our previous findings in NSCLC-3 cells (39), the apoptotic response was dependent on the sequence of PS-341 and SN-38 treatment (Fig. 1A). Based on criteria for determining interaction between biologically active agents (43), the results showed that SN-38->PS-341 treatment led to a synergistic response in HCT116 (p53+/+) cells. In contrast, similar analysis in PS-341->SN-38–treated cells revealed the combination to be antagonistic. However, in HCT116 (p53–/–) cells, the combination of PS-341 with SN-38 either pretreatment or post-treatment with PS-341 (PS-341->SN-38 or SN-38->PS-341) did not significantly alter apoptosis induced by individual agents. Enhanced apoptosis observed in HCT116 (p53+/+) cells treated with SN-38->PS-341 was correlated with increased expression of active cleaved caspase-3 (Fig. 1B). Thus, our data support a role for p53 in mediating the apoptotic response of HCT116 cells to PS-341 either alone or in combination with SN-38.



View larger version (25K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. p53-dependent induction of apoptosis following treatment with PS-341, SN-38, or the combination of PS-341 and SN-38. A, HCT116 (p53–/–) or HCT116 (p53+/+) cells were treated in the absence (Control) or presence of PS-341, SN-38, PS-341->SN-38, or SN-38->PS-341 as described in Materials and Methods. Apoptosis was determined following treatment and reincubation in drug-free medium for 16 h. B, HCT116 (p53+/+) cells were treated in the absence (Control) or presence of 1 µmol/L PS-341 (P), 0.2 µmol/L SN-38 (S), 1 µmol/L PS-341 followed by 0.2 µmol/L SN-38 (P->S), and 0.2 µmol/L SN-38 followed by 1 µmol/L PS-341 (S->P), and active caspase-3 was determined following treatment and reincubation in drug-free medium for 16 h by Western blot analysis using an antibody that recognizes the cleaved, active form of caspase-3. Determination of {alpha}-tubulin expression served as a control for equal loading. Consistent with the immunoblotting data, caspase-3 activity (based on cleavage of substrate DEVD-AFC) in the p53+/+ cells treated with PS-341 alone or post-treated with PS-341 was 1.5- to 2.3-fold higher compared with other treatments in p53+/+ cells. C, expression of p53 protein by Western blot analysis using p53-specific antibodies in cell extracts of wild-type HCT116 (p53+/+) cells or HCT116 (p53+/+) cells transfected with siRNA to GFP (si-GFP) or p53 (si-p53) incubated in the absence or presence of 0.25 µg/mL doxorubicin (DOX) for 24 h. Determination of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression served as a control for equal loading. D, HCT 116 (si-GFP) and HCT116 (si-p53) cells were treated in the absence or presence of PS-341, SN-38, PS-341->SN-38 or SN-38->PS-341 and apoptosis was determined as described above. Columns, mean of triplicate experiments; bars, SD. *, P ≤ 0.007, significantly different from HCT116 (p53–/–) or HCT116 (si-p53).

 
Because results with p53 knockout cells could be influenced by potential clonal variabilities that may arise during the selection of p53-null clones, the role of p53 in mediating apoptosis induced by SN-38->PS-341 treatment was tested in HCT116 (p53+/+) stably transfected with p53-directed siRNA oligonucleotide. Cells transfected with p53 siRNA expressed significantly lower levels of p53 (Fig. 1C) and were less susceptible to apoptosis following treatment with PS-341 alone or SN-38->PS-341 compared with HCT116 cells transfected with GFP siRNA (Fig. 1D). This finding further validates the role of p53 in mediating apoptosis by post-treatment with PS-341.

Treatment with PS-341 Alone or following SN-38-Induced DNA Damage Leads to Prolonged Inhibition of Proteasomal Activity and Stabilization of p53 Protein in HCT116 (p53+/+) Cells
A major mechanism regulating stability of p53 protein involves ubiquitination and proteasomal degradation. Therefore, we determined the effect of PS-341 alone or in combination (either pretreatment or post-treatment with PS-341) with SN-38 on the activity of 20S proteasome and p53 protein levels in HCT116 (p53–/–) and HCT116 (p53+/+) cells. Treatment of HCT116 (p53–/–) or HCT116 (p53+/+) cells with PS-341 or SN-38->PS-341 led to continuous inhibition (80%) of proteasomal activity up to 16 hours after treatment (Fig. 2 ). In contrast, only a transient inhibition in proteasomal activity at 3 hours after treatment followed by recovery to >70% of control by 16 hours after treatment was observed in PS-341->SN-38–treated cells (Fig. 2). Because treatment with PS-341, PS-341->SN-38, or SN-38->PS-341 led to similar effects on proteasomal activity in both HCT116 (p53–/–) and HCT116 (p53+/+) cells, these results suggest that the key proteasomal target(s) that is involved in enhancing apoptosis is p53 and/or a p53-regulated protein(s).



View larger version (34K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. Inhibition of proteasome activity by PS-341 alone or in combination with SN-38 in HCT116 (p53–/–) and HCT116 (p53+/+) cells. HCT116 (p53–/–) and HCT116 (p53+/+) cells were treated in the absence or presence of 1 µmol/L PS-341, 0.2 µmol/L SN-38, 1 µmol/L PS-341 followed by 0.2 µmol/L SN-38, and 0.2 µmol/L SN-38 followed by 1 µmol/L PS-341 as described in Materials and Methods. Proteasome activity was determined in cell extracts prepared following treatment and reincubation in drug-free medium for 3 or 16 h after treatment. Columns, mean of replicate experiments.

 
We next determined whether differential inhibition of proteasomal activity with the various treatments affected accumulation and/or subcellular distribution of p53 protein. Examination of p53 levels by Western blot analysis revealed that p53 was induced following treatment of HCT116 (p53+/+) cells with PS-341, SN-38, PS-341->SN-38, and SN-38->PS-341 (Fig. 3A ). However, maximal induction was observed at 16 hours after treatment following treatment with PS-341 alone or SN-38->PS-341. Longer exposure of the Western blot revealed the presence of higher molecular weight forms that were more abundant in cell extracts obtained from PS-341- or SN-38->PS-341–treated cells (Fig. 3B). Increased presence of the higher molecular weight forms of p53, which likely correspond to ubiquitinated p53, corroborates with persistent inhibition of proteasomal activity in cells treated with PS-341 or SN-38->PS-341. Because compartmentalization of p53 to different subcellular fractions is being recognized as an important mechanism regulating the pleiotropic functions of p53 (16, 17), we determined the relative amounts of p53 protein in the cytosolic and nuclear fractions at 3 and 16 hours after treatment (Fig. 3C). At 3 hours after treatment, p53 was equally distributed between cytosolic and nuclear fractions of cells treated with PS-341, SN-38, PS-341->SN-38, or SN-38->PS-341 (Fig. 3C). However, at 16 hours after treatment, p53 was significantly more abundant in the nuclear fraction compared with the cytosolic fraction in cells treated with SN-38 or PS-341->SN-38, treatments that do not alter apoptosis. In contrast, p53 was almost equally distributed in the cytosolic and nuclear fractions in cells treated with PS-341 or SN-38->PS-341. Comparison of the amount of p53 in the cytosolic fractions at 16 hours after treatment revealed that significantly higher levels of this protein were present in cells treated with PS-341 or SN-38->PS-341 compared with those treated with SN-38 or PS-341->SN-38. The preponderance of {alpha}-tubulin in the cytosolic fraction and of topoisomerase I in the nuclear fraction (Fig. 3D) ruled out cross-contamination between these two subcellular fractions for the observed differences in p53 distribution in Fig. 3C. This result implies that extranuclear p53 may be important for the apoptotic function of this protein. However, increased level of cytosolic p53 does not seem to be the sole determinant of apoptosis, because the levels of total and cytosolic p53 in cells treated with PS-341 alone or SN-38->PS-341 were comparable despite differences in the degree of apoptosis induced by these treatments. Thus, additional signaling mechanisms activated by the DNA damage pathway are required for inducing maximal apoptosis.



View larger version (20K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Induction and subcellular distribution of p53 protein in HCT116 (p53+/+) cells treated with PS-341, SN-38, or the combination of PS-341 and SN-38. HCT116 (p53+/+) cells were treated in the absence or presence of 1 µmol/L PS-341, 0.2 µmol/L SN-38, 1 µmol/L PS-341 followed by 0.2 µmol/L SN-38, and 0.2 µmol/L SN-38 followed by 1 µmol/L PS-341. Following incubation of control and treated cells in drug-free medium, cell extracts were prepared for determining expression of p53 by Western blot analysis using antibodies specific for p53. Determination of actin expression served as a control for equal loading. A and B, total cell lysates prepared in radioimmunoprecipitation assay buffer at 16 h after treatment: shorter exposure of the blot (A) and longer exposure of the same blot to visualize the higher molecular weight p53 species (B). C, cytosolic (c) and nuclear (n) fractions prepared at 3 or 16 h after treatment. An aliquot of the cytosolic fraction and nuclear fraction corresponding to an equivalent amount of cells was analyzed. D, the purity of the cytosolic and nuclear fractions was determined based on the distribution of the cytosolic and nuclear proteins, tubulin and topoisomerase I (topo I), respectively.

 
Enhancement of DNA Damage–Induced p53-Dependent Apoptosis by PS-341 Does Not Require Arrest of Cells in the G2 + M Phase of the Cell Cycle
Evaluation of cell cycle perturbations in HCT-116 (p53+/+) cells revealed significant differences in the cell cycle distribution profiles of cells treated with various combinations of PS-341 and SN-38 (Fig. 4 ). Although all four treatment protocols led to some degree of accumulation of cells in the G2 + M phase of the cell cycle, maximal G2 + M arrest was observed in cells treated with PS-341->SN-38. In cells treated with SN-38->PS-341, a significant sub-G1 population, representative of apoptotic cells, was observed. However, in p53-null cells treated with SN-38->PS-341, no sub-G1 peak was seen; instead, a significant G2 + M fraction comparable with that observed in HCT-116 (p53+/+) or HCT-116 (p53–/–) cells treated with PS-341->SN-38 was observed (data not shown). These findings suggests that post-treatment with PS-341 preferentially activates the apoptotic function of p53 in favor of cell cycle arrest in the G2 + M phase, whereas pretreatment with PS-341 leads to G2 arrest that allows for repair of damaged DNA.



View larger version (13K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. Cell cycle traverse analysis of HCT-116 (p53+/+) cells treated with PS-341, SN-38, or the combination of SN-38 and PS-341. HCT-116 (p53+/+) cells were treated in the absence or presence of PS-341, SN-38, PS-341->SN-38, or SN-38->PS-341. Following incubation of the cells in drug-free medium for 16 h, cells were washed and stained with propidium iodide for flow cytometric analysis. Results from a representative experiment show the cell cycle phase distribution profile and the relative percent of cells in the sub-G1 fraction.

 
Reduced Expression of 14-3-3{sigma} Correlates with Decreased Accumulation of Cells in the G2 + M Phase of the Cell Cycle and Induction of Apoptosis in HCT116 (p53+/+) Cells Treated with SN-38->PS-341
The differential cell cycle distribution profiles observed in HCT116 (p53+/+) cells treated with various combinations of PS-341 and/or SN-38 prompted us to evaluate whether the p53-regulated protein, 14-3-3{sigma}, which is involved in preventing G2 + M progression and mitotic catastrophe, plays a role in mediating SN-38->PS-341–induced apoptosis. We first determined whether expression of 14-3-3{sigma} was altered in HCT116 (p53+/+) cells treated with PS-341 and/or SN-38. Although 14-3-3{sigma} was induced in HCT116 (p53+/+) cells treated with SN-38 alone or PS-341->SN-38 (Fig. 5A ), treatment with SN-38->PS-341 did not markedly alter the levels of 14-3-3{sigma} compared with the untreated control cells. In HCT116 (p53–/–) cells, no change in 14-3-3{sigma} levels was observed (data not shown). Because these data did not provide a clear correlation of 14-3-3{sigma} expression, apoptosis, and accumulation of cells in G2 + M phase, we subsequently sought to determine whether 14-3-3{sigma} is important for mediating the growth arrest/DNA repair function of p53 in response to DNA damage by comparing apoptosis induced by PS-341, SN-38, PS-341->SN-38, or SN-38->PS-341 in HCT116 (14-3-3{sigma}+/+) and HCT116 (14-3-3{sigma}–/–) cells. Our results show that apoptosis induced by PS-341, SN-38, PS-341->SN-38, or SN-38->PS-341 is significantly higher in 14-3-3{sigma}-null cells compared with 14-3-3{sigma} wild-type cells (Fig. 5B). In 14-3-3{sigma}-null cells, apoptosis is rapidly induced and can be observed as early as 12 hours after treatment. Because HCT116 (14-3-3{sigma}–/–) cells treated with SN-38->PS-341 undergo massive apoptosis, very few cells with intact cellular morphology are observed at 16 hours after treatment. The apoptosis induced by PS-341->SN-38 in HCT116 (14-3-3{sigma}–/–) cells is likely due to reduced accumulation of cells in the G2 + M phase of the cell cycle compared with HCT116 (14-3-3{sigma}+/+) cells (Fig. 5C). The reduced accumulation of cells in the G2 + M phase was accompanied by an increase in the sub-G1 population, indicative of apoptosis. No remarkable differences in cell cycle distribution profile were observed in HCT116 (14-3-3{sigma}–/–) and HCT116 (14-3-3{sigma}+/+) cells treated with SN-38.



View larger version (20K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. 14-3-3{sigma}-dependent response to treatment with PS-341, SN-38, or the combination of PS-341 and SN-38. HCT116 (p53+/+), HCT116 (14-3-3{sigma}–/–), or HCT116 (14-3-3{sigma}+/+) cells were treated in the absence or presence of 1 µmol/L PS-341 alone, 0.2 µmol/L SN-38 alone, 1 µmol/L PS-341 followed by 0.2 µmol/L SN-38, and 0.2 µmol/L SN-38 followed by 1 µmol/L PS-341 and incubated in drug-free medium for 12 to 16 h. A, Western blot analysis of 14-3-3{sigma} and actin expression in HCT116 (p53+/+) cell lysates prepared in radioimmunoprecipitation assay buffer at 16 h after treatment. B, apoptosis at 12 h after treatment. Columns, mean of triplicate experiments; bars, SD. C, cell cycle traverse perturbations analyzed by flow cytometry at 16 h after treatment. Results from a representative experiment show the cell cycle phase distribution profile and the relative percent of cells in the sub-G1 fraction.

 
Potentiation of DNA Damage Induced Apoptosis by Posttreatment with PS-341 Involves Down Regulation of Survivin
To identify downstream mediators of p53-dependent apoptosis induced by SN-38->PS-341 treatment, we examined changes in the levels of two p53-regulated antiapoptotic proteins, Bax and PUMA, which are involved in activating the caspase cascade. Western blot analysis carried out on cell extracts prepared 16 hours after treatment with PS-341, SN-38, PS-341->SN-38, and SN-38->PS-341 revealed no changes in expression of Bax protein (data not shown). Induction of PUMA was observed following all different treatment protocols, but no difference in the expression that correlated with apoptosis was seen between the various treatments (data not shown). These data suggest that up-regulation of Bax or PUMA is possibly not involved in p53-dependent apoptosis induced by SN-38->PS-341. However, we cannot rule out the involvement of these proteins in apoptosis via an oligomerization or mitochondrial translocation mechanism.

Our previous data with MG-132 (39) suggested that the effect of proteasome inhibition on enhancing DNA damage–induced apoptosis was NF-{kappa}B independent. Because most inhibitors of apoptosis proteins, with the possible exception of survivin (33, 44), are NF-{kappa}B regulated, we focused our efforts on this antiapoptotic protein, especially because this protein is reported to be negatively regulated by wild-type p53 (29, 30). To evaluate the role of survivin in the apoptotic response of proteasome inhibitors, we first examined changes in the level of survivin protein in HCT116 (p53–/–) and HCT116 (p53+/+) cells treated with PS-341, SN-38, PS-341->SN-38, or SN-38->PS-341. Western blot analysis with antibodies directed against survivin protein showed that treatment with only SN-38->PS-341, but not PS-341, SN-38, or PS-341->SN-38, led to down-regulation of survivin protein levels in HCT116 (p53+/+) cells (Fig. 6A ). The decrease in survivin protein levels observed in HCT116 (p53+/+) cells was not seen in HCT116 (p53–/–) cells (Fig. 6A), suggesting that down-regulation of survivin protein is likely dependent on p53. Thus, these data show a correlation between p53-dependent potentiation of DNA damage–induced apoptosis by PS-341 and down-regulation of survivin. To evaluate the functional role of survivin in apoptosis induced by SN-38->PS-341 treatment, we used two different strategies. In the first strategy, the functional activity of survivin was attenuated by transfecting HCT116 (p53+/+) cells with dominant-negative mutant T34A survivin. In these cells, survivin cannot be phosphorylated at amino acid residue 34, which is required for the antiapoptotic activity. In the second strategy, survivin protein was down-regulated with survivin siRNA. Figure 6B shows the apoptotic response of T34A survivin mutant transfected HCT116 (p53+/+) cells to PS-341, SN-38, PS-341->SN-38, and SN-38->PS-341. A modest but significant increase (P < 0.001) in apoptosis was observed in HCT116/T34A survivin cells compared with HCT116/neo cells, when these cells were treated with PS-341 or SN-38->PS-341. Some increase was also seen with PS-341->SN-38. This is not surprising, because the dominant-negative mutant survivin protein is thought to decrease phosphorylation and stability of endogenous wild-type protein (44).



View larger version (26K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. Survivin-dependent response to treatment with PS-341, SN-38, or the combination of PS-341 and SN-38. A, expression of survivin in HCT116 (p53+/+) cells treated in the absence (C) or presence of 1 µmol/L PS-341 alone (P), 0.2 µmol/L SN-38 alone (S), 1 µmol/L PS-341 followed by 0.2 µmol/L SN-38 (P->S), and 0.2 µmol/L SN-38 followed by 1 µmol/L PS-341 (S->P). Following incubation of the cells in drug-free medium for 16 h, cell extracts were prepared in radioimmunoprecipitation assay buffer and analyzed for survivin expression by Western blot analysis using specific antibodies. Determination of actin expression served as a control for equal loading. B, determination of apoptosis in HCT116 (p53+/+) cells transfected with the dominant-negative survivin mutant T34A. Apoptosis was determined following treatment without (Control) or with 1 µmol/L PS-341 alone (P), 0.2 µmol/L SN-38 alone (S), 1 µmol/L PS-341 followed by 0.2 µmol/L SN-38 (P->S), and 0.2 µmol/L SN-38 followed by 1 µmol/L PS-341 (S->P) as described in Materials and Methods. C, down-regulation of survivin protein in HCT116 (p53–/–) and HCT116 (p53+/+) cells transfected with survivin siRNA. Western blot analysis of survivin and actin expression in cell extracts of HCT116 (p53–/–) and HCT116 (p53+/+) cells transfected with LipofectAMINE 2000, scrambled siRNA, or survivin siRNA. D, effect of targeted down-regulation with survivin siRNA on the induction of apoptosis in HCT116 (p53–/–) and HCT116 (p53+/+) cells treated with PS-341, SN-38, or the combination of PS-341 and SN-38. HCT116 (p53–/–) and HCT116 (p53+/+) cells transfected with LipofectAMINE 2000, scrambled siRNA, or survivin siRNA were treated 24 h later with drug diluent (Control), 1 µmol/L PS-341 alone (P), 0.2 µmol/L SN-38 alone (S), 1 µmol/L PS-341 followed by 0.2 µmol/L SN-38 (P->S), and 0.2 µmol/L SN-38 followed by 1 µmol/L PS-341 (S->P). Following incubation of the treated cells in drug-free medium for 16 h, apoptosis was determined as described in Materials and Methods. Columns, mean of triplicate experiments; bars, SD.

 
For the second strategy, HCT116 (p53–/–) and HCT116 (p53+/+) cells were transfected with LipofectAMINE 2000 alone, scrambled siRNA, or survivin siRNA and the relative amounts of survivin in each of these cells were first determined. The results in Fig. 6C show that survivin siRNA, but not scrambled siRNA, oligonucleotide down-regulates survivin protein expression in HCT116 (p53–/–) and HCT116 (p53+/+) cells. Determination of apoptosis in transfected HCT116 (p53–/–) cells treated with PS-341, SN-38, PS-341->SN-38, or SN-38->PS-341 revealed no differences in the apoptotic response between cells transfected with survivin siRNA or scrambled siRNA (Fig. 6D). In contrast, apoptosis was significantly (P ≤ 0.002) enhanced in HCT116 (p53+/+) cells transfected with survivin siRNA compared with cells transfected with scrambled siRNA (Fig. 6D). However, maximal apoptosis was observed in HCT116 (p53+/+) cells treated with SN-38->PS-341. These results complement the observations in HCT116 (p53+/+) cells transfected with dominant-negative T34A mutant survivin and support the functional role of survivin in p53-dependent apoptosis. The increase in apoptosis observed in HCT116 (p53+/+) cells for all different treatments, including control cells, indicates that decreased levels of survivin protein are important for inducing p53-dependent apoptosis. Our earlier observation that treatment of HCT116 (p53+/+) cells with only SN-38->PS-341, but not PS-341, SN-38, or PS-341->SN-38, leads to down-regulation of survivin protein implies a functional role for survivin in mediating SN-38->PS-341–induced p53-dependent apoptosis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recent clinical studies have reported the chemotherapeutic potential of proteasome inhibitors alone or in combination with other antitumor agents (e.g., CPT-11 or docetaxel) for the treatment of solid tumors. Although it is generally presumed that the therapeutic efficacy of proteasome inhibitors involves inhibition of the antiapoptotic activity of NF-{kappa}B, our earlier study (39) in non–small cell lung carcinoma cell lines, NSCLC-3 or NSCLC-5, showed that potentiation of DNA damage–induced apoptosis by proteasome inhibitors was NF-{kappa}B independent and required post-treatment with the proteasome inhibitor. To identify the mechanism for the sequence-dependent synergistic interaction of proteasome inhibitors and DNA-damaging agents, in this study, we evaluated the role of p53, a key intermediate in the DNA damage response pathway, which is regulated by the 26S proteasome ubiquitin degradation pathway. Our results show that p53 is required for sensitization of DNA damage–induced apoptosis due to proteasome inactivation, because cells that are null for p53 do not exhibit enhanced apoptosis in the presence of proteasome inhibitors. Interestingly, sensitization of DNA damage–induced apoptosis by proteasome inhibitors occurs only when the proteasomal activity is repressed after induction of DNA damage by the topoisomerase I–targeting drug, SN-38. Inhibition of proteasomal activity before DNA damage does not alter the apoptotic response; rather, it leads to an antagonistic effect. The recovery of proteasomal activity when PS-341 treatment precedes exposure to SN-38 may be due to the antagonistic effect of the subsequent DNA damage. In contrast, post-treatment with PS-341 (SN-38->PS-341) or PS-341 alone [no subsequent drug treatment-induced effects (e.g., DNA damage) that could affect proteasome function] maintains the inhibitory effects of PS-341 on proteasome activity. The p53-dependent apoptotic response corresponds, at least in part, to induction of different molecular weight forms of p53 and to altered subcellular distribution of these species between the cytosol and the nucleus. Further, we show that suppression of the downstream p53 target proteins, 14-3-3{sigma}, and the antiapoptotic protein survivin is required for enhancing apoptosis.

The 26S proteasome, which plays an important role in regulating the cellular levels of key intermediates of several signal transduction pathways, is critical for modulating the intracellular levels of the multifunctional protein, p53. In our studies evaluating the effect of the proteasome inhibitor, PS-341, on DNA damage–induced apoptosis by SN-38, we found that both agents, either alone or in combination, led to induction of p53. However, treatment with PS-341 alone and post-treatment with PS-341 after SN-38-induced DNA damage led to maximal induction of p53, including higher molecular weight, potentially ubiquitinated species of this protein. Increased accumulation of p53 observed at 16 hours after treatment was likely due to prolonged inhibition of proteasomal activity. The transient inhibition of proteasomal activity (~3 hours after treatment) observed in cells pretreated with PS-341 before treatment with SN-38 probably resulted in degradation of p53 in these cells.

Coincident with differences in the level of p53 at 16 hours, the subcellular distribution of this protein also varied. In cells treated with PS-341 alone or post-treated with PS-341 following treatment with SN-38, a significant amount of p53 was present in the cytosolic fraction compared with that present in cells treated with SN-38 alone or pretreated with PS-341 before SN-38. In contrast, no difference in subcellular distribution of p53 was observed at 3 hours after treatment. This finding suggests that, in cells pretreated with PS-341 followed by treatment with SN-38, p53 may either be preferentially degraded in the cytosol due to recovery of proteasomal activity in that compartment or be shuttled from the cytosol to the nucleus. Increased presence of cytosolic p53 in cells post-treated with PS-341 after SN-38 implicates the importance of extranuclear p53 in the apoptotic response. This finding corroborates with recent studies establishing a role for mitochondrial p53 in apoptosis (17, 18).

The functional role of p53 in response to DNA damage is to invoke either cell cycle arrest or apoptosis. Whereas cell cycle arrest would allow for repair of DNA damage, apoptosis will lead to cell death for removal of cells with damaged DNA. Based on the flow cytometry data analyzing cell cycle perturbations induced by DNA damage and proteasome inactivation, distinct differences were, however, apparent when proteasome inhibition either preceded or followed DNA damage induced by SN-38. Specifically, the cell cycle distribution with arrest in the G2 + M fraction was similar for p53-null and p53 wild-type cells pretreated with PS-341 followed by SN-38. However, cells expressing wild-type p53 post-treated with PS-341 following DNA damage exhibited a 2-fold reduction in the G2 + M fraction. The reduced accumulation in the G2 + M fraction for cells post-treated with PS-341 following DNA damage was accompanied by the presence of cells with sub-G1 DNA content, indicative of apoptosis. Thus, targeting the 26S proteasomal pathway before or following DNA damage leads to activation of distinct functions of p53. Post-treatment with PS-341 preferentially activates the apoptotic function of p53, in favor of cell cycle arrest, whereas pretreatment with PS-341 stimulates the cell cycle arrest and DNA repair functions of p53.

Because the two p53-regulated genes p21 and 14-3-3{sigma} are involved in the growth arrest and checkpoint control function of p53, we evaluated their role in the cellular response to PS-341 and/or SN-38. Although induction of p21 protein paralleled induction of p53, the presence of this protein did not influence the cellular responses to PS-341 and/or SN-38, because no differences in cell cycle traverse perturbations or apoptosis were observed in p21–/– and p21+/+ HCT116 (p53+/+) cells (data not shown). These results with p21–/– and p21+/+ HCT116 (p53+/+) cells differ from that reported by Yu et al. (45) and could be related to continuous treatment for 48 hours with PS-341 compared with the 30-minute exposure used in the present study. In contrast to the results with p21, expression of the 14-3-3{sigma} protein varied reciprocally with induction of p53 following treatment with PS-341 and/or SN-38. The 14-3-3{sigma} protein was significantly induced in cells treated with the DNA-damaging agent, SN-38, alone or pretreated with PS-341 followed by SN-38. Thus, increased expression of 14-3-3{sigma} in cells pretreated with PS-341 followed by treatment with SN-38 results in inhibition of G2 progression, which would be expected to facilitate DNA repair. On the contrary, reduced expression of this protein in cells post-treated with PS-341 following treatment with SN-38 could potentially prevent G2 arrest and impair checkpoint control mechanisms, which would facilitate enhanced cell death due to inadequate repair of the damaged DNA. Indeed, deletion of the 14-3-3{sigma} gene in HCT116 (p53+/+) cells by homologous recombination significantly enhances apoptosis and decreases the G2 block. The rapid onset and significantly higher magnitude of apoptosis induced by PS-341 or by post-treatment with PS-341 following DNA damage in 14-3-3{sigma}-null cells compared with wild-type cells suggest that the presence of even small amounts of 14-3-3{sigma} in wild-type cells delays exit of the damaged cells from G2. Our data corroborate with published results showing a role for 14-3-3{sigma} in prevention of mitotic catastrophe after DNA damage (21) and those correlating decreased expression of some isoforms of 14-3-3 with increased sensitivity of human lung cancer cells to ionizing radiation (46).

It is interesting to note that the apoptotic response to PS-341 alone or post-treatment with PS-341 following SN-38-induced DNA damage differs despite similar changes in p53 downstream events. The significant induction of p53-dependent apoptosis only in cells post-treated with PS-341 following SN-38 suggests that, although the changes induced by proteasomal inhibition on subcellular distribution of p53 are important for apoptosis, maximal induction requires, in addition, activation of the DNA damage signal transduction pathway. Cooperation of these two pathways likely occurs at some downstream step that leads to maximal induction of apoptosis.

The apoptotic function of p53 has been shown to involve transcriptional activation or repression of several genes involved in apoptosis. p53 activates several proapoptotic genes, including Bax, PUMA, and NOXA, and down-regulates the antiapoptotic protein survivin. Although no differences were observed for the induction of Bax and PUMA with SN-38 and/or PS-341 treatment, down-regulation of survivin was seen only in cells post-treated with PS-341 following treatment with SN-38. This finding implicates an essential role for down-regulation of survivin in apoptosis induced by post-treatment with PS-341. Because down-regulation of survivin is observed only with PS-341 post-treatment, which leads to significant apoptosis, this event might represent one of the distal downstream steps that is modulated just before activation of the caspase cascade. Indeed, attenuation of survivin activity or down-regulation of this protein has been shown to activate caspase-9 (44), which in turn activates caspase-3, the universal effector of apoptosis. In our studies, we have observed maximal activation of caspase-3 in cells post-treated with PS-341 after SN-38-induced DNA damage. In addition, down-regulation of survivin may also disrupt the spindle formation and spindle checkpoint functions of the cells, resulting in the activation of the apoptotic pathway.

The functional significance of down-regulation of survivin, which exerts its antiapoptotic effects by inhibiting caspase-9 activity following phosphorylation on Thr34 (44), was confirmed by our data showing significant enhancement of apoptosis in HCT116 (p53+/+) cells transfected with dominant-negative mutant (T34A) survivin or survivin siRNA. In HCT116 (p53–/–) cells transfected with survivin siRNA, none of the treatments led to any significant increases in apoptosis. The observation that decreasing survivin levels using a siRNA strategy in HCT116 (p53–/–) cells did not promote apoptosis with PS-341 and/or SN-38 suggests that additional signals activated by p53 are also required to stimulate apoptosis.

DNA-damaging strategies to induce apoptosis and cell death are widely employed for the treatment of different cancers. However, a major obstacle to these treatment strategies is the development of resistance. In this regard, modulation of apoptosis induced by DNA-damaging agents, by proteasomal inhibitors, provides an alternative approach to treat chemoresistant tumors. The newer insights into the mechanisms regulating the differential modulation of DNA damage–induced apoptosis based on pretreatment or post-treatment with PS-341 to inactivate proteasomes are important in the design of clinical trials using the sequential combination of PS-341 and a DNA-damaging agent in drug-resistant tumors.


   Acknowledgments
 
We thank Dr. Bert Vogelstein for generously providing the wild-type and knockout HCT116 cells as well as key reagents and Terri O'Brian (Art Medical Illustrations and Photography Department) for skillful preparation of the artwork.


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

Received 7/ 5/05; revised 9/30/05; accepted 10/19/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wang JC. Cellular roles of DNA topoisomerases: a molecular perspective. Nat Rev Mol Cell Biol 2002;3:430–40.[CrossRef][Medline]
  2. Kellner U, Sehested M, Jensen PB, Giesler F, Rudolph P. Culprit and victim—DNA topoisomerase II. Lancet Oncol 2002;3:235–43.[CrossRef][Medline]
  3. Froelich-Ammon SJ, Osheroff N. Topoisomerase poisons: harnessing the dark side of enzyme mechanism. J Biol Chem 1995;270:21429–32.[Free Full Text]
  4. Li T-K, Liu LF. Tumor cell death induced by topoisomerase-targeting drugs. Annu Rev Pharmacol Toxicol 2001;41:53–77.[CrossRef][Medline]
  5. Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature 2000;408:307–10.[CrossRef][Medline]
  6. Balint E, Vousden K. Activation and activities of the p53 tumour suppressor protein. Br J Cancer 2001;85:1813–23.[CrossRef][Medline]
  7. Sax JK, El-Diery WS. p53 downstream targets and chemosensitivity. Cell Death Differ 2003;10:413–7.[CrossRef][Medline]
  8. Alarcon-Vargas D, Ronai Z. p53-Mdm2—the affair that never ends. Carcinogenesis. 2002;23:541–7.[Abstract/Free Full Text]
  9. Woods DB, Vousden K. Regulation of p53 function. Exp Cell Res 2001;264:56–66.[CrossRef][Medline]
  10. Haupt S, Louria-Hayon I, Haupt Y. p53 licensed to kill? Operating the assassin. J Cell Biochem 2003;88:76–82.[CrossRef][Medline]
  11. Brooks CL, Gu W. Ubiquitination, phosphorylation and acetylation: the molecular basis for p53 regulation. Curr Opin Cell Biol 2003;15:164–71.[CrossRef][Medline]
  12. Xu Y. Regulation of p53 responses by post-translational modifications. Cell Death Differ 2003;10:400–3.[CrossRef][Medline]
  13. Leng RP, Lin Y, Ma W, et al. Pirh2, a p53-induced ubiquitin-protein ligase, promotes p53 degradation. Cell 2003;112:779–91.[CrossRef][Medline]
  14. Sakaguchi K, Herrera JE, Saito S, et al. DNA damage activates p53 through a phosphorylation-acetylation cascade. Genes Dev 1998;12:2831–41.[Abstract/Free Full Text]
  15. Appella E, Anderson CW. Post-translational modifications and activation of p53 by genotoxic stresses. Eur J Biochem 2001;268:2764–72.[Medline]
  16. Marchenko ND, Zaika A, Moll UM. Death signal-induced localization of p53 protein to mitochondria. A potential role in apoptotic signaling. J Biol Chem 2000;275:16202–12.[Abstract/Free Full Text]
  17. Mihara M, Erster S, Zaika A, et al. p53 has a direct apoptogenic role at the mitochondria. Mol Cell 2003;11:577–90.[CrossRef][Medline]
  18. Bunz F, Dutriaux A, Lengauer C, et al. Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 1998;282:1497–501.[Abstract/Free Full Text]
  19. El-Diery WS. Regulation of p53 downstream genes. Semin Cancer Biol 1998;8:345–57.[CrossRef][Medline]
  20. Hermeking H, Lenhauer C, Polyak K, et al. 14-3-3{sigma} is a p53-regulated inhibitor of G2-M progression. Mol Cell 1997;1:3–11.[CrossRef][Medline]
  21. Chan TA, Hermeking H, Lengauer C, Kinzler KW, Vogelstein B. 14-3-3{sigma} is required to prevent mitotic catastrophe after DNA damage. Nature 1999;401:616–20.[CrossRef][Medline]
  22. Weber JD, Zambetti GP. Renewing the debate over the p53 apoptotic response. Cell Death Differ 2003;10:409–12.[CrossRef][Medline]
  23. Yousefi S, Conus S, Simon H-U. Cross-talk between death and survival pathways. Cell Death Differ 2003;10:861–3.[CrossRef][Medline]
  24. Oren M. Decision making by p53: life, death and cancer. Cell Death Differ 2003;10:431–42.[CrossRef][Medline]
  25. Zhang L, Yu J, Park BH, Kinzler KW, Vogelstein B. Role of BAX in the apoptotic response to anticancer agents. Science 2000;290:989–92.[Abstract/Free Full Text]
  26. Yu J, Zhang L, Hwang PM, Kinzler KW, Vogelstein B. PUMA induces the rapid apoptosis of colorectal cancer cells. Mol Cell 2001;7:673–82.[CrossRef][Medline]
  27. Nakano K, Vousden K. PUMA, a novel proapoptotic gene, is induced by p53. Mol Cell 2001;7:683–94.[CrossRef][Medline]
  28. Villunger A, Michalak EW, Coultas L, et al. p53- and drug-induced apoptotic responses mediated by BH3-only proteins puma and noxa. Science 2003;302:1036–8.[Abstract/Free Full Text]
  29. Hoffman WH, Biade S, Zilfou JT, Chen J, Murphy M. Transcriptional repression of the anti-apoptotic survivin gene by wild type p53. J Biol Chem 2002;277:3247–57.[Abstract/Free Full Text]
  30. Mirza A, McGuirk M, Hockenberry TN, et al. Human survivin is negatively regulated by wild-type p53 and participates in p53-dependent apoptotic pathway. Oncogene 2002;21:2613–22.[CrossRef][Medline]
  31. Mesri M, Wall NR, Li J, Kim RW, Altieri DC. Cancer gene therapy using a survivin mutant adenovirus. J Clin Invest 2001;108:981–90.[CrossRef][Medline]
  32. Shankar SL, Mani S, Q'Guin KN, Kandimalla E, Agrawal S, Shafit-Zagardo B. Survivin inhibition induces human neural tumor cell death through caspase-independent and -dependent pathways. J Neurochem 2001;79:426–36.[CrossRef][Medline]
  33. Altieri DC. Survivin and apoptosis control. Adv Cancer Res 2003;88:31–52.[CrossRef][Medline]
  34. Ciechanover A. The ubiquitin-proteasome pathway: on protein death and cell life. EMBO J 1998;17:7151–60.[CrossRef][Medline]
  35. Adams J, Palombella VJ, Sausville JJ, et al. Proteasome inhibitors: a novel class of potent and effective antitumor agents. Cancer Res 1999;59:2615–22.[Abstract/Free Full Text]
  36. Sunwoo JB, Chen Z, Dong G, et al. Novel proteasome inhibitor PS-341 inhibits activation of nuclear factor-{kappa}B, cell survival, tumor growth, and angiogenesis in squamous cell carcinoma. Clin Cancer Res 2001;7:1419–28.[Abstract/Free Full Text]
  37. Desai S, Li T-K, Rodriquez-Bauman A, Rubin EH, Liu L. Ubiquitin/26S proteasome-mediated degradation of topoisomerase I as a resistance mechanism to camptothecin in tumor cells. Cancer Res 2001;61:5926–32.[Abstract/Free Full Text]
  38. Zhang H-F, Tomida A, Koshimizu R, Ogiso Y, Lei S, Tsuruo T. Cullin 3 promotes proteasomal degradation of the topoisomerase I-DNA covalent complex. Cancer Res 2004;64:1114–21.[Abstract/Free Full Text]
  39. Tabata M, Tabata R, Grabowski DR, Bukowski RM, Ganapathi MK, Ganapathi R. Roles of NF-{kappa}B and 26S proteasome in apoptotic cell death induced by topoisomerase I and II poisons in human nonsmall cell lung carcinoma. J Biol Chem 2001;276:8029–36.[Abstract/Free Full Text]
  40. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002;296:550–3.[Abstract/Free Full Text]
  41. Gurova KV, Hill JE, Razorenova OV, Chumakov PM, Gudkov AV. p53 pathway in renal cell carcinoma is repressed by a dominant mechanism. Cancer Res 2004;64:1951–8.[Abstract/Free Full Text]
  42. Li M, Brooks CL, Wu-Baer F, Chen D, Baer R, Gu W. Mono- versus polyubiquitination: differential control of p53 fate by Mdm2. Science 2003;302:1972–5.[Abstract/Free Full Text]
  43. Momparler RL. In vitro systems for evaluation of combination chemotherapy. Pharmacol Ther 1980;8:21–35.[Medline]
  44. Wall NR, O'Connor DS, Plescia J, Pommier Y, Altieri DC. Suppression of survivin phosphorylation on Thr34 by flavopiridol enhances tumor cell apoptosis. Cancer Res 2003;63:230–5.[Abstract/Free Full Text]
  45. Yu J, Tiwari S, Steiner P, Zhang L. Differential apoptotic response to the proteasome inhibitor bortezomib (VelcadeTM, PS-341) in bax-deficient and p21-deficient colon cancer cells. Cancer Biol Ther 2003;2:694–9.[Medline]
  46. Qi W, Martinez JD. Reduction of 14-3-3 proteins correlates with increased sensitivity to killing of human lung cancer cells by ionizing radiation. Radiat Res 2003;160:217–23.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Am J Health Syst PharmHome page
K. N. Utecht and J. Kolesar
Bortezomib: A novel chemotherapeutic agent for hematologic malignancies
Am. J. Health Syst. Pharm., July 1, 2008; 65(13): 1221 - 1231.
[Abstract] [Full Text] [PDF]

Home page
 J Biomol ScreenHome page
L. Rickardson, M. Wickstrom, R. Larsson, and H. Lovborg
Image-Based Screening for the Identification of Novel Proteasome Inhibitors
J Biomol Screen, March 1, 2007; 12(2): 203 - 210.
[Abstract] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles