| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Cell and Tumor Biology |
Drug Resistance Group, Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, Northern Ireland, United Kingdom
Requests for reprints: Daniel B. Longley, Cancer Research Centre, Queen's University Belfast, University Floor, Belfast City Hospital, Lisburn Road, Belfast, Northern Ireland, United Kingdom BT9 7AB. Phone: 44-2890-263960; Fax: 44-2890-263744; E-mail: d.longley{at}qub.ac.uk.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Colorectal cancer is the second highest cause of cancer mortality in the Western world. Despite improvements in the efficacy of chemotherapy drugs used in the treatment of colorectal cancer, response rates in the advanced disease setting are of the order of 45% to 50% for the most effective drug combinations, with a median survival of up to 21 months (5). The most frequently used chemotherapeutic agents are the fluoropyrimidine 5-fluorouracil (5-FU), the topoisomerase I inhibitor irinotecan (CPT-11), and the platinum agent oxaliplatin. The antifolate tomudex is also used in the treatment of advanced colorectal cancer. Any strategies that target the Fas receptor must take account of the fact that 
45% to 61% of colon cancers harbor mutations in the tumor suppressor gene p53 (6, 7). Fas is a well-documented p53 target gene (4), and loss of p53 function has been shown to affect the ability of cancer cells to up-regulate the Fas receptor in response to chemotherapy (8). However, there is increasing evidence that not all p53 mutations result in absolute loss of function (9, 10). Functional activities or properties of mutant proteins include retained wild-type (WT) activity (11), loss of function (12), gain of function (13, 14), and dominant-negative effects (15). The high frequency of p53 mutations in colorectal cancer increases the clinical significance of p53-independent mechanisms of apoptosis. IFN-
has been shown to induce Fas expression in p53 mutant cell lines, and this induction was dependent on the signal transducer and activator of transcription 1 (STAT1) protein (16). IFN- activates STAT1 by triggering phosphorylation at two distinct sites: Tyr701 and the COOH-terminal Ser727. The tyrosine phosphorylation results in STAT1 dimerization, translocation to the nucleus, and activation of target genes, whereas the serine phosphorylation strongly increases the transcriptional activity of STAT1. Recent data have indicated that some STAT1-dependent genes are still activated when Tyr701 is mutated to a nonphosphorylatable phenylalanine residue (17, 18). The induction of p53 responsive genes, such as Fas, Bax, and Noxa, has been reported to be reduced in STAT1-deficient cells (19). Moreover, STAT1 can interact directly with p53, an association that is enhanced following DNA damage (19).
The aim of the present study was to investigate the roles of p53 and STAT1 in regulating Fas-mediated apoptosis in response to chemotherapy in colorectal cancer.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Western blotting. Western blots were done as described previously (6). The Fas (Santa Cruz Biotechnology, Santa Cruz, CA), p53 (Santa Cruz Biotechnology), caspase-8 (Oncogene Research Products, Merck Biosciences Ltd., Nottingham, United Kingdom), and poly(ADP-ribose) polymerase (PARP; PharMingen, BD Bioscience, Franklin Lake, NJ) mouse monoclonal antibodies and STAT1, phospho-STAT1 (Ser727), and phospho-STAT1 (Tyr701) rabbit polyclonal antibodies (Cell Signaling, Beverly, MA) were used in conjunction with a horseradish peroxidase–conjugated sheep anti-mouse or anti-rabbit secondary antibody (Amersham Biosciences, Bucks, United Kingdom). Equal loading was assessed using a ß-tubulin mouse monoclonal primary antibody (Sigma-Aldrich, Dorset, United Kingdom). Densitometry was done using a Chemidoc XRS imager and QuantityOne software (Bio-Rad Laboratories, Hertfordshire, United Kingdom).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability assay. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma, St. Louis, MO). Cells were seeded at 1,800 cells per well on 96-well plates 24 hours before drug treatment and were treated with a range of concentrations of 5-FU (Faulding Pharmaceuticals, Warwickshire, United Kingdom), tomudex (AstraZeneca, Macclesfield, United Kingdom), CPT-11 (Pharmacia and Upjohn, Kalamazoo, MI), and oxaliplatin (Sanofi-Synthelabo, Malvern, PA) for 24 hours, after which the agonistic Fas monoclonal antibody, CH-11 (MBL International, Woburn, MA), was added (25-100 ng/mL) for 48 hours. MTT was added to a final concentration of 500 µg/mL and the cells incubated at 37°C for a further 3 hours. The culture medium was removed and formazan crystals were reabsorbed in 200 µL DMSO. Cell viability was determined by reading the absorbance of each well at 570 nm using an ELISA plate reader (Molecular Devices, Wokingham, United Kingdom).
Flow cytometric analysis. Cells were seeded in a six-well tissue culture plate at a density of 1 x 105 cells per well. After 24 hours, 5-FU, tomudex, CPT-11, or oxaliplatin were added to the medium and the cells were cultured for a further 24 hours. CH-11 (10-100 ng/mL) was then added for 24 hours. DNA content of harvested cells was evaluated following propidium iodide staining of cells using the EPICS XL Flow Cytometer (Beckman Coulter UK, High Wycombe, United Kingdom). A FITC-conjugated monoclonal anti-human Fas antibody (DakoCytomation, Cambridgeshire, United Kingdom) was used to determine cell surface expression of the receptor. Cells were collected and washed twice in PBS before incubation with the antibody (1:20 dilution) at 4°C for 30 minutes. A nonreactive FITC-conjugated mouse IgG1 antibody was used as a negative control for each sample. Following incubation, the cells were washed twice with PBS containing 2% bovine serum albumin and fixed in 0.3 mL of 4% paraformaldehyde. Samples were then analyzed on the EPICS XL Flow Cytometer.
Caspase-8 assay. A fluorimetric caspase-8 assay kit was used (Sigma) to measure caspase-8 activity. This assay is based on the release of 7-amino-4-methyl coumarin (AMC) by caspase-8. Cells were seeded in a six-well tissue culture plate at a density of 105 cells per well and treated with the indicated doses of 5-FU, tomudex, CPT-11, and oxaliplatin the following day. After 24 hours, 100 ng/mL CH-11 was added for an additional 12 hours and the samples were suspended in 500 µL of the provided lysis buffer. Lysate (20 µL) was transferred to a 96-well plate and the fluorimetric assay was carried out according to the manufacturer's instructions. Fluorescence detection was carried out using a Microplate Fluorimeter (CytoFluor, Applied Biosystems, Foster City, CA) using excitation and emission wavelengths of 360 and 440 nm, respectively, at 5-minute intervals. Caspase-8 activity was calculated using an AMC calibration curve analyzed on the same plate. All samples were analyzed in duplicate, and the specificity of the reaction for caspase-8 was confirmed by analyzing a second set of duplicate lysates for each sample in the presence of a caspase-8 inhibitor (Ac-IETD-CHO).
Real-time PCR. Real-time reverse transcription-PCR (RT-PCR) was done sing a DNA Engine Opticon 2 (Genetic Research Instrumentation Ltd., Essex, United Kingdom). RNA was collected using the RNA STAT-60 reagent (Tel-Test (Friendswood, TX) according to the manufacturer's instructions, and cDNA was synthesized using 1 µg RNA and a reverse transcriptase kit (Life Technologies Invitrogen). Gene expression was analyzed using the DyNAmo SYBR Green qPCR kit (Finnzymes, Espoo, Finland). PCR conditions consisted of an initial denaturation step of 95°C for 10 minutes followed by 39 cycles of 94°C for 10 seconds, 55°C for 20 seconds, and 72°C for 20 seconds, with a final extension of 72°C for 10 minutes. A melting curve was included at the end of each run to check the specificity of the amplified product. On completion of the run, the PCR products were electrophoresed on a 2% agarose, 0.001% ethidium bromide gel to confirm that their size matched that of the expected amplicon. Primer sequences were as follows: Fas (forward) 5'-AAAGGCTTTGTTCGAAAG-3', Fas (reverse) 5'-CACTCTAGACCAAGCTTTGG-3', 18S (forward) 5'-CATTCGTATTGCGCCGCTA-3', and 18S (reverse) 5'-CGACGGTATCTGATCGTCT-3'. Experiments were done in triplicate.
STAT1 small interfering RNA. The STAT1-targeted and nonsilencing control sequences used were 5'-AACTAGTGGAGTGGAAGCGGA-3' and 5'-AAGGGTCGAGAGAGAGCTGTA-3', respectively (Dharmacon RNA Tech, Dallas, TX). The oligonucleotide was diluted in Opti-MEM-1 (Life Technologies Invitrogen) and transfected with Oligofectamine (Life Technologies Invitrogen) according to the manufacturer's instructions.
Statistical analyses. The nature of the interaction between the chemotherapeutic drugs and CH-11 was determined by calculating the R index (RI) as described by Kern (20) and modified by Romanelli (21). The RI is calculated as ratio of the expected cell survival (Sexp, defined as the product of the survival observed with drug A alone and the survival observed with drug B alone) to the observed cell survival (Sobs) for the combination of A and B (RI = Sexp / Sobs). Synergism is then defined as a RI of greater than unity. This method was selected because treatment with CH-11 alone had little effect on cell viability, which meant that other methods, such as the median effect principle (22) and isobologram methods (23), were not suitable. To further assess the statistical significance of the interactions, we designed a univariate ANOVA using an additive model based on the null hypothesis that there was no interaction between the drugs (SPSS Software, SPSS, Inc., Chicago, IL). The unpaired t test was used to determine statistically significant differences between treatment effects. Significance was defined as P < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
38 and 48 kDa in agreement with the findings of others (25). The observed induction of Fas protein in this cell line was associated with induction of p53 (Fig. 1B and C). In the p53-null cell line, treatment with 5-FU and oxaliplatin did not significantly induce Fas protein expression; however, a small increase in Fas expression (38-kDa band) was observed in response to both CPT-11 and tomudex (Fig. 1B and C).
|
1.5 (P < 0.01), and the extent of this synergy was significantly less than in the p53 WT cells at this concentration (Fig. 2A).
|
31% (P < 0.001); however, the increase in apoptosis in response to 5-FU alone in the p53-null setting was not significant. When CH-11 was added to 5-FU-treated p53 WT cells, the induction of apoptosis was dramatic with 76% of cells in the sub-G0-G1 fraction. In contrast, only a modest increase in apoptosis was observed in the p53-null cell line when CH-11 was added to 5-FU pretreated cells (18% compared with 11% and 7% for CH-11 alone and 5-FU alone, respectively). An almost identical pattern was observed in response to oxaliplatin, with the p53-null cells being much more resistant to apoptosis induced by oxaliplatin alone and, more significantly, oxaliplatin in combination with CH-11. In contrast, the levels of apoptosis induced by CPT-11 alone were similar in the p53 WT and null cell lines (25% and 19%, respectively). More importantly, combined treatment with CPT-11 and CH-11 also resulted in similar levels of apoptosis in the p53 WT and null cell lines (52% and 57%, respectively). Results with tomudex were similar to those with CPT-11, with relatively high levels of apoptosis induced in the p53-null cells following tomudex and CH-11 treatment; however, the induction of apoptosis in tomudex/CH-11–treated cells was still significantly higher in the p53 WT cell line (57% compared with 40%; P = 0.02).
|
|
CPT-11 and tomudex cause a p53-independent induction of Fas cell surface expression. Flow cytometric analysis of immunostained cells showed higher constitutive cell surface expression of the Fas receptor in the HCT116 p53 WT cell line compared with the p53-null cell line. This is probably the basis for the greater sensitivity of the p53 WT cells to CH-11 in the absence of chemotherapy (Fig. 3A and B). Dramatic increases in Fas cell surface expression were observed in the p53 WT cell line following treatment with each of the chemotherapeutic agents (Fig. 4). However, in the p53-null setting, the induction of cell surface Fas expression following treatment with 5-FU and oxaliplatin was significantly lower than in the p53 WT cell line. In contrast, high levels of cell surface Fas were expressed in response to CPT-11 and tomudex in the p53-null cell line. Of note, the increased cell surface expression of Fas in response to CPT-11 and tomudex in the HCT116 p53-null cell line was accompanied by only a modest increase in total Fas expression (Fig. 1B and C), suggesting that these agents may trigger p53-independent trafficking of Fas to the cell surface. Thus, CPT-11 and tomudex induce p53-independent up-regulation of Fas at the cell surface.
Chemotherapy-induced Fas-mediated apoptosis in other colorectal cancer cell lines. We extended our study to examine whether these effects were observed in other colorectal cancer cell lines. Fas-mediated cell death in response to chemotherapy was examined in the p53 WT RKO and p53 mutant H630 cell lines. Induction of the Fas receptor at the cell surface in the H630 cell line was seen in response to CPT-11 and tomudex and to a lesser extent 5-FU but not oxaliplatin (Fig. 5A). In the p53 WT RKO cell line, there was induction of the Fas receptor at the cell surface in response to IC60 doses of all four chemotherapeutic agents (Fig. 5A). When the H630 cell line was treated with these chemotherapy drugs for 24 hours followed by CH-11 for an additional 48 hours, significant synergy was evident in cells treated with CPT-11 and tomudex (P < 0.001) but not 5-FU or oxaliplatin (Fig. 5B). In the RKO cell line, 5-FU, tomudex, and CPT-11 all synergized with CH-11 (P < 0.001; Fig. 5B). Surprisingly, oxaliplatin and CH-11 did not synergise in RKO cells despite the high levels of Fas cell surface expression in oxaliplatin-treated RKO cells (Fig. 5A). The synergy (or lack of synergy) between each drug and CH-11 in each cell line was reflected in the extent of apoptosis in chemotherapy and CH-11 cotreated cells as determined by flow cytometry (Fig. 5C). At high concentrations of chemotherapy, lower RI values were sometimes observed than at low concentrations (Fig. 5B). This may be due to more efficient induction of cell death in response to chemotherapy alone at these higher concentrations, with the result that the synergistic effects of CH-11 are less marked.
|
|
|
and ß isoforms of STAT1 in the HCT116 cell lines (Fig. 8A). The STAT1 splice form is the transcriptionally active form containing the Ser727 phosphorylation site; the ß splice form lacks this site (27). Interestingly, no changes in chemotherapy-induced up-regulation of Fas mRNA were observed following STAT1 down-regulation in either cell line (Fig. 8B). Despite this, STAT1 down-regulation surprisingly resulted in a significant decrease in Fas cell surface expression in the HCT116 p53-null cell line following treatment with CPT-11 and tomudex for 48 hours (Fig. 8C). Importantly, down-regulation of both the isoforms of STAT1 was still evident at this time point (72 hours after transfection with siRNA; Fig. 8D). In contrast, induction of Fas cell surface expression in response to CPT-11 and tomudex (and also 5-FU and oxaliplatin; data not shown) was not affected by STAT1 knockdown in the p53 WT setting. To determine whether the reduction in Fas expression seen with this siRNA approach abrogated the induction of apoptosis in response to CPT-11 and CH-11, we cotreated STAT1 siRNA-transfected p53-null cells with CPT-11 and CH-11 (Fig. 8E). We found that STAT1 down-regulation inhibited PARP cleavage and caspase-8 activation in response to cotreatment with CPT-11 and CH-11 (10-25 ng/mL). Higher concentrations of CH-11 (>50 ng/mL) still induced apoptosis in STAT1 siRNA-transfected cells, which may reflect the fact that STAT1 siRNA did not completely abrogate the induction of Fas cell surface expression by CPT-11 (Fig. 8C). This suggests that either STAT1 silencing was incomplete or that alternative STAT1-independent signaling pathways operate to regulate CPT-11-induced Fas cell surface expression. Collectively, these results suggest that phosphorylation of STAT1 at Ser727 results in transcription of a cofactor(s) that mediates Fas cell surface trafficking in response to CPT-11 and tomudex in the HCT116 p53-null cell line.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Both agonistic anti-Fas antibodies and Fas-specific peptides have been explored for their potential as targeted therapies against the Fas death receptor. Initial attempts to develop a therapeutic anti-Fas antibody were confounded by severe hepatic toxicity in the three murine models investigated (32). However, a novel agonistic Fas monoclonal antibody has recently been developed, which lacks the severe hepatic toxicity seen previously, suggesting that it may be possible to develop therapeutically useful nontoxic Fas-targeted antibodies (33). Other approaches to activate Fas-mediated apoptosis include local delivery of recombinant FasL and FasL gene therapy. In addition, targeting of cell surface Fas constitutes a major mechanism by which immune effector cells, such as circulating T lymphocytes, can eliminate tumor cells, which suggests that clinical studies combining immunotherapy with chemotherapy might be very effective. It is therefore important to understand the mechanisms regulating Fas expression in response to the different chemotherapeutic agents currently used in the treatment of colorectal cancer.
Many genes regulated by p53 have been shown to participate in apoptotic pathways, including Bax, DR5, Puma, Noxa, and Fas. Owen-Schaub et al. have shown that introduction of WT p53 into the p53 mutant HT29 cell line enhanced cell surface expression of the Fas receptor (34). A strong correlation has also been shown between WT p53 status and induction of Fas receptor expression in cancer cell lines exposed to chemotherapeutic drugs (4, 8, 35). Recent studies have described a p53-responsive element in intron 1 of the human Fas gene as well as three putative elements in the promoter (4, 24). Our findings support the concept that for some chemotherapy agents (i.e., 5-FU and oxaliplatin) the ability to induce the Fas receptor is highly dependent on p53-mediated transcriptional up-regulation. However, we have shown for the first time that for other chemotherapies (tomudex and CPT-11) a different p53-independent mechanism can also operate to up-regulate Fas cell surface expression. Furthermore, this mechanism seems to be largely independent of transcriptional up-regulation. Although 5-FU can act as a thymidylate synthase inhibitor, RNA-directed cytotoxicty has been reported to be the more important cytotoxic mechanism in the HCT116 cell line (35). This may be the cause of the differences in the ability of 5-FU and tomudex to induce Fas cell surface expression in the p53-null HCT116 cell line. In support of this, we have recently found that p53 mutant HT29 cells induce Fas cell surface expression in response to both 5-FU and tomudex, and this results in synergistic induction of apoptosis following the addition of CH-11 to both drugs (data not shown). This is notable because the cytotoxic effects of 5-FU in the HT29 cell line have been reported to be predominantly due to thymidylate synthase inhibition (35). The anticancer effects of 5-FU in vivo are believed to be predominantly due to thymidylate synthase inhibition; therefore, in this setting, 5-FU treatment may also induce Fas in a p53-independent manner.
Mutations in p53 are present in at least 50% of all colorectal tumors, and specific mutations at codons 175 and 248 account for more than a third of all the p53 mutations in these tumors. We therefore analyzed the effects of these clinically relevant p53 mutations on chemotherapy-induced Fas signaling. Previous reports have found that p53-responsive elements of the Fas gene are still capable of being activated by p53 mutants, indicating that some mutant p53 proteins may retain the ability to up-regulate Fas transcription in response to chemotherapy (24, 36). However, we found that cell lines stably expressing the p53 mutations, R175H and R248W, behaved in an almost identical fashion to the p53-null cell line with respect to drug sensitivity, induction of Fas cell surface expression in response to chemotherapy, and synergy between chemotherapy and CH-11. The ability of tomudex and CPT-11 to induce Fas cell surface expression in the p53-null and mutant cell lines suggests that, in patients with p53 mutant tumors, the use of therapies directed against the Fas receptor would be best combined with either of these two drugs.
IFN- has been shown to induce Fas expression in p53 mutant cell lines and this induction was dependent on STAT1 (16). IFN- stimulates gene expression through phosphorylation of the Tyr701 residue of STAT1 by Janus-activated kinases associated with the IFN- receptor. Tyr701 phosphorylation induces dimerization of STAT1, nuclear translocation, and binding to IFN--activated sequence (GAS) elements in the promoters of target genes. The absence of a GAS element in the promoter region of Fas makes it possible that additional factors are transcriptionally activated by STAT1 to regulate Fas expression. In addition, STAT1 Ser727 phosphorylation can transactivate genes independently of Tyr701 phosphorylation (17). Initially, Tyr701 phosphorylation was thought to be necessary for subsequent Ser727 phosphorylation of STAT1 to occur. However, stress stimuli, such as UV, lipopolysaccharides, TNF-, and osmotic stress, have been shown to cause phosphorylation of STAT1 at Ser727 without phosphorylation at Tyr701, and this has been reported to be mediated by p38 mitogen-activated protein kinase (MAPK; ref. 37). The phosphorylation of STAT1 at Ser727 that we observed in response to tomudex and CPT-11 in the p53-null HCT116 cell line suggested that a STAT1-dependent mechanism was responsible for the modulation of Fas cell surface expression, although neither drug activated STAT1 Tyr701. Intriguingly, we observed that STAT1 siRNA significantly reduced CPT-11- and tomudex-induced Fas cell surface expression in the p53-null cell line despite having no direct effect on Fas mRNA expression. This suggests that STAT1 up-regulates expression of a gene (or genes) involved in trafficking of Fas to the cell surface in response to CPT-11 and tomudex. Furthermore, it would seem that activation of this target gene(s) is dependent on STAT1 phosphorylation at Ser727 but not at Tyr701.
At present, it is unclear why CPT-11- and tomudex-induced Fas cell surface expression in HCT116 p53 WT cell line is largely STAT1 independent but STAT1 dependent in p53-null cells. p38 MAPK has been reported to induce STAT1 Ser727 phosphorylation (38), and p38 is negatively regulated by the p53 target gene WIP-1 (39). It is possible that p53 induction in HCT116 p53 WT cells, which we observed in response to each chemotherapy, stimulates induction of WIP-1, which then inhibits p38-mediated Ser727 phosphorylation of STAT1. p53 can also up-regulate PTEN, which inhibits accumulation of phosphatidylinositol-3,4,5-triphosphate (PIP3). Reduced levels of PIP3 may then inhibit phosphoinositide-dependent kinase-1–mediated activation of protein kinase C
(PKC), a serine kinase that has been shown to phosphorylate STAT1 (40, 41). In the absence of p53, p38- and/or PKC-mediated STAT1 Ser727 phosphorylation may occur in response to CPT-11 and tomudex resulting in transcription of a gene (or genes) that mediate translocation of Fas to the cell surface. In the p53 WT setting, the increased Fas cell surface expression following chemotherapy may simply reflect the higher levels of total Fas expression resulting from increased Fas gene transcription. It is also possible that p53 regulates alternative Fas trafficking mechanisms that are STAT1 independent. These possibilities are currently under investigation.
In conclusion, we have found that of the chemotherapies used to treat colorectal cancer CPT-11 and tomudex up-regulate Fas cell surface expression in a p53-independent, STAT1-dependent manner. Moreover, the mechanism for this up-regulation does not involve increased Fas gene expression, suggesting that these agents induce trafficking of Fas to the cell membrane. These findings suggest that tomudex and CPT-11 may be more effective anticancer drugs than 5-FU and oxaliplatin in the treatment of p53 mutant colorectal tumors. Furthermore, our data suggest that Fas-targeted approaches may be most effective in colorectal cancer when used in combination with CPT-11 and tomudex.
|
Acknowledgments |
|---|
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.
|
Footnotes |
|---|
1 http://www-p53.iarc.fr/index.html 
Received 3/22/05. Revised 5/26/05. Accepted 7/22/05.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
induces cell growth inhibition by Fas-mediated apoptosis: requirement of STAT1 protein for up-regulation of Fas and FasL expression. Cancer Res 1998;58:2832–7. and - signal transduction pathways. EMBO J 1993;12:4221–8.[Medline]
uses a different signaling pathway. Proc Natl Acad Sci U S A 1999;96:13956–61. regulates apoptosis via activation of STAT1. J Biol Chem 2004;279:45603–12.This article has been cited by other articles:
|
|
 |
|
  A. Zaanan, P. Cuilliere-Dartigues, A. Guilloux, Y. Parc, C. Louvet, A. de Gramont, E. Tiret, S. Dumont, B. Gayet, P. Validire, et al. Impact of p53 expression and microsatellite instability on stage III colon cancer disease-free survival in patients treated by 5-fluorouracil and leucovorin with or without oxaliplatin Ann. Onc., October 15, 2009; (2009) mdp383v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Proutski, L. Stevenson, W. L. Allen, A. McCulla, J. Boyer, E. G. McLean, D. B. Longley, and P. G. Johnston Prostate-derived factor--a novel inhibitor of drug-induced cell death in colon cancer cells Mol. Cancer Ther., September 1, 2009; 8(9): 2566 - 2574. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Mitchell, P. Kabolizadeh, J. Ryan, J. D. Roberts, A. Yacoub, D. T. Curiel, P. B. Fisher, M. P. Hagan, N. P. Farrell, S. Grant, et al. Low-Dose BBR3610 Toxicity in Colon Cancer Cells Is p53-Independent and Enhanced by Inhibition of Epidermal Growth Factor Receptor (ERBB1)-Phosphatidyl Inositol 3 Kinase Signaling Mol. Pharmacol., September 1, 2007; 72(3): 704 - 714. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Wilsker and F. Bunz Loss of ataxia telangiectasia mutated- and Rad3-related function potentiates the effects of chemotherapeutic drugs on cancer cell survival Mol. Cancer Ther., April 1, 2007; 6(4): 1406 - 1413. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Bagnoli, E. Balladore, E. Luison, P. Alberti, F. Raspagliesi, B. Marcomini, S. Canevari, and D. Mezzanzanica Sensitization of p53-mutated epithelial ovarian cancer to CD95-mediated apoptosis is synergistically induced by cisplatin pretreatment Mol. Cancer Ther., February 1, 2007; 6(2): 762 - 772. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. L. Allen, E. G. McLean, J. Boyer, A. McCulla, P. M. Wilson, V. Coyle, D. B. Longley, R. A. Casero Jr., and P. G. Johnston The role of spermidine/spermine N1-acetyltransferase in determining response to chemotherapeutic agents in colorectal cancer cells Mol. Cancer Ther., January 1, 2007; 6(1): 128 - 137. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Smakman, D. J.M. van den Wollenberg, S. G. Elias, T. Sasazuki, S. Shirasawa, R. C. Hoeben, I. H.M. Borel Rinkes, and O. Kranenburg KRASD13 Promotes Apoptosis of Human Colorectal Tumor Cells by ReovirusT3D and Oxaliplatin but not by Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand. Cancer Res., May 15, 2006; 66(10): 5403 - 5408. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Boyer, W. L. Allen, E. G. McLean, P. M. Wilson, A. McCulla, S. Moore, D. B. Longley, C. Caldas, and P. G. Johnston Pharmacogenomic identification of novel determinants of response to chemotherapy in colon cancer. Cancer Res., March 1, 2006; 66(5): 2765 - 2777. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 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 |
) and null cell lines (
). Mean of experiments carried out in triplicate. The RI is calculated as ratio of the expected cell survival (Sexp, defined as the product of the survival observed with drug A alone and the survival observed with drug B alone) to the observed cell survival (Sobs) for the combination of A and B (RI = Sexp / Sobs). Synergism is defined as RI > 1. Representative of at least three separate experiments. Univariate ANOVA was used to determine the statistical significance of the interaction between drug and CH-11.
) and R248W (
) cell lines. Univariate ANOVA was used to determine the statistical significance of the interaction between each drug and CH-11.
