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Objective@#To investigate the effect of Δ40p53, an alternative spliced isoform of p53 lacking the N-ter minus, on the pro-apoptotic function of p53.@*Methods@#The wild-type p53 was ectopically expressed in HCT116-p53-/- (endogenous Δ40p53 expression), HCT116-p53+ /+ (wild-type p53) and H1299 (p53-null) cells by adenoviral delivery, while Δ40p53 plasmid were transfected into these cells to overexpress Δ40p53. The levels of Δ40p53 and p53 mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR. The expression of related proteins was deter mined by Western blotting. The interaction of p53 and Δ40p53 was observed by co-immunoprecipitation assay. Calcein-AM/propidium iodide (PI) staining and flow cytometry were used to detect the apoptotic rate of tested cells in each group.@*Results@#HCT116-p53-/- cells expressed endogenous Δ40p53 isoform. Neither transcription nor protein expression of wild-type p53 was interfered by the increased expression of Δ40p53. Full length p53 and Δ40p53 could bind to each other. Calcein-AM/PI staining showed that the apoptotic rates of H1299-Control, HCT116-p53-/- -Control, H1299+ p53, HCT116-p53-/-+ p53, H1299+ oxaliplatin (Oxa), HCT116-p53-/-+ Oxa, H1299+ p53+ Oxa and HCT116-p53-/-+ p53+ Oxa groups were (2.50±0.47)%, (2.40±0.32)%, (5.20±0.58)%, (4.10±0.18)%, (22.40±1.73)%, (19.30±1.11)%, (29.90±1.15)% and (39.30±2.26)%, respectively. It was statistically significant between H1299+ p53+ Oxa and HCT116-p53-/-+ p53+ Oxa groups (t=3.721, P=0.0205). Moreover, the apoptotic rates of H1299-Control, H1299+ Δ40p53, H1299+ p53, H1299+ p53+ Δ40p53, H1299+ Oxa, H1299+ Δ40p53+ Oxa, H1299+ p53+ Oxa and H1299+ p53+ Δ40p53+ Oxa groups were (2.60±0.35)%, (2.20±0.17)%, (4.80±0.49)%, (4.90±1.10)%, (20.30±1.10)%, (19.60±1.45)%, (27.90±1.39)%, (35.20±1.43)%, respectively. Furthermore, flow cytometry assay showed that the apoptotic rates of above cells were (2.70±0.32)%, (2.20±0.24)%, (4.60±0.48)%, (3.90±0.67)%, (19.30±1.11)%, (17.70±0.66)%, (28.30±2.76)% and (37.50±1.51)%, respectively. H1299+ p53+ Δ40p53+ Oxa cells showed higher cell apoptosis than H1299+ p53+ Oxa cells (t=2.930, P=0.042).@*Conclusion@#Δ40p53 isoform can bind to full-length p53, and enhance its pro-apoptotic function in tumor cells.
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<p><b>OBJECTIVE</b>To summarize the treatment status of gastric gastrointestinal stromal tumor (GIST) in Shandong province,by analyzing the clinicopathological features and prognostic factors.</p><p><b>METHODS</b>Clinicopathological and follow-up data of 1 165 patients with gastric GIST between January 2000 and December 2013 from 23 tertiary referral hospitals in Shandong Province were collected to establish a database. The risk stratification of all cases was performed according to the National Institutes of Health(NIH) criteria proposed in 2008. Kaplan-Meier method was used to calculate the survival rate. Log-rank test and Cox regression model were used for univariate and multivariate prognostic analyses.</p><p><b>RESULTS</b>Among 1 165 cases of gastric GIST, 557 were male and 608 were female. The median age of onset was 60 (range 15-89) years. Primary tumors were located in the gastric fundus and cardia in 623 cases(53.5%), gastric body in 346 cases(29.7%), gastric antrum in 196 cases(16.8%). All the cases underwent resection of tumors, including endoscopic resection (n=106), local resection (n=589), subtotal gastrectomy(n=399), and total gastrectomy(n=72). Based on the NIH risk stratification, there were 256 cases (22.0%) at very low risk, 435 (37.3%) at low risk, 251 cases (21.5%) at intermediate risk, and 223 cases (19.1%) at high risk. A total of 1 116 cases(95.8%) were followed up and the median follow-up period was 40 (range, 1-60) months. During the period, 337 patients relapsed and the median time to recurrence was 34 (range 1-60) months. The 1-, 3-, and 5-year survival rates were 98.6%, 86.1% and 73.4%, respectively. The 5-year survival rates of patients at very low, low, intermediate, and high risk were 93.1%, 85.8%, 63.0% and 42.3% respectively, with a statistically significant difference (P=0.000). Multivariate analysis showed that primary tumor site (RR=0.580, 95%CI:0.402-0.835), tumor size (RR=0.450, 95%CI:0.266-0.760), intraoperative tumor rupture(RR=0.557, 95%CI:0.336-0.924), risk classification (RR=0.309, 95%CI:0.164-0.580) and the use of imatinib after surgery (RR=1.993, 95%CI:1.350-2.922) were independent prognostic factors.</p><p><b>CONCLUSIONS</b>The choice of surgical procedure for gastric GIST patients should be based on tumor size. All the routine procedures including endoscopic resection, local excision, subtotal gastrectomy and total gastrectomy can obtain satisfactory curative outcomes. NIH classification has a high value for the prediction of prognosis. Primary tumor site, tumor size, intraoperative tumor rupture, risk stratification and postoperative use of imatinib are independent prognostic factors in gastric GIST patients.</p>
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<p><b>OBJECTIVE</b>To investigate the role of apoptosis stimulating protein 2 of p53 (ASPP2) phosphorylation status in the regulation of ASPP2-p53 apoptotic pathway activity.</p><p><b>METHODS</b>Cells were individually transfected with green fluorescent protein (GFP)-encoding vector, constitutively non-phosphorylatable ASPP2 mutant-ASPP2 (Am)-encoding vector, and wild type ASPP2 (Aw)-encoding vector) plasmids, respectively, to make them overexpressing phosphorylated and non-phosphorylated ASPP2 proteins, respectively. Cell apoptosis was induced by oxaliplatin. The apoptosis rate of cells was determined by flow cytometry after staining with FITC-conjugated annexin V and PI. ASPP2 protein level and its phosphorylation status were observed by Western blot. The interaction between ASPP2 and p53 was observed by immunoprecipitation assay.</p><p><b>RESULTS</b>Oxaliplatin induced cell apoptosis and caused phosphorylation of ASPP2 at ser92/ser361 in the HCT116 cells. The apoptosis rate of Aw and Am plasmids-transfected cells were (3.8 ± 1.0)% and (3.9 ± 1.2)% respectively, statistically with a non-significant difference (P > 0.05) in comparison with that of the GFP plasmid-transfected cells [(4.0 ± 0.8)%]. After oxaliplatin treatment, the apoptosis rate of Aw plasmid-transfected cells was (46.7 ± 3.9)%, significantly higher than that of the Am and GFP plasmid-transfected cells [(40.1 ± 10.2)% and (37.1 ± 6.9)%, respectively, P < 0.05], however, there was no statistically significant difference (P > 0.05) between Am and GFP plasmid-transfected cells. These results indicate that phosphorylated ASPP2 promoted the oxaliplatin-induced apoptosis of HCT116 cells through a p53-dependent pathway. Phosphorylation status of ASPP2 influenced its binding activity to p53.</p><p><b>CONCLUSION</b>Phosphorylation status of ASPP2 modulates p53 apoptotic function in oxaliplatin-induced apoptosis of colorectal cancer HCT116 cells.</p>
Subject(s)
Humans , Apoptosis , Apoptosis Regulatory Proteins , Metabolism , Colorectal Neoplasms , Metabolism , HCT116 Cells , Organoplatinum Compounds , Phosphorylation , Tumor Suppressor Protein p53 , Genetics , MetabolismABSTRACT
Objective To investigate the role of apoptosis stimulating protein 2 of p53 (ASPP2)in the apoptosis,cell cycle and autophagy of starvation-induced colorectal cancer HCT116 p53 +/+ (p53 wild-type) cell line.Methods Six groups were included:(1) control group; (2) green fluorescent protein adenovirus (rAd-GFP) infection group; (3)ASPP2 adenovirus (rAd-ASPP2) infection group; (4)starvation group; (5)rAd-GFP + starvation group; (6) rAd-ASPP2 + starvation group.HCT116 cells were infected with ASPP2 adenovirus (rAd-ASPP2),resulting ASPP2 gene over-expression.The apoptosis,autophagy and cell cycle changes were induced by culturing with serum-free medium for 24 h.Apoptosis was evaluated by Calcein/PI uptaking test,and autophagy was observed by counting the red fluorescent protein autophagy plasmid CFP-Lc3 which was transfected into cytoplasm.Cell cycle was detected by flow cytometry.Statistical analysis was performed by one-way analysis of variance (ANOVA).Results Over-expressed ASPP2 was found to significantly promote starvation-induced HCT116 apoptosis and autophagy.The cell apoptosis rate in rAd-GFP + starvation group was 10.00％ ± 1.42％,and 18.44％ ±2.06％ in rAd-ASPP2 + starvation group(q =9.548,P =0.000).The cell autophagy rate in rAd-GFP+ starvation group and rAd-ASPP2 + starvation group was 35.00％ ± 5.34％ and 57.61％ ± 6.06％ respectively(q =7.657,P =0.000).Over-expressed ASPP2 accelerated HCT116 G2/M arrest under starvation,but resulted in both G0/G1 and G2/M arrest without starvation.Conclusion These results suggest that ASPP2 can promote starvation-induced HCT116 p53 +/+ cells apoptosis and autophagy,and affect the cell cycle.
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Objective To investigate the role of ASPP2 (apoptosis stimulating protein 2 of p53,ASPP2) in starvation-induced autophagy and apoptosis of colorectal cancer HCT116 p53-/-(p53 gene deletion) cell line.Methods The study included three experiment groups:green fluorescent protein adenovirus (rAd-GFP) infection group,autophagy inhibitor LY294002 treatment group and ASPP2 adenovirus (rAd-ASPP2) infection group.Celluar autophagy and apoptosis were induced by coculturing with serum-free medium for 0 h,24 h,48 h.Apoptosis level was detected by Calcein/PI uptaking test.Autophagy level was observed under the fluorescence microscope via transfection with cerise fluorescent protein autophagy plasmid CFP-Lc3.Results In control group,starvation for 24 hours significantly promoted autophagy of HCT116 cells (0 h:1.04 ±0.24; 24 h:12.17 ±0.86,P ＜0.05),while apoptosis was not increased (0 h:2.01％ ±0.06％; 24 h:3.23％ ±0.34％,P ＞0.05).With 48 h starvation,autophagy(0 h:1.04 ±0.24; 48 h:21.09 ±3.32) and apoptosis(0 h:2.01％ ±0.06％ ; 48 h:30.20％ ±3.18％)of HCT116 increased (P ＜ 0.05).With the use of LY294002 apoptosis induced by 24 h starvation significantly increased (rAd-GFP group:3.23％ ± 0.34％ ; LY294002 group:15.68％ ± 1.24％,P ＜0.01),but aopotosis under 48 h starvation decreased (rAd-GFP group:30.20％ ± 3.18％; LY294002group:25.44％ ± 3.01％,P ＜ 0.05).With ASPP2 transfection,autophagy under 24 h starvation significantly declined (rAd-GFP group:12.17 ± 0.86,ASPP2 group:1.45 ± 0.45,P ＜ 0.01),and apoptosis increased(rAd-GFP group:3.23％ ± 0.34％ ; ASPP2 group:10.45％ ± 0.81％,P ＜ 0.05).Both autophagy (rAd-GFP group:21.09 ± 3.32; ASPP2 group:29.93 ± 3.48) and apoptosis (rAd-GFP group:30.20％ ±3.18％ ; ASPP2 group:36.72％ ±2.74％) were higher than that in controls under 48 h starvation (P ＜ 0.05).Conclusions ASPP2 probably promotes apoptosis of colorectal cancer cells by two-way regulated autophagy.
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P53 protein plays a crucial role in inhibition tumor development.It will accumulate in cells in the condition of DNA damage,oncogenes activation or stress.As a nuclear transcription factor,P53 can transactivate the genes which correlate with apoptosis,cell cycle control and other procedures. Current research reveals P53 outside the nucleus can induce apoptosis and inhibit autophagy which contributes to its function of tumor suppression.The study of the extranuclear function of P53 is beneficial to further understanding the mechanism of P53 in the genesis and development of tumor.