外源性PTEN增强放射线诱导胰腺癌ASPC-1细胞G2/M期阻滞

EXOGENOUS PTEN ENHANCES RADIATION-INDUCED G2/M ARREST OF PANCREATIC CANCER CELL LINE ASPC-1

目的观察外源性PTEN在乏氧及放射前后对胰腺癌细胞系ASPC-1细胞周期及克隆形成的影响。方法将质粒pEAK8和pEAK8-PTEN分别转染ASPC-1细胞,获得ASPC-1-pEAK8细胞及ASPC-1-pEAK8-PTEN细胞,将ASPC-1、ASPC-1-pEAK8和ASPC-1-pEAK8-PTEN细胞各分成常氧组、乏氧组、照射组、乏氧照射组。乏氧组施加乏氧(1%O2)处理,乏氧时间为24 h,照射组接受4Gy单次照射,乏氧照射组在乏氧下进行照射。应用Western印迹杂交、流式细胞术、成克隆分析法检测外源性PTEN对ASPC-1细胞PTEN蛋白表达、细胞周期分布及细胞克隆形成能力的影响。结果ASPC-1-pEAK8-PTEN细胞较ASPC-1、ASPC-1-pEAK8细胞PTEN蛋白增加明显。常氧下,ASPC-1-pEAK8-PTEN细胞较ASPC-1、ASPC-1-pEAK8细胞G2/M期细胞增多,并且放射线进一步增强了G2/M期细胞阻滞。乏氧8h后,ASPC-1-pEAK8-PTEN细胞较ASPC-1、ASPC-1-pEAK8细胞凋亡比例显著提高。常氧及放射后,ASPC-1、ASPC-1-pEAK8、ASPC-1-pEAK8-PTEN细胞克隆形成率分别为33.33±8.38%、31.67±4.32%、24.00±3.90%和5.53±0.52%、5.33±0.74%、3.73±1.20%。乏氧及放射后,细胞克隆形成率分别为29.67±4.97%、29.50±3.39%、19.83±5.12%和12.08±0.78%、11.17±0.73%和7.38±0.58%。结果表明,ASPC-1-pEAK8-PTEN细胞较ASPC-1,ASPC-1-pEAK8细胞在照射及乏氧前后克隆形成率均明显降低。结论外源性PTEN可使胰腺癌ASPC-1细胞阻滞在G2/M期,增强乏氧诱导细胞凋亡,增强放射线诱导ASPC-1细胞G2/M期阻滞的能力,提高放射线对ASPC-1细胞在常氧及乏氧下的细胞杀伤。

Objective To investigate the effect of phosphatase and tensin homologue deleted on chro- mosome ten （PTEN） on cell cycles and plating efficiency （PE） in human pancreatic cancer cell line ASPC- 1 under hypoxia and/or radiation exposure. Methods After transfection with a eukaryotic expression plasmid （pEAKS） containing PTEN or not in vitro by lipofectin, the ASPC-1 cells were named ASPC-1- pEAK8-PTEN or ASPC-1-pEAK8. ASPC-1, ASPC-I-pEAK8 and ASPC-1-pEAK8-PTEN cells were assigned to the normal oxygen, hypoxia, radiation, and hypoxia radiation groups, respectively. The radiation group and hypoxia （1% O2 for 24h） radiation group were treated with 4Gy X-ray. Western blotting, flow cytometry and clonogenic survival assay were adopted to measure the expression of PTEN protein, the effect of PTEN on cell cycle and plating efficiency of cells, respectively. Results Weak expression of PTEN protein was found in ASPC-1 and ASPC-I-pEAK8 cells while significantly excessive PTEN proteins were found in ASPC-1-pEAK8-PTEN ceils. Under normal oxygen, the cell cycle analysis demonstrated an increase of the G2/M phase in the ASPC-1-pEAK8-PTEN cells than in the ASPC-1 or ASPC-1-pEAK8 cells. Moreover, the cell blockage of G2/M phase was obviously increased in the ASPC-1-pEAK8-PTEN cells than in the ASPC-1 or ASPC-1-pEAK8 cells after exposure to 4Gy X-ray. The cellular apoptosis of ASPC-1-pEAK8-PTEN cells afte. 8h hypoxia was remarkably higher than that of ASPC-1 or ASPC-1-pEAK8 cells. The ASPC-1, ASPC-1-pEAK8 and ASPC-1-pEAK8- PTEN cell plating efficiency was 33. 33±8.38%, 31. 67±4. 32%, 24. 00±3.90% and 5. 53±0. 52%, 5. 33±0.74%, 3. 73±1.20% under normal oxygen and after radiation, respectively; and it was 29. 67± 4.97%, 29.50±3.39%, 19. 83±5.12% and 12. 08±0. 78%, 11.17±0.73%, 7.38±0.58% under hypoxia and after radiation, respectively. The results showed that the plating efficiency of ASPC-1-pEAK8- PTEN cells was lower than that of ASPC-1 or ASPC-1-pEAK8 cells whether under hypoxia and radiation or not. Conclusion Exogenous PTEN blocks pancreatic cancer ASPC-1 cells in the G2/M phase, enhances the cell apoptosis induced by hypoxia, increases the block in the G2/M phase of cell cycle after radiation, and decreases the plating efficiency of ASPC-1 cells under normal oxygen or hypoxia.