Study On Apoptosis Of Hep-2 Cells And Apoptosis-Related Genes Induced By Ionizing Radiation

ObjectiveRadiation-induced apoptosis has been a hot spot in the study of tumorigenesis since the last decade and the recognition to it as one of the parts of radiation-induced cell death has initiated a series of investigations aiming at exploring the apoptotic response and radiosensitivity of cells to radiation. These strategies require the integration of both radiobiological concepts and the knowledge of the biochemical and molecular biology involved in apoptosis.Some previous studies have indicated a link between radiation and apoptosis, however the mechanism is not clear. The aim of this study is to characterize the patterns and possible mechanisms of ionizing radiation-induced apoptosis.Materials and Methods1. Cell cultureThe human laryngeal cancer cell line Hep-2,obtained from Cell Biology Institute of Shanghai, Chinese Academy of Science, was grown in RPMI 1640 supplemented with 10% FBS, penicilion (100U/ml) and streptomycin (100μg/ml) at 37℃in a 5% CO₂ humidified atmosphere. All the cells in log phase was used for the following experiments.2. Ionizing radiation treatmentCells were cultured in complete medium and treated with different doses of radiation from 0 (negative control) to 15Gy for various time intervals (0, 12, 24, 48, 72 hours).3. Apoptosis analysis by flow cytometry (FCM)Analysis of Annexin V/PI Positive cells: Flow cytometric analysis was per- formed on a FACSCalibur flow cytometer. Data acquisition and analysis were performed by the CellQuest software.4. Western blot and RT-PCR analysisThe protein and RNA expression levels of BCL-2, BAX, C-MYC, P15, P16, P21, P27 and mutant P53 were detected by Western blot and RT-PCR, respectively.5. Statistical analysisData were indicated as (?)±s. The data were analyzed by SPSS for Windows Version. 11.5 package. P＜0.05 was considered to be significant.Results1. Transmission electron microscope showed Hep-2 cell shrinkage and compaction of the nuclear chromatin, with the formation of sharply delineated, uniformly finely granular masses that became marginated against the nuclear envelope.2. Radiation-induced apoptosis was characterized as a time and dose dependent manner.In the negative control, few cells had the traits of apoptosis, while apoptosis in many cells under the treatment of radiation happened. With the extension of the culture time and increase of radiation doses, more cells underwent apoptosis. DNA fragmentation was another typical property of the apoptotic cells. The pattern of DNA fragmentation was match to the result of Annxin V/PI double staining which revealed radiation-induced apoptosis in a time and dose dependent manner. At 48 hour, apoptosis induced by 10Gy reached the peak compared to the other time and intensity points.3. Cell cycle analysis by FCMThe result showed that the cell cycle was blocked at G₁/G0 stage when treated with high dose of radiation (10Gy), In contrast,when treated with a lower dose (2.5Gy), it was blocked at G₂/M stage.4. Immunohistochemistry, Western blot and RT-PCR analysisThe results of Immunohistochemistry and Western blot revealed that BCL- 2, C-MYC, P15, P16, P21 and P27 levels were significant difference between the negative control group and the experimental ones, while BAX showed no significant difference. The mRNA levels of BCL-2, C-MYC and mutant P53 in experimental group by RT-PCR were down-regulated dependent on the time of radiation, while P15, P16, P21 and P27 in experimental group by RT-PCR were up-regulated.ConclusionIonizing radiation could induce both apoptosis and necrosis in a time and dose dependent manner. The expression levels of P15, P16, P21 and P27 were up-regulated, while those of BCL-2 and C-MYC down-regulated BCL-2, P15, P16, P21 and P27 participated the process of radiation-induced apoptosis, probably independent of the tumor suppressor gene P53. While different expression of BCL-2, P15, P16, P21 and P27 in Hep-2 cells suggested that there would be more than one pathway in radiation induced-apoptosis. The rapid development and application of novel molecular radiobiological techniques should, in the near future, contribute to a better understanding of these mechanisms, which would in turn provide a basis strategy for more effective anti-cancer treatment.