The Role Of Cell Cycle-related Proteins In The Regulation Of Hypofrationated Radiosensitivity Of NSCLC

Objectives:1. To obtain a stable non-small cell lung cancer radioresistant cell lines by conventional fractionated radiotherapy(A54-2R) or hpofractionated radiotherapy(A54-4R).2. To investigate the effects of cell cycle redistribution in the establishment of radioresistance in NSCLC, as well as the expression of cell cycle-related proteins.Methods:1. NSCLC cell line A549 cells were treated with 6MV X-ray irradiation with conventional fractionated(2Gy/d, 5f/w, 17f) or hypofractionated irradiation(4Gy/d, 3f/w, 7f), which were named as A54-2R and A54-4R respectively.2. Flow cytometry were used to exam the distribution of cell cycle after the A549-NC, A549-2R and A549-4R cells were challenged by 0 Gy, 2 Gy and 4 Gy,respectively.3. m RNA micro-array screening were used to exam the expression of cell cycle-related genes.4. Western Blot were used to exam the expression of cell cycle-related proteins 12 hours after the A549(A549-NC), A549-2R and A549-4R cells were challenged by0 Gy, 2 Gy and 4 Gy.5. Immunohistochemical staining were used to detect expression of Aurora A、P53and P21 in 58 tumor samples from resected primary NSCLCs, and the prognostic value of Aurora A、P53 and P21 expression in curatively resected non-small cell lung cancer(NSCLC) were investigated.Results:1. After a period of irradiation of the A549 cell line, we finally obtained two radiation resist cells(A549-2R and A549-4R). The irradiated A549 cells demonstrated changes in their morphologies. Compared with parental A549 cells,The A549 cells with irradiation developed a cobblestone-like morphology,disrupted cell-cell adhesion and more scattered appearance.2. The G1-phase fraction was almost the same in size in A549-NC and A549-2Rcells, while the G1-phase fraction was decreased and S-phase was increased in A549-4R cells. Exposure to 2 Gy increased the G1-phase fraction in A549-NC cells with the decrease in the S-phase fraction, a typical G1/S check point response. The G1/S block was not observed in A549-2R and A549-4R cells.Instead of G1 arrest, long-term FR cells were arrested in S-phase or G2-phase after 2 Gy.3. The result of m RNA micro-array screening showed that compared with A549-NC the transcription of cyclin D and cyclin E in A549-2R increased more than 2times,while the transcription of P53、P21、CDK2、CDK4、CDK6 were near the same between A549-NC and A549-2R. Compared with A549-NC, the transcription of CDK4、CDK6 and cyclin E in A549-4R increased more than 2times,while the transcription of P53、P21、CDK2 and cyclin D were near the same between A549-NC and A549-4R. Compared with A549-2R, the transcription of P21 in A549-4R decreased more than 2 times, while the transcription of P53、CDK2、CDK4、CDK6、cyclin D and cyclin E were near the same between the two groups.4. The expression of P53、P53 Ser15、P53 Ser20 and total P21 were near the same between A549-NC, A549-2R and A549-4R, while cytoplasm p21 were increasing with higher radiation in A549-NC, A549-2R and A549-4R. Compared with A549-NC, the expression of cyclin D1 and CDK6 was increased significantly in A549-2R and A549-4R, while the expression of CDK2、CDK4 and cyclin D3 were near the same between the two groups. The expression of cell cycle related proteins were near the same between A549-2R and A549-4R.5. The positive rates of Aurora A、P53 and P21 expression were 89.7%(52/58)、46.6%(27/58) and 31.0%(18/58) respectively. Over survival(OS) was shorter with higher Aurora A expression, with3-year survival rates 75% vs.46%(P=0.039).Patients with higher p53 expression showed shorter OS with3-year survival rates80.6% vs. 55.6%( χ2=4.39, P=0.036). Patients with higher p21 expression showed shorter OS with 3-year survival rates 77.5% vs. 50%(χ2=4.56,P=0.033).Conclusions:1. The mechanism of redioresistance in A549-2R might be cyclin D1 overexpressionwhich result in the invalidation of G1/S chechpoint, and the overexpression of cytoplasm p21 do some roles in the invalidation of G1/S chechpoint.2. The mechanism of redioresistance in A549-4R is due to the invalidation of G1/S chechpoint and accumulation of cells in S-phase. The reason which result in the invalidation of G1/S chechpoint was the same to A549-4R, which include the overexpression of cyclin D1 and cytoplasm p21.3. The p53-p21 medicated G2/M checkpoint was normal in A549-NC, A549-2R and A549-4R.4. Detection of positive Aurora A 、 P53 and P21 expression may be a useful predictive indicator of prognosis. Overexpression of Aurora A 、 P53 and P21 showed an independent association with shorter OS in NSCLC patients.