Abstract
中期染色體被認為和細胞的輻射敏感機制有關， 在一株中國倉鼠卵巢細 胞 (CHO) 對於輻射線敏感的突變細胞株 xrs-5中發現， 其中期染色體的 形狀比起在野生型 K1 細胞中看到的要來的緊密而縮短。 研究者認為這 種染色體的異常現象可能會使得當 DNA 遭到破壞時， 許多修補蛋白無法 進入。 然而在我的實驗中發現，另一株同樣也對輻射敏感的突變株 xrs-6 卻仍有約 30% 的中期染色體呈現和野生型相同的細長狀。 而且在 一株具有和 K1 細胞相同抗性的自然回復株， SR-16中仍然有染色體較 K1 細胞緊縮的現象， 因此我們認為染色體的形狀和輻射敏感並沒有直接 的關係。最近研究發現 Ku 蛋白質的一個次單位： Ku80是造成 CHO輻射 敏感突變株對於輻射敏感的原因。 Ku 蛋白質是一個叫做需 DNA 蛋白質 激脢 (DNA-PK) 的一部份。 Ku80 蛋白質的缺陷使 DNA-PK 失去活性， 導致 xrs mutants 對輻射線特別敏感。 DNA-PK 參與在 DNA 修補的途徑 可能是在細胞受輻射照射時磷酸化一些修補蛋白。 我們試圖找出當細胞 受輻射時， 在 K1細胞中有那些蛋白質磷酸化的程度較 xrs mutant 高。 這些蛋白質可能就是 DNA-PK 的 in vivo 受質。 但是我們用 SDS-PAGE 分析並沒有發現有任何差異， 可能由於 SDS-PAGE 解析度的限制， 我們 將改用 2-D SDS-PAGE 來得到較佳的解析度。在許多人類的細胞中發現， X-ray 會誘導 G1 arrest 和 p53的增加，G1 arrest 的產生需要野生型 p53 的增加。 在我的實驗發現 X-ray 不會誘導 CHO 的 G1 arrest， 也 沒有使 p53 蛋白質增加。 我們仍須確定是否因為 CHO 的 p53 是突變型 所以不會被 X-ray 所誘導， 抑或 X-ray 對 CHO 的 p53 有不同的影響 The morphology of metaphase chromosome morphology was thought to be related to the radiosensitivity. It has been found that metaphase chromosomes in X-ray sensitive CHO mutant xrs-5 cellswere more contract and shorter than in the wild type K1 cells. It wassuggested that this abnormality might inhibit repair enzymes from entering the damaged DNA. In this work, I found about 30% of xrs-6 cells which are as sensitive to X-ray as xrs-5 cells, had metaphase chromosomes like those in K1 cells. Furthermore, a spontaneous revertant SR-16, which is as resistant to X-ray as the wild type K1 cells, has been found to have short and contract chromosomes. Our results therefore indicate that chromosome morphology is not necessarily related to radiosensitivity. Previous results indicated that the defect in Ku80, a subunit of Ku protein, was the reason for the X-ray sensitive phenotype of CHO xrsmutants. Ku protein is a component of DNA-dependent protein kinase (DNA-PK). The defect in Ku80 diminished the DNA-PK activity and resulted in hyper- radiosensitivity of xrs mutants. DNA-PK was thought to participate in the repair pathway by phosphorylating some repair factors when cells were irradiated. I tried to find ifcertain proteins were hyper-phosphorylated in K1 but not in xrs mutants after irradiation. These proteins could be the repair factors and in vivo substrates of DNA-PK. No difference has been found when SDS-PAGE was used for analysis. Because of the limited resolution of SDS-PAGE, 2-D PAGE will be employed later to get better resolution. Exposure of certain human cells to X-ray will cause growth arrest at the G1 phase of the cell cycle. This X-ray induced G1 growth arrest requires wild type p53 gene product and is accompanied by the increase of p53 protein. My results showed X-ray did not induce G1 arrest in CHO cells and did not cause the increase of p53 protein. It is to be determined whether the p53 in CHO cells is the mutant form therefore the level can not be increased