New Assay Methods for Monitoring Radiation Effects.

一种新出现的辐射后的p41蛋白被鉴定为可能的机制(S)和作为辐射效应的标志物的可行性。P41的含量具有辐射剂量和时间依赖性。新制备的抗p41多克隆抗体也对不同肿瘤细胞系的p42进行了免疫印迹分析。对p41和p42的氨基酸分析表明，p41是p42的切割产物。P42通过蛋白质合成在体外产生，并以此为底物鉴定MOLT4细胞X射线照射后激活的特定caspase(未发表数据)。分析1、3、6、10GyX射线照射后HT-29细胞数天内MUC1粘蛋白的表达。糖基化的MUC1粘蛋白(500 kDa和390 kDa)和低糖基化的MUC1粘蛋白(350 kDa和240 kDa)随剂量增加而增加，在4d时达到最大值。含MUC1基因5‘侧翼序列的CAT法检测MUC1基因的转录活性。在照射后第2天至第4天，HT-29细胞CAT活性的诱导率升高。这些发现和RT-PCR检测到的mRNA增加表明，X射线照射上调了HT-29细胞中MUC1基因的转录活性。在实际使用这些标记来监测辐射效应之前，还需要进一步的表征，尽管目前的结果表明这种可能性。

A newly appearing p41 protein after X-irradiation of MOLT4 cells was characterized to access the mechanism(s) and feasibility as a marker of radiation effect. Amount of p41 was radiation dose and time dependent. Newly prepared polyclonal antibody against p41 also stained p42 on western blotting analysis of various tumor cell lines. Amino acid analysis of p41 and p42 showed that p41 was a cleavage product of p42. P42 was generated in vitro by protein synthesis and used as a substrate to identify a particular caspase (unpublished data) activated after X-irradiation of MOLT4 cells.Expression of MUC1 mucin of HT-29 cells after X-irradiation of 1, 3, 6, 10 Gy were analyzed for several days during cell death. Glycosylated MUC1 mucins (500 kDa and 390 kDa) and underglycosylated MUC1 mucins (350 kDa and 240 kDa) were found increased in dose-dependent manner reaching maximum level in 4 days. Transcriptional activity of MUC1 gene was analyzed by CAT assay containing the 5'-flanking sequence of MUC1 gene. Induction of CAT activity in HT-29 cells was found elevated from day 2 to 4 of X-irradiation. These findings with increased mRNA determined by RT-PCR indicate that transcriptional activity of MUC1 gene in HT-29 cells was upregulated by X-irradiation.Further characterization is required prior to practical use of these markers to monitor radiation effect, although the present results indicate such possibility.