Studies on cytokines and proteases which regulate UV-induced mutation in human cells

为了阐明紫外线(主要是250 nm波长)照射的人体细胞中调节突变性的分子机制，特别是那些受蛋白酶和细胞因子调节的分子机制。对紫外光诱导的蛋白水解酶进行了部分纯化和鉴定，并从不同细胞的裂解产物中获得了具有较高活性的蛋白水解酶样品。样本中含有约20,000个MR的分子，其活性被止痛剂抑制，但被ALLNa1.2增强。利用聚合酶链式反应-差异显示技术，我们已经在人类干扰素(HuIFN)-β处理的Cockayne综合征(CS)成纤维细胞中寻找了特异性表达的基因。其中6个克隆的cDNA与数据库中未知基因的表达标签序列相似，其余1个克隆与着色性干皮病XPG基因的cDNA同源性。结合我们先前的发现，用人干扰素-β…预处理的CS细胞对紫外线(UV)杀伤的抵抗力增强更早于紫外线照射，提示XPG可能是HuIFN-β诱导的紫外线抗性基因之一。从对紫外线敏感的人RSB细胞中建立了耐紫外线的UV‘-10细胞。我们在这里使用差异显示方法寻找UV‘-10细胞和RSB细胞之间差异表达的基因，以阐明紫外线抗性的分子机制。从首次筛选的514个基因片段中获得了13个候选基因片段。Northern分析表明，在这些片段中，有3个片段在UV‘-10细胞中的表达高于RSB细胞。核苷酸序列分析表明，3个片段中有2个编码核蛋白(NPM)和T-纤溶酶。紫外线照射可诱导UV‘-10细胞NPM mRNA的表达，但对RSB细胞无明显影响。UV’-10细胞经反义NPM基因和T-纤溶酶基因转染后，其对紫外线杀伤的抵抗力增强，至少在受试的人细胞中是如此。较少

In order to clarify molecular mechanisms which regulate mutability in UV(mainly 250nm wavelength) - irradiated human cells, particularly those regulated by proteases and cytokines.1. Partial purification and characterization of UV-induced proteases were preformed and protease samples were obtaind from cell lysates of variant cells with increased levels of the induced protease activity. The samples contained molecules with about 20,000 Mr, the activity of which is inhibited by antipain but enhanced by ALLNa1.2. Using PCR-differential display, we have searched for genes expressed specifically in human interferon (HuIFN)- beta -treated Cockayne syndrome (CS) fibroblast cells. The cDNAs of six clones were similar to expression tagged sequences from unknown genes in databases and the remaining one was identical to the cDNA of the xeroderma pigmentosum XPG gene. Together with our previous finding of increased resistance to ultraviolet (UV) cell-killing of CS cells pretreated with HuIFN- beta … More prior to UV irradiation, suggest that XPG might be one of the genes possibly involved in the HuIFN- beta-induced UV-resistance.3. UV-resistant UV'-10 cells were established from UV-sensitive human RSb cells. We searched here for genes expressed differentially between UV'-10 and RSb cells using a differencial display method to elucidate the molecular mechanisms underlying the UV-resistance. Thirteen candidate cDNA fragment were obtained from 514 mRNA species first screened. Among the cDNA fragments, 3 revealed increased mRNA expression in UV'-10 cells compared with RSb cells by Northern analysis. Nucleotide sequence analysis identified 2 of the 3 cDNA fragments as encoding nucleophosimin (NPM) and T-plastin. The expression of NPM mRNA was induced after UV irradiation in UV'-10 cells but not in RSb cells, whereas irradiation did not affect the expression of T-plastin mRNA.UV'-10 cells transfected with antisense cDNA for NPM mRNA and possibly T-plastin genes may contribute to the incresed resistance to UV cell-killing, at least in the human cells tested. Less