Tumor suppression by using gelsolin mutants

1.从人活化的c-Ha-ras癌基因转化的NIH/3 T3细胞（EJ-NIH/3 T3）的平坦回复突变体R1中分离到一种突变型凝溶胶蛋白[His 321] gelsolin。[His 321]凝溶胶蛋白在位置321处具有组氨酸而不是脯氨酸残基，并且当其组成型表达时抑制EJ-NHI/3 T3细胞的致瘤性。[His 321]凝溶胶蛋白在体外与野生型凝溶胶蛋白相比具有降低的肌动蛋白丝切断活性和增加的成核活性。[His 321]凝溶胶蛋白在体外比野生型凝溶胶蛋白更强地抑制磷脂酶C γ 1对PtdInsP 2的水解，因为其对磷酸肌醇脂质的结合能力更高。我们的研究结果表明，节段S3包含突变是功能相关的凝溶胶蛋白的活动的调节，即使相关的肌动蛋白结合域是在节段1，2，4-6，和周围的区域残基321可能含有磷酸肌醇脂质结合位点。[His 321]凝溶胶蛋白酶的功能改变 关于我们 可能是ras转化细胞致瘤性丧失的重要原因.我们研究了凝溶胶蛋白在一些人膀胱癌细胞系和组织中的表达。在所有6个细胞系和18个肿瘤组织中的14个（77.8%），凝溶胶蛋白的表达与其在正常膀胱上皮细胞中的表达相比是不可检测的或极低的。此外，在将外源性人或小鼠真实凝溶胶蛋白cDNA导入人膀胱癌细胞系UMUC-2后，UMUC-2的凝溶胶蛋白转染子大大降低了体内的集落形成能力和致瘤性。这些结果提示凝溶胶蛋白在人膀胱癌的发生发展中起着重要的抑癌作用. PDGF或EGF的刺激在表达His 321的两个NIH/3 T3克隆中诱导的DNA合成远低于用单独的载体转染的两个克隆。这些结果表明，通过对PDGF和/或EGF的信号转导途径的影响，His 321突变的凝溶胶蛋白抑制NIH/3 T3的生长。少

1. A mutant gelsolin, [His321] gelsolin, was isolated from R1, a flat revertant of human activated c-Ha-ras oncogene-transformed NIH/3T3 cells (EJ-NIH/3T3). [His321] Gelsolin has a histidine instead of a proline residue at position 321 and suppresses the tumorigenicity of EJ-NHI/3T3 cells when it is constitutively expressed. [His321] Gelsolin has decreased actin-filament-severing activity and increased nucleating activity compared with wild-type gelsolin in vitro. [His321] gelsolin inhibits PtdInsP2 hydrolysis by phospholipase C gamma 1 more strongly than wild-type gelsolin in vitro because of its higher binding capacity for phosphoinositol lipid. Our results suggest that the segment S3 which contains the mutation is functionally relevant for regulation of gelsolin's activities even though the relevant actin-binding domains are in segments 1,2, and 4-6, and that the region around the residue 321 may contain a phosphoinositol-lipid-binding site. Altered functions of [His321] gelsolin mi … More ght be important for the loss of tumorigenicity of the ras-transformed cells.2. We examined the expression of gelsolin in a number of human bladder cancer cell lines and tissues. In all 6 cell lines and in 14 of the 18 tumor tissues (77.8%), gelsolin expression was undetectable or extremely low in comparison with its expression in normal bladder epithelial cells. Furthermore, upon the introduction of the exogenous human or mouse authentic gelsolin cDNA into a human bladder cancer cell line, UMUC-2, gelsolin transfectants of UMUC-2 greatly reduced the colony-forming ability and the tumorigenicity in vivo. These results suggest that gelsolin plays a key role as a tumor suppressor in human urinary bladder carcinogenesis.3. Stimulation by PDGF or EGF induced far less DNA synthesis in two NIH/3T3 clones expressing His321 that in two clones transfected with the vector alone. These results suggest that through the effects on the signal transduction pathway of PDGF and/or EGF His321-mutated gelsolin inhibits the growth of NIH/3T3. Less