Tumor suppression by using gelsolin mutants

1. 从人活化的c-Ha-ras癌基因转化的NIH/3T3细胞（EJ-NIH/3T3）的平坦逆转物R1中分离到一种突变凝胶[His321]凝胶。[His321] Gelsolin在321位有组氨酸而不是脯氨酸残基，当Gelsolin组成性表达时，Gelsolin可抑制EJ-NHI/3T3细胞的致瘤性。[His321]与野生型凝胶相比，Gelsolin的肌动蛋白丝切断活性降低，成核活性增加。[His321] gelsolin对磷酸肌醇脂的结合能力更高，因此在体外比野生型gelsolin更能抑制磷脂酶C γ 1对PtdInsP2的水解。我们的研究结果表明，尽管相关的肌动蛋白结合结构域位于第1、2和4-6段，但包含突变的片段S3在功能上与凝胶蛋白活性的调节有关，并且残基321周围的区域可能含有磷酸肌醇-脂结合位点。[His321]凝胶蛋白功能的改变可能是ras转化细胞致瘤性丧失的重要原因。我们检测了凝胶蛋白在许多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%）中，与正常膀胱上皮细胞相比，gelsolin的表达无法检测到或极低。此外，将外源人或小鼠凝胶蛋白cDNA导入人膀胱癌细胞系UMUC-2后，转染UMUC-2的凝胶蛋白大大降低了其在体内的集落形成能力和致瘤性。这些结果提示凝胶蛋白作为肿瘤抑制因子在人膀胱癌变中起关键作用。在两个表达His321的NIH/3T3克隆中，PDGF或EGF刺激诱导的DNA合成远低于单独转染载体的克隆。这些结果表明，通过对PDGF和/或EGF信号转导通路的影响，his321突变的gelsolin抑制了NIH/3T3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