Functional analysis in the search for a putative tumor suppressor gene based upon the changes in the expression of membrane proteins in human tumor cells

以前对HeLa和正常成纤维细胞杂交细胞的研究表明，肿瘤的发生可能是由11号染色体上一个可能的肿瘤抑制基因控制的。在本研究中，我们发现致瘤细胞杂交瘤细胞表达葡萄糖转运蛋白亚型GLUT1和GLUT3，而非致瘤细胞仅表达GLUT1。在从非致瘤细胞分离的伽玛射线诱导的致瘤克隆中，这些异构体的共同表达也很明显。GLUT3mRNA在致瘤细胞杂交瘤细胞中特异表达。当GLUT3稳定过表达时，对2-脱氧葡萄糖的亲和力显著增强。这些结果表明，随着对葡萄糖亲和力的增加，HeLa细胞中GLUT3的表达增强，这是由肿瘤抑制基因的功能障碍在转录水平上调节的。利用含有与荧光素酶基因相连的小窝蛋白启动子的表达载体，研究了可能的肿瘤抑制基因在基因表达中的作用，该载体的活性与小窝蛋白在这些杂交细胞中的表达平行。进一步研究了小窝蛋白启动子控制基因表达的功能区。在本项目期间，还开发了一种新型阳离子脂质体，可通过血清有效地将基因输送到体外和体内的人肿瘤细胞中。

Previos studies on human cell hybrids of HeLa and normal fibroblasts indicates that the tumorigenicy may be controlled by a putative tumor suppressor gene on chromosome 11. In this study, we found that tumorigenic cell hybrids express the glucose transporter isoforms GLUT1 and GLUT3 while non-tumorigenic cells express GLUT1 alone. The co-expression of these isoforms was also evident in gamma-ray-induced tumorigenic clones isolated from a non-tumorigenic cell. GLUT3 mRNA was specifically expressed in tumorigenic cell hybrids. When GLUT3 was stably overexpressed, the affinity for 2-deoxyglucose markedly increased. These results suggest that the enhanced GLUT3 expression in HeLa cell hybrids accompanied with the increased affinity for glucose is regulated at the transcriptional level by the dysfunction of the tumor suppressor gene. A possible role of the putative tumor suppressor gene in control of gene expression has been studied using an expression vector containing caveolin promoter linked to the luciferase gene, whose activities were parallel to the caveolin expression in these hybrid cells. Functional regions of the caveolin promoter to control the gene expression are further examined. A new cationic liposome for efficient gene delivery with serum into human tumor cells in vitro and in vivo was also developed during this project.