Role of overexpression of the Sp1 and Sp3 transcription factors in the malignant transformation of human fibroblasts.

摘要

Two kinds of genes are involved in the malignant transformation of a normal cell: oncogenes with dominant gain of function and tumor suppressor genes with recessive loss of function. Alteration in functions of these genes breaks the tight control over the proliferation and death of normal cells, and finally leads to the progressive conversion to cancer cells. Sp1 is a transcription factor for many genes, including genes that play an important role in tumorigenesis. Recently, overexpression or higher binding activity of Sp1 has been found in several types of human cancers, including pancreatic adenocarcinoma, breast carcinoma, gastric carcinoma, and thyroid carcinoma.; To investigate the role of overexpression of Sp1 in the malignant transformation of human fibroblasts, I transfected an Sp1 U1snRNA/Ribozyme into two human fibrosarcoma cell lines, malignantly transformed in culture by a carcinogen or overexpression of an oncogene, and into a patient-derived fibrosarcoma cell line. Several cell strains expressing the Sp1 U1snRNA/Ribozyme showed significant decrease in Sp1 level. When injected into athymic mice, these cell strains with near normal levels of Sp1 failed to form tumors or did so only at a greatly reduced frequency and with a much longer latency. They showed spindle-shaped morphology and exhibited increased apoptosis and decreased expression of several genes linked to cancer, viz., epithelial growth factor receptor, urokinase plasminogen activator (uPA), uPA receptor, and vascular endothelial growth factor. These results strongly suggest that overexpression of Sp1 plays a causal role in malignant transformation of human fibroblasts and that for cancers in which it is overexpressed, Sp1 constitutes a target for therapy.; To determine how expression of Sp3 is regulated and the basis for the coordinated expression of Sp1 and Sp3 in human fibrosarcoma cell lines, I isolated 2.1 kb of the 5'-flanking region of the human Sp3 gene and inserted it upstream of the firefly luciferase reporter gene. Deletion analysis showed that the fragment (minimal promoter) spanning nt -339 to -39 (relative to the ATG translation start codon) conferred the same activity as that of the 2.1 kb promoter. Within the minimal promoter there are two putative Sp1/Sp3 binding sites. Mutation studies demonstrated that each of the two putative Sp1/Sp3 binding sites is required for promoter activity. Gel shift assays showed that both Sp1 and Sp3 bound to these two sites. In human embryonic kidney (HEK 293) cells and SL2 insect cells, Sp1 caused strong activation of the Sp3 minimal promoter. In contrast, in insect cells, Sp3 was a weak activator for the Sp3 minimal promoter, and in HEK 293 cells, it repressed activation. This suggests that Sp3 requires secondary modification(s) in order to function as a repressor.