| Abstract: |
Eukaryotic mRNAs contain 3'-untranslated regions (UTR) that are involved in posttranscriptional control of gene expression. AU-rich octanucleotide repeats, UUAUUUAU, present in the 3'-UTR of mature lymphokine and other cytokine transcripts, have been implicated in the regulation of mRNA stability and translational efficiency. For example, previous evidence suggests that AU-rich element (ARE) present in the 3'-UTR of murine tumor necrosis factor-α (TNF-α) can affect the posttranscriptional regulation of murine TNF-α gene expression in hematopoietic cells. Although cytokines are produced in epithelial cells, little is known about the regulation of TNF-α and other cytokine gene expression by 3'-UTR elements in human malignant epithelial cells. To better understand the function of the 3'-UTR of the human TNF-α gene in the regulation of TNF-α protein production in human epithelial cancer cells, a series of luciferase reporter constructs with portions of the 3'-UTR of human TNF-α was transfected into human breast carcinoma cell lines ZR-75-1 and ZR-75-1R (which overexpress TNF-α). The 3'- UTR of TNF-α markedly suppressed luciferase activity in both cell lines, and the suppression of activity was reversed by deletion of the AU-rich sequences. This suppression was quantitative, with six repeats causing more inhibition than two repeats. Increased levels of luciferase activity were observed 3 h after TNF-α stimulation in ZR-75-1 cells transfected by constructs containing AU-rich repeats. In addition, cytoplasmic extracts from both cell lines were assayed for factors that bind to the 3'-UTR of human TNF-α mRNA. RNA-protein binding activities were found in both cell lines. Competition studies showed that these proteins specifically bound to AU-rich repeats present in the 3'-UTR of TNF-α. No binding activity was observed when the AU-rich repeats were deleted. TNF-α exposure markedly increased activity of several RNA-binding proteins, especially a novel M(r) 50,000- 55000 RNA-binding protein. The binding activity in untreated ZR-75-1R was higher than that in untreated ZR-75-1 cells, suggesting that the level of RNA-protein binding correlates with the expression level of TNF-α in human epithelial cancer cells and that the RNA-binding proteins may control expression of TNF-α in ZR-75-1 cells. We conclude that the AU-rich repeats in the 3'-UTR of human TNF-α mRNA may regulate gene expression in human epithelial cancer cells by binding to AU sequence-specific proteins, including a previously undescribed M(r), 50000-55,000 protein not observed in hematopoietic cells. |
