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Posttranscriptional Regulation of Protein Expression in Human Epithelial Carcinoma Cells by Adenine-Uridine-Rich Elements in the 3'-Untranslated Region of Tumor Necrosis Factor- Messenger RNA¹

Developmental Chemotherapy Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

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 the AU-rich element (ARE) present in the 3'-UTR of murine tumor necrosis factor- (TNF-) can affect the posttranscriptional regulation of murine TNF- gene expression in hematopoietic cells. Although cytokines are produced in epithelial cells, little is known about the regulation of TNF- 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- gene in the regulation of TNF- protein production in human epithelial cancer cells, a series of luciferase reporter constructs with portions of the 3'-UTR of human TNF- was transfected into human breast carcinoma cell lines ZR-75-1 and ZR-75-1R (which overexpresses TNF-). The 3'-UTR of TNF- 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- 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- 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-. No binding activity was observed when the AU-rich repeats were deleted. TNF- exposure markedly increased activity of several RNA-binding proteins, especially a novel M_r 50,000–55,000 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- in human epithelial cancer cells and that the RNA-binding proteins may control expression of TNF- in ZR-75-1 cells. We conclude that the AU-rich repeats in the 3'-UTR of human TNF- mRNA may regulate gene expression in human epithelial cancer cells by binding to AU sequence-specific proteins, including a previously undescribed M_r 50,000–55,000 protein not observed in hematopoietic cells.

¹ This work was supported by Grant R01-CA 63831 from the National Cancer Institute.

² To whom requests for reprints should be addressed, at Developmental Chemotherapy Service, Memorial Sloan-Kettering Cancer Center, Box 67, 1275 York Avenue, New York, NY 10021. Phone: (212) 639-2203; Fax: (212) 717-3272.

Received 8/ 4/97. Accepted 10/17/97.

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