Characterization of the human L-plastin gene promoter in normal and neoplastic cells.

Abstract

Plastins are a family of human actin-binding proteins (isoforms) which are abundantly expressed in all normal replicating mammalian cells. One isoform, L-plastin, is constitutively expressed at high levels in hemopoietic cell types while T-plastin is constitutively expressed in all non-hemopoietic cells of solid tissues that have replicative potential (fibroblasts, endothelial cells, epithelial cells, melanocytes, etc.). L-plastin is, however, constitutively synthesized in many types of malignant human cells of solid tissues suggesting that its expression is induced during tumorigenesis. The frequency of L-plastin induction in some cancers of the steroid-regulated female reproductive tract (breast, ovary, uterus, and placenta) appears to be especially high (79% in a limited survey). To learn the mechanism of L-plastin gene activation accompanying tumorigenesis, we have begun to characterize the promoter and regulatory elements of the L-plastin gene. Transcription initiation from this promoter was found to occur at multiple sites and as near as 10 base pairs from the 3'-side of the TATAAA box. The promoter and its flanking DNA were cloned and sequenced to identify potential regulatory elements that participate in the induction of the L-plastin gene in neoplastic cells. Examination of upstream sequences revealed the existence of two potential progesterone, one potential estrogen, and four potential Ets-1 responsive elements flanking the promoter. A 315-base pair fragment spanning the TATAAA box and a potential Sp1-binding site exhibited maximum promoter activity using CAT as a reporter while longer promoter fragments extending into upstream flanking sequences spanning the hormone receptor-response elements exhibited reduced promoter activity. An expression vector, pHLPPr-1-neo, was constructed using a 5.1-kilobase pair EcoRI-HindIII fragment of the L-plastin gene that contained the potential upstream regulatory elements, the TATAAA box, and part of the first exon. This promoter could direct the constitutive expression of the reporter beta-galactosidase at high frequency in transfected colonies of transformed cells that express L-plastin constitutively; by contrast, this promoter was virtually inactive in transfected colonies of normal fibroblasts and it exhibited a low frequency of constitutive activation in transfected colonies of in vitro SV40-transformed fibroblasts which did not exhibit L-plastin expression. The utility of this recombinant promoter in determining the mechanism(s) that leads to activation of the L-plastin gene in tumor cells is discussed. The potential significance of regulation of the L-plastin gene by reproductive hormones in cancers arising in hormone-responsive tissues is also discussed.