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Signaling and Regulation

Cell Type-Dependent Regulation of hMLH1 Promoter Activity Is Influenced by the Presence of Multiple Redundant Elements¹

Departments of ¹ Medicine and ² Pathology, Laboratory for Molecular Oncology, LDS Hospital, Salt Lake City, UT

Requests for reprints: Kevin A. Strait, Cancer Research Laboratory, LDS Hospital, 325 8th Avenue, Salt Lake City, UT 84143. Phone: (801) 408-1558; Fax: (801) 408-5822. E-mail: ldkstrai{at}ihc.com

Immunohistochemical analysis confirmed the presence of MLH1 protein in A2780 ovarian cancer cells and its absence in this same cell line on acquired resistance to cisplatinum (A2780/CP). Transfection of a -1781-bp hMLH1 promoter construct into either A2780 or A2780/CP cells produced similar (30-fold) induction of luciferase, an indication that the transcriptional machinery for hMLH1 expression remains intact. hMLH1-luciferase activity was also unaffected by re-expression of hMLH1 following treatment of A2780/CP cells with the methylase inhibitor 2'-deoxy-5-azacytidine. Serial 5'-deletion studies of the hMLH1 promoter region in ovarian cancer cells localized transcriptional enhancers to a region (-250 to -151 bp) that excludes the previously identified CCAAT element (-282) active in HeLa cells. When these same deletion constructs were transfected into HeLa cells, deletion of the CCAAT-containing region caused a significant loss of promoter activity, an indication of cell-specific use of enhancer elements. Finally, a series of internal deletion and linker mutation studies of the -250 to -151 bp ovarian enhancer region revealed that the hMLH1 promoter contains multiple redundant enhancer elements capable of independent promoter activation and may explain the association of this region with methylation silencing of hMLH1.

Key Words: hMLH1 • CpG methylation • Redundant elements • Mismatch • Repair