Regulation of expression of N-methylpurine DNA glycosylase in human mammary epithelial cells: role of transcription factor AP-2.
The DNA repair enzyme, N-methylpurine DNA glyclosylase (MPG), is overexpressed in breast cancer as compared with its expression in normal breast epithelial cells. In an effort to determine the mechanism responsible for this difference in expression, we studied rates and regulation of transcription of the MPG gene in normal (HMEC), spontaneously immortalized (MCF10A), and malignant (T47D) mammary epithelial cells. Steady state levels of MPG mRNA are 3-4-fold greater in T47D cells than in MCF10A cells. Nuclear "run-off" transcription measurements revealed MPG transcription rates to be approximately 3-fold greater in the tumor cells than in normal cells. Characterization of the MPG promoter by deletion analysis and transient transfection experiments revealed that all basal promoter activity resided between nucleotides -227 and -81 upstream from the ATG translation start site. Constructs containing this region were expressed at 4-fold greater levels when transfected into malignant T47D cells (56 x baseline) than in MCF10A cells (14 x baseline). Computer database analysis of the region of nucleotides -227 to -81 revealed multiple overlapping Sp1 consensus binding sites and two overlapping consensus AP-2 binding sites located between bases -181 and -169. Electrophoretic mobility shift assays indicated that while Sp1 bound this region of the promoter, nuclear extracts from both cell types contained equal Sp1 binding activity. In contrast, AP-2 binding activity was significantly greater in T47D cells, and Western blots confirmed increased AP-2 protein levels in these cells. Cotransfection into MCF10A cells of the MPG promoter construct and an AP-2 expression plasmid increased MPG promoter activity 2.1-fold. Cotransfection of a dominant negative mutant of AP-2 into T47D cells reduced the extent of MPG promoter-driven transcription by 50%. To investigate the functional significance of the two overlapping AP-2 consensus binding sites, each site was mutated separately. Mutation of the upstream site decreased promoter activity by 15%, but mutation of the downstream site decreased promoter activity by 45% and abolished AP-2 binding to the promoter sequence. These data suggest that AP-2 is important in regulating MPG expression in breast cancer cells, and that the increased amount of AP-2 in these cells plays a major role in directing the increased expression of MPG.
Cerda
S R
SR
Division of Hematology/Oncology, Department of Medicine, and Department of Micro/Immunology, Lurie Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Chu
S S
SS
Garcia
P
P
Chung
J
J
Grumet
J D
JD
Thimmapaya
B
B
Weitzman
S A
SA
D015153:Blotting, Western; D001940:Breast / Q000201:enzymology* / Q000473:pathology; D001943:Breast Neoplasms / Q000201:enzymology* / Q000473:pathology; D045647:DNA Glycosylases; D004260:DNA Repair / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000502:physiology*; D004847:Epithelial Cells / Q000201:enzymology*; D005260:Female; D005609:Free Radicals; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic / Q000235:genetics; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000502:physiology*; D014162:Transfection
D004268:DNA-Binding Proteins; D005609:Free Radicals; D007202:Indicators and Reagents; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D008156:Luciferases; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases