© 1995 Oxford University Press
research-article |
Cell type-specific expression of human 8-oxo-7,8-dihydroguanosine triphosphatase in normal breast and skin tissues in vivo
1The Division of Radiobiology, The Department of Radiology Columbus, OH 43210, USA
2Department of Pharmacology, The College of Medicine, The Ohio State University Columbus, OH 43210, USA
3To whom correspondence should be addressed at: Room 103, Wiseman Hall, 400 West 12th Avenue, Columbus, OH 43210, USA
8-Oxo-7,8-dihydroguanosine triphosphate (8-oxo-dGTP) is formed from the oxidation of GTP in the nucleotide pools of cells during normal cellular metabolism and from exogenous sources. 8-Oxo-dGTP is a potent mutagenic substrate for DNA synthesis causing transversion muta tions. In human cells this oxidized base is hydrolyzed to 8-oxo-7,8-dihydroguanosine monophosphate by 8-oxo-7,8-dihydroguanosine triphosphatase (8-oxo-dGTPase) to pre vent the misincorporation of 8-oxo-dGTP into cellular DNA. In order to better understand specific human tissue and cell type responses to oxidative stress, we used colon- metric in situ hybridization, with an 8-oxo-dGTPase- specific antisense oligomer probe, to map, for the first time, the cellular distribution of 8-oxo-dGTPase mRNA in tissue sections of normal neonatal foreskin and adult human breast tissues. Paraffin embedded tissue sections were hybridized with a digoxigenin-labeled 39 base oligomer, antisense to 8-oxo-dGTPase cDNA. Hybridization of the probe to cells expressing the 8-oxo-dGTPase gene was visualized following immunodetection with an alkaline phosphatase-conjugated anti-digoxigenin antibody. Follow ing color development, we were able to simultaneously identify tissue architecture and cell types with expression of the 8-oxo-dGTPase gene. There was no hybridization- specific color when sections were ‘mock’ hybridized, hybridized with a sense probe or treated with RNase. In skin dermis, fibroblasts express high levels of 8-oxo- dGTPAse mRNA. Within the epidermis, a gradient of expression was observed, from high to moderate levels in the replicating basal epithelial cells to undetectable in the non-mitotic suprabasal and granular epithelial cells. In the breast tissue, fibroblasts in the loosely connective tissue and myoepithelial cells expressed high levels of 8-oxo- dGTPase mRNA, while expression in the luminal epithelial cells was not detectable. Our data suggest that expression of 8-oxo-dGTP is heterogenous between cell types within an organ and may help to explain cell type-specific responses to oxidative stress, especially in replicating and potentially replicating cells with low levels of this protective protein.