Elevation of transforming growth factor‐α mRNA and protein expression by diverse tumor promoters in sencar mouse epidermis

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Abstract
The study presented here was designed to further investigate the role of transforming growth factor-α (TGFα) in skin tumor promotion by examining the ability of 12-O-tetradecanoylphorbol-13-acetate (TPA) and several non–phorbol ester promoters to alter TGFα mRNA and protein levels in mouse epidermis. Total RNA was isolated from SENCAR mouse epidermis at various times after single topical treatments with TPA (3.4 nmol), chrysarobin (220 nmol), okadaic acid (2.5 nmol), and thapsigargin (8.5 nmol). Northern analyses of these isolated RNA samples revealed that all four tumor promoters transiently elevated TGFα mRNA levels. Whereas TPA, okadaic acid, and thapsigarin elevated TGFα mRNA levels over similar time courses (peak at 4–8 h), chrysarobin elevated TGFα mRNA levels with a markedly delayed time course (peak at 24–48 h). More detailed studies with TPA also revealed that multiple treatments (four over a 2-wk period) transiently elevated TGFα mRNA in both the epidermis and the dermis. The time courses for changes in TGFα mRNA after multiple TPA treatments were similar for both tissues. To facilitate studies of altered TGFα mRNA expression in mouse epidermis and possibly other mouse tissues, a semiquantitative reverse transcriptase–polymerase chain reaction method was developed. This method faithfully revealed changes in TGFα mRNA levels with all four tumorpromoting agents similar to those determined by northern blot analyses. Immunofluorescence analysis of frozen sections from promoter-treated skin revealed elevated TGFα protein levels in both epidermis and dermis, although staining was most intense in the epidermal layer. Immunofluorescence analysis of epidermal hyperplasia adjacent to a full-thickness wound also demonstrated significant epidermal TGFα staining. Collectively, these results indicate that mechanistically diverse tumor promoter stimuli elevate TGFα mRNA and protein in SENCAR mouse epidermis. Elevated levels of TGFα may play an essential role in mitogenic stimulation during tumor promotion by diverse promoting stimuli. © 1995 Wiley-Liss Inc.

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