Differential promoter activity in benign and malignant human cells of skin origin.
In order to develop systems to express mammalian proteins in human skin-derived cells, we tested 6 different viral and 1 eukaryotic promoter (pCMV, pRSV, pSV, pMMTV, pPoly E, pPoly L, pHMT) for their ability to drive the expression of the chloramphenicol acetyltransferase (CAT) enzyme in different human skin-derived cells. DNA was transfected in human keratinocytes derived from normal foreskin and cervix, in the HPV-negative cervical cancer line HT-3 and in malignant melanoma cell lines (SK-Mel 23, SK-Mel 37) using a liposome-based technique or calcium precipitation. Transfection efficacy was controlled by cotransfection of a beta-galactosidase gene construct. The enzymatic activity of the CAT-gene expression was determined by incubation of the cell extract prepared from the transfected cells with 14 C-labeled chloramphenicol. The CMV-promoter was highly active in all skin- or mucosal-derived cells. In contrast to the strong CMV-promoter, the RSV-, SV-, and HMT-promoter were less active and varied in dependence of the cell type. The pattern of the promoter activity differed between benign and transformed genital keratinocytes. Only the SV-promoter showed a comparable strong basal activity, which was restricted to the SK-Mel 37 cells. In conclusion, the promoter activity has to be tested for each cell type depending on the aims of the gene expression.
Artuc
M
M
University Hospital Rudolf Virchow, Dept. of Dermatology, FU Berlin.
Nürnberg
W
W
Platzer
M
M
Czarnetzki
B M
BM
Schadendorf
D
D
D002277:Carcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D011401:Promoter Regions, Genetic; D012016:Reference Values; D012867:Skin / Q000378:metabolism / Q000473:pathology; D012878:Skin Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D012879:Skin Physiological Phenomena; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D014780:Viruses / Q000254:growth & development
D015500:Chloramphenicol O-Acetyltransferase