A novel approach to analysis of transcriptional regulation in human cells: initial application to melanocytes and melanoma cells.
An assay system for transcriptional profile analysis of cultured eukaryotic cells has been developed to simultaneously handle multiple samples in a rapid, sensitive, and internally controlled manner. The methodology incorporates a microtiter plate assay system, a rapid cell-harvest enzyme-assay technique, and the bacterial reporter genes beta-glucuronidase and beta-galactosidase. We demonstrate, using beta-actin and SV40 (late) transcription promoting sequences, that this technically refined microtiter-triton-lysate (MTL) assay methodology can readily differentiate between the transcriptional states of human melanocytes before and after pharmacologic stimulation and malignantly transformed versus normal cell environments. Differences in the transcriptional environments are revealed by the relative expression of transcription element probes. The transcriptional activity ratio of the beta-actin compared to the SV40 late transcription promoting sequences was approximately 1:2 in primary cultured melanocytes, 2:1 in 12-0-tetradecanoyl phorbol-13-acetate (TPA)-treated melanocytes and 1:4 in the Tang melanoma cell line. Because this MTL assay methodology can accommodate a panel of transcription element probes, we anticipate that the resultant transcriptional profiles will prove useful in deciphering the diverse transcriptional changes that occur within normally regulated and malignantly transformed cells.
Grichnik
J M
JM
United States Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts 02111.
Gilchrest
B A
BA
D000199:Actins / Q000494:pharmacology; D002478:Cells, Cultured; D005966:Glucuronidase / Q000032:analysis; D006801:Humans; D008544:Melanocytes / Q000378:metabolism*; D008545:Melanoma / Q000378:metabolism*; D013539:Simian virus 40 / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000032:analysis
D000199:Actins; D001616:beta-Galactosidase; D005966:Glucuronidase; D013755:Tetradecanoylphorbol Acetate