Determination of tissue-type plasminogen-activator mRNA in human and non-human cell lines by dot-blot hybridization.
A labelled cDNA clone was used in DNA-RNA hybridization on nitrocellulose filter paper (dot-blot technique) to detect and quantify mRNA for endogenous tissue plasminogen activator (PA) in cell extracts and samples of RNA purification runs. Although, for detection purposes, the assay was less sensitive than translation in Xenopus oocytes, it was at least as reliable and much more convenient for the purpose of quantitative determination. In particular, the technique was used to study the kinetics of PA mRNA formation in a human melanoma cell line (Bowes) after exposure to the tumour promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). Incubation of the cells with TPA resulted in a 15-20-fold increase in cellular PA mRNA content. The effect was time- and dose-dependent: the increase in PA-specific mRNA was clearly visible as early as 4 h after initiation of TPA treatment in Bowes cells. It was blocked completely by pretreatment of the cells with actinomycin D, indicating that TPA caused enhancement of synthesis of PA mRNA rather than inhibition of PA mRNA degradation. The use of the nitrocellulose dot-blot technique also revealed that two non-human cell lines produce mRNAs which cross-react with the human PA mRNA, namely the mouse melanoma cell line B16 and the rat brain-tumour cell line, RT4-71-1. TPA was found to exert similar stimulatory effects on the synthesis of mRNAs in these cell lines as in Bowes cells.
D000818:Animals; D001345:Autoradiography; D002460:Cell Line; D003101:Collodion; D003429:Cross Reactions; D003609:Dactinomycin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D010959:Tissue Plasminogen Activator / Q000378:metabolism*
D012333:RNA, Messenger; D003609:Dactinomycin; D003101:Collodion; D010959:Tissue Plasminogen Activator