Endogenous gene and amplifiable cDNA construct both produce unstable t-PA mRNA in Bowes melanoma cells.

Bowes melanoma cells, which naturally produce tissue-type plasminogen activator (t-PA), were transfected with a plasmid containing a human t-PA cDNA under transcriptional control of the promoter/enhancer of the major immediate early gene of human cytomegalovirus (CMV) plus genes expressing geneticin (G418) resistance and dihydrofolate reductase (DHFR). In one of the initial geneticin-resistant transformants, t-PA mRNA transcribed from the chromosomally integrated plasmid had the same short half-life, 20-30 min, as did mRNA transcribed from the endogenous t-PA gene compared to 7-8 h for total poly(A)+ mRNA. After subsequent selection of such cells with methotrexate, a cell line was obtained in which the t-PA cDNA construct was co-amplified with the DHFR gene and which produced 10 times more t-PA protein than the original Bowes melanoma cells.