CLONING OF TETRADECANOYL PHORBOL ESTER-INDUCED PRIMARY RESPONSE SEQUENCES AND THEIR EXPRESSION IN DENSITY-ARRESTED SWISS 3T3 CELLS AND A TPA NONPROLIFERATIVE VARIANT

  • 1 January 1987
    • journal article
    • research article
    • Vol. 1  (3) , 263-270
Abstract
The tumor promoter tetradecanoyl phorbol acetate (TPA) is also a potent mitogen for murine 3T3 cells. We have previously described the isolation of variant Swiss 3T3 cell lines unable to proliferate in response to TPA. In this report we sought to identify genes that are stimulated by TPA as ''primary responses'', i.e., without intervening protein synthesis. We constructed a cDNA library in .lambda.gt10, using RNA from quiescent 3T3 cells treated with TPA in the presence of cycloheximide (CHX). Of 50,000 recombinant phages, we identified 50 isolates that demonstrated preferential hybridization to cDNA probes generated from TPA-stimulated cells. One of the clones contains a fragment of the proto-oncogene c-fos. Twenty-nine of the remaining 49 clones fall into six cross-hybridization families. All the characterized clones detected mRNAs that are also inducible by epidermal growth factor, fibroblast growth factor or elevated serum. TPA induction of all the characterized messages is rapid and transient. All these mRNAs are superinduced by a combination of mitogens and CHX. Induction of these messages following TPA addition also occurs in subconfluent 3T3 cultures; expression of these genes is, therefore, not restricted to the G0/G1 transition. Expression of all six clones was also induced by TPA and other mitogens in 3T3-TNR9 cells, a TPA non-proliferative variant.