SPECIFICITY OF ACTION OF A HERPES-VIRUS VP16 TETRACYCLINE-DEPENDENT TRANSACTIVATOR IN MAMMALIAN-CELL CULTURES

In this work, we have studied the activity of a tetracycline modulatab le trans-activator (tTA) generated by fusing the DNA binding domain of the tetracycline repressor to the trans-activation domain of the Herp es simplex virus protein 16 (HSV VP16) (plasmid pUHD15-1Neo), In the t hree different cell lines studied (HTC, rat hepatoma; T47D, human brea st cancer; SK-N-BE, human neuroblastoma), the expression of the lucife rase gene under the control of a tetracycline operator sequence (plasm id pUHC13-3) was used as a control of the incorporation and the functi onality of the trans-activator. Clones selected from these cells respo nded in a time and dose-dependent manner to the withdrawal of tetracyc line, In all these clones, the tTA trans-activator not only modulates the activity of the luciferase gene, but also modulates the activity o f a number of endogenous proteins, including C/EBP beta, the glucocort icoid receptor (GR), and SP1, In the transfected cells, the level of t hese transcription factors was strongly inhibited in the presence of t etracycline and was highly increased after tetracycline removal, Elect rophoresis mobility shift assay (EMSA) and footprint experiments prove d that the induced proteins are perfectly efficient in binding the DNA , Their transcriptional activity was also determined, In HTC/A9 cells, the level of the chloramphenicol acetyltransferase (CAT) expression d riven by the promoter of the alpha(1)-glycoprotein (AGP) gene was stro ngly enhanced at 72-84 hr following removal of tetracycline from the g rowth media, The accumulation of the endogenous AGP mRNA also increase d at 84 hr. In the T47D/TA11 and SK-N-BE/C2.6 cells, a general activat ion of protein synthesis was also evidenced.