Normal human T cells as a model system for the study of molecular events at the G0/G1 interface.

Abstract

Relatively few studies have focused on gene products specific for the G$\sb0$ state and their inhibition following cellular activation. Since protooncogenes play important roles in the regulation of cell growth, we postulated that similar genes may be involved in the maintenance of cellular quiescence. ets-1, the cellular homolog of the v-ets oncogene of the avian acute leukemia virus, E26, is selectively expressed in quiescent T cells. The ets-1 promoter was repressed 10 to 20-fold in activated T cells as detected by nuclear run-on assays. This difference in transcriptional activity correlated with alterations in higher order chromatin structure as reflected by changes in the patterns of DNase I hypersensitivity (DH) at the 5$\sp\prime$ end of the ets-1 transcription unit. In vitro binding studies demonstrated specific binding of factor(s) present in activated but not quiescent T-cell nuclear extracts to an Ets binding site (EBS) which mapped to an inducible DH site. In addition, transfection studies defined a negative regulatory element in the 5$\sp\prime$-untranslated region of the ets-1 gene which repressed CAT reporter activity 30-fold in Jurkat T cells. These two elements may be involved in repressing ets-1 expression in cycling T cells. In our initial attempts to assess ets-1 promoter function in primary T cells, we were unable to express exogenous DNA in both resting and cycling primary T cells using ETS-1-CAT and other reporter constructs. The latter was a surprising result since proliferation has been associated with efficient transfection and expression of exogenous DNA. Reporter gene expression required T-cell receptor (TCR)-dependent signal transduction of cells prior to but not after introduction of DNA. Thus, we present evidence for an active mechanism, repressible by TCR-dependent signal transduction, which protects quiescent and cycling T cells from the expression of exogenous DNA. Consistent with the potential participation of wild-type (wt) p53 in this mechanism, overexpression of wtp53 resulted in the 25-fold inhibition of RSV-CAT expression in normal T cells. TCR-dependent stimulation resulted in the inhibition of wtp53 suggesting that this tumor suppressor may be involved in the recognition and inactivation of exogenous DNA.