CLONING OF SENESCENT CELL-DERIVED INHIBITORS OF DNA-SYNTHESIS USING AN EXPRESSION SCREEN

We here describe a rapid and simple expression screen method that has allowed us to isolate cDNAs coding for inhibitors of DNA synthesis fro m senescent human diploid fibroblasts. The assay involved transient tr anscriptional overexpression of a gene product encoded by a cDNA in a proliferating cell, on the assumption that this would be sufficient to block DNA synthesis in a short-term assay using tritiated thymidine a utoradiography. Three cDNAs, referred to as senescent cell-derived inh ibitors (sdi), that exhibit DNA synthesis-inhibitory activity when int roduced into young cycling cells, were successfully identified. Expres sion of one of the cDNAs, sdi1, increased 10- to 20fold in senescent c ompared with young cells and the increase in RNA closely paralleled th e onset of the senescent phenotype and loss of cell proliferation. sdi 1 expression was also increased in young cells made nondividing (quies cent) by deprivation of growth factors or contact inhibition. Followin g serum stimulation, RNA levels of sdi1 in quiescent cells were initia lly increased, but then declined to low levels just prior to the entry of the cells into S phase. In contrast, RNA levels of sdi1 in senesce nt cells failed to decline, suggesting a role for this gene in maintai ning the senescent phenotype. The sdi1 gene has been mapped to the p a rm of chromosome 6. (C) 1994 Academic Press, Inc.