NFκB-responsive genes are differentially regulated in senescent and non-senescent fibroblast cells that are growth arrested by cyclin-dependent kinase inhibitors

4997 Expression of cyclin-dependent kinase inhibitor (CDKI) proteins mediates cell cycle arrest in various physiological programs including senescence, which is defined as permanent cell cycle arrest associated with characteristic changes in cell morphology (enlarged and granular cells expressing senescence-asociated β-galactosidase activity). Senescent fibroblasts may contribute to the development of cancers by secreting factors that suppress apoptosis and support proliferation and angiogenesis. The genes for such factors and their corresponding paracrine activities can be induced in HT1080 fibrosarcoma cells by the expression of a CDKI, p21 (Chang et al., PNAS 97, 4291-4296, 2000). p21 is known to activate NFκB-dependent transcription, and promoter induction for several p21-responsive genes is mediated by NFκB (Poole et al., Cell Cycle 3, 931-940, 2004). We used Affymetrix Human Genome U133 Plus 2.0 microarrays to compare the effects of p21 and NFκB inducer TNFα on gene expression in HT1080 cells. We found by immunostaining of HT1080 cells that both TNFα and p21 induce the translocation of NFκB p65/RelA from cytoplasm to the nucleus. Interestingly, a prominent subset of TNFα-activated genes was not induced by p21, and only a minority of p21-induced genes were upregulated by TNFα. We also compared the effects of p21 on gene expression in HT1080 cells and in WI-38 normal human lung fibroblasts. HT1080 and WI-38 cells induced overlapping but non-identical sets of genes in response to p21. Paradoxically, a greater fraction of genes that were induced by TNFα in HT1080 cells was activated by p21 in WI-38 fibroblasts than in HT1080 cells. We also compared the effects of p21 with those of two other CDKIs, p16 and p27, in HT1080 cells. The overall effects of all three CDKIs on gene expression were very similar. However, several TNFα-inducible genes, including cytokines, were inhibited by p21 and p16 in HT1080 cells but induced by p27 in HT1080 and by p21 in WI-38 cells. Remarkably, p21- or p16-expressing HT1080 cells develop the senescent phenotype, whereas p27-expressing HT1080 and p21-expressing WI-38 cells undergo growth arrest but do not display the senescent morphology. We speculate that differential expression of NFκB-inducible cytokines could be causally related to the development of the senescent phenotype in CDKI-expressing cells.