Abstract
Despite the importance of the INK4a/ARF locus in tumor suppression, its modulation by histone deacetylase inhibitors (HDACis) remains to be characterized. Here, we have shown that the levels of p16INK4a are decreased in human and murine fibroblasts upon exposure to relatively high concentrations of trichostatin A and sodium butyrate. Interestingly, the levels of p19ARF are strongly upregulated in murine cells even at low concentrations of HDACis. Using ARF-deficient cells, we have demonstrated that p19ARF plays an active role in HDACi-triggered cytostasis and the contribution of p19ARF to this arrest is of higher magnitude than that of the well established HDACi target p21Waf1/Cip. Moreover, chemically induced fibrosarcomas in ARF-null mice are more resistant to the therapeutic effect of HDACis than similar tumors in wild type or p21Waf1/Cip-null mice. Together, our results have established the tumor suppressor ARF as a relevant target for HDACi chemotherapy.
Highlights
Given the key role of cell cycle integrity in tumor suppression and cancer therapy, attention has focused on the ability of histone deacetylase inhibitors (HDACis) to alter the levels of cell cycle regulatory proteins
Effects of HDACis on Histone Acetylation and Heterochromatin—The effects of the HDACis Trichostatin A (TSA) and NaB on histone acetylation and heterochromatin have been characterized for the most part in immortal cancer cell lines, which invariably have deregulated expression of the INK4a/ARF locus
We have focused on the analysis of normal cells, and we have first validated some of the known effects of TSA and NaB on primary fibroblasts
Summary
Cell Culture and Drug Treatment—Normal human lung diploid fibroblasts (IMR90: ATCC CCL-186; WI38: ATCC CCL-75) and mouse. Stock solutions of TSA (Sigma) and lactacystin (Sigma) were prepared in Me2SO; NaB (Sigma) and nicotinamide (NAM) (Sigma) stocks were in phosphate-buffered saline (PBS) Aliquots of these drugs were stored at Ϫ20 °C and added to the culture medium as 1000-fold concentrated stock solutions 16 –24 h after cell plating. Subsequent to blocking for 12 h with PBS and 1% fetal calf serum, cells were incubated with a p19ARF rabbit polyclonal antibody (R562; AbCam) at a 1:100 dilution or with a HP1␥ mouse monoclonal antibody (Chemicon) at a 1:1000 dilution. Promoter Constructs and Reporter Assays—Human p16INK4a (hp16INK4a) promoter constructs cloned into the pGL2-basic luciferase reporter vector (Promega) and containing, respectively, 3017, 869, and 798 nucleotides upstream the ATG start codon were a generous gift from Dr Gordon Peters (Cancer Research UK, London). Experimental data were fitted to concentration response curves by the Hill equation using the KaleidaGraph 3.6 graphing and data analysis package from Synergy software
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