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DNA demethylating agents are used to reverse epigenetic silencing of tumor suppressors in cancer therapeutics. Understanding of the molecular and cellular factors involved in DNA demethylation-induced gene desilencing and senescence is still limited. We have tested the involvement of two stress chaperones, Pex19p and mortalin, in 5-Aza-2' deoxycytidine (5AZA-dC; DNA demethylating agent)-induced senescence. We found that the cells overexpressing these chaperones were highly sensitive to 5AZA-dC, and their partial silencing eliminated 5AZA-dC-induced senescence in human osteosarcoma cells. We demonstrate that these chaperones modulate the demethylation and chromatin remodeling-dependent (as accessed by p16(INK4A) expression) and remodeling-independent (such as activation of tumor suppressor p53 pathway) senescence response of cells. Furthermore, we found the direct interactions of 5AZA-dC with these chaperones that may alter their functions. We conclude that both mortalin and Pex19p are important mediators, prognostic indicators, and tailoring tools for 5AZA-dC-induced senescence in cancer cells.

Background & Objective: Histone deacetylation is associated with transcriptional activation controlled by DNA methylation. It is important to investigate changes of tumor cells treated with agent through two kinds of mechanisms. This study was designed to investigate the synergic effect of histone deacetylase inhibitor, sodium phenylbutyrate(SPB), and demethylating agent, 5-Aza-2'-deoxycytidine(5-Aza-CdR), on cell growth and explore the possibility of re-expression of the hypermethylated and silenced p16 gene in the myeloma cell line U266., Methods: The cell cycle was analyzed by flow cytometry. Apoptosis was observed by transmission electron microscopy, DNA ladder, fluorescence-activated cell sorter (FACS). The expression level of p16 was detected by RT-PCR and Western blot analysis., Results: The apoptotic rates of U266 cells induced by 5-Aza-CdR(1 mumol/L), SPB(1 mmol/L) alone and combination of 5-Aza-CdR and SPB were 15.09%, 89.19%, and 85.18%, respectively. The G1 phase was arrested and sub-G1 phase(50%) was induced by combination of 5-Aza-CdR and SPB. There was no G1 phase arrested when SPB or 5-Aza-CdR was used alone. The proportion of cells in G2 phase was increased with SPB alone. SPB was not able to induce the expression of p16. The expression level of p16 was induced with 5-Aza-CdR. The expression level of both mRNA and protein of p16 was increased significantly by synergy of SPB and 5-Aza-CdR., Conclusions: p16 gene in U266 cell line could be reactivated markedly with synergy of 5-Aza-CdR and SPB with cell cycle arresting in G1 phase. Meanwhile, the cell cycle phase occurring apoptosis that induced by combination of 5-Aza-CdR with SPB is different from that induced by each alone.

We have previously reported that a set of oral squamous cell carcinoma lines express specifically elevated cdk6 activity. One of the cell lines, SCC4, contains a cdk6 amplification and expresses functional p16ink4a, the other cell lines express undetectable levels of p16ink4a, despite a lack of coding-region mutations. Two of the cell lines, SCC15 and SCC40 have a hypermethylated p16ink4A promoter and a third cell line, SCC9, has a mutation in the p16ink4a promoter. Using the demethylation agent 5-aza-2'-deoxycytidine, we showed that the p16ink4a protein was re-expressed after a 5-day treatment with this chemical. One cell line, SCC15 expressed high levels of p16ink4a. In this line, cdk6 activity was decreased after 5-aza-2'deoxycytidine treatment, and the hypophosphorylated, growth suppressive form of the retinoblastoma tumor suppressor protein pRB was detected. Expression of p16ink4a persisted, even after the drug was removed and the cells expressed senescence-associated beta-galactosidase activity. Ectopic expression of p16ink4a with a recombinant retrovirus in this cell line also induced a similar senescence-like phenotype. Hence, it was possible to restore a functional pRB pathway in an oral squamous cell carcinoma line by inducing re-expression of endogenous p16ink4a in response to treatment with a demethylating agent.

Alterations in DNA methylation patterns accompany the establishment of immortal cell lines. De novo methylation of CpG islands within the control regions of growth-regulatory genes may inactivate their transcription, giving cells selective growth advantages in culture. We exposed seven human tumor cell lines and two human fibroblast cell strains to the demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-CdR), to determine whether the silencing of growth-regulatory genes by de novo methylation in immortalized cell lines could be reversed, possibly restoring growth control. After recovery from the immediate cytotoxic effects of 5-Aza-CdR, this agent suppressed cellular growth in all seven tumor lines but not in either fibroblast strain. Because alterations in the p16 (CDKN2/MTS1) cell cycle regulatory gene are associated with numerous cancers, we analyzed expression of this gene before and after 5-Aza-CdR treatment. The gene was reactivated by 5-Aza-CdR treatment in three of four tumor cell lines not expressing p16, whereas the fourth tumor line contained a p16 homozygous deletion. p16 was shown to be hypermethylated only in the cell lines and its up-regulation by 5-Aza-CdR was associated with demethylation of the p16 promoter. The remaining tumor lines expressed p16 at constant levels before and after 5-Aza-CdR treatment and showed minimal p16 promoter methylation, suggesting that other growth-regulatory genes may have been silenced by de novo methylation in these cells. p16 expression, cell growth inhibition, and G1 cell cycle arrest by 5-Aza-CdR in the T24 bladder tumor cell line were also heritable after prolonged passage in culture. Furthermore, a dormant p16 gene was reactivated in T24 cells growing in nu/nu rats, and 5-Aza-CdR treatment of T24 cells before inoculation into nu/nu mice decreased the rate of tumor growth. These results suggest that 5-Aza-CdR may slow the growth of tumor cells by reactivating growth-regulatory genes silenced by de novo methylation.