Study Of The Mechanism Of Over-expressing P21^waf1/CIP Induced P16^Ink4a Demethylation In ALT Tumor Cells

Aging is a double-edged sword, it can inhibit the tumorigenesis, or become a promoting factor for tumorigenesis. On one hand, aging is a significant barrier in the process of tumorigenesis. When DNA damage or abnormal activation of oncogene occurs in the process of cell proliferation, cells will age and stop proliferation to prevent the cells from cancinogenesis Senescent cells, on the other hand, could accumulate negative mutations, and accelerate telomere dysfunction, which result in the decrease of genome stability. The changed microenvironment caused by senescent cells is inducive for cell tumorigenesis.Werner syndrome(WS) is a good model to study the relationship between aging and cancer. WS is one kind of human autosomal recessive hereditary disease manifesting geriatrics syndrome of premature aging and shorten life span, along with frequent tumor occurrence. Double knockout of telomerase RNA(mTerc) and Wrn gene (mTR-/-Wrn-/-) establishs the Werner syndrome mouse model successfully, the very model faithfully manifests the tumor susceptibility of human premature aging symptom.Previous studies have found that the MEF (mouse embryonic fibroblast) of Werner syndrom mouse model (G5 mTR-/-Wrn-/-)shown premature aging, but some of the cells could escape senescence and become immortalized cell lines (395-3B-1? 395-3B-2?395-9B-1?395-6B-1?395-7A-1?395-7A-3). Some of them(395-3B-1? 395-3B?395-9B-1) could form tumors in SCID mice. These cells, due to the lack of telomerase, referred as the ALT tumor cells (Alternative Lengthening of Telomere, ALT). Our previous data have shown that the CpG islands in the promoter region of p16 gene are highly methylated during the process of escaping aging and becoming tumorigenesis. The expression of p16 is significantly down-regulated. We have also shown that the expression of exogenous p21WAF1/CIP1 could induce partial demethylation of p16 promotor, regain p16 expression and promote the tumor cells senescence.By Western blot and real-time PCR, we detected the expression level of DNA methyltransferase DNMT1?DNMT3A?DNMT3B in ALT tumor cells with p21WAF1/CIP1 over-expression. The results showed that the expression of DNMT1 had been significantly down-regulated at both mRNA and protein level. Meanwhile, the expression of p300, a transcription activating factor of DNMT1 had been down-regulated as well.We also used immunoblotting to detect the overall histone modifications in p21 overexpressed cell line 395-3B-2.. We found that:transcription inhibiting modification of histone H3K9me3 was reduced and transcription activating modification of histone H3K4me3 was increased. Then, chromatin immunoprecipitation technique was used to further confirm the histone modifications in the p16 promoter region. The data showed that the transcription activating histone modification H3K9ace significantly increased in p16 locus. The inhibition of transcription modifications H3K9me3 and H3K27me3 were significantly reduced, and the amount of Dnmtl binding to the p16 promoter region was reduced. Finally, we detected the level of essential proteins associated with histone modification,such as PcGs Ezh2 and Bmi-1, found that their expression level was significantly reduced.This study investigated the mechanism of the p21WAF1/CIP1-induced CpG island demethylation in p16 gene promoter region. We found that p300 and DNMT1 expression were downregulated in 395-3B-2 cells after overexpression of p21WAF1/CIP1; The type of histone modifications of p16 promoter region turned from inhibition form H3K9me3 and H3K27me3 into activation form H3K9ace. These data suggest that the induction of p16Ink4a demethylation by p21WAF1/CIP1 could be achieved in this way:p21WAF1/CIP1 inhibits DNMT1 expression by inhibiting p300 expression; At the same time, p21 also inhibits the expression of Ezh2 and Bmi-1 and result in the change of histone modification in p16 promoter region from H3K9me3/H3K27me3 to H3K9ace; p21WAF1/CIPI may prevent the Dnmtl from binding to p16 promoter region, by which the p16 promoter methylation pattern can not be maintained, results in the demethylation of p16 promoter. p21WAF1/CIP1 plays an important role in epigenetic regulation of p16Ink4a. It has been indicated that p16Ink4a is frequently methylated in tumors, especially in advanced tumors, which greatly affects the survival of cancer patients. This study provided a new potential target for treatment of tumor with P16Ink4a methylation.