The Molecular Mechanisms Of Cell Cycle Arrest And Apoptosis Induced By Histone Deacetylase Inhibitors In Neoplasia Cells

Epigenetics is defined as any heritable influence on chromosome or geneexpression that is not accompanied by changes in DNA sequences. And now,epigenetic changes of special genes, mainly DNA methylation and co-modificationof histones more recently, were recognized as additional mechanisms contributingto the tumorigenisis.Although histone proteins themselves come in generic or specialized forms, exquisitevariation is provided by covalent modifications (phosphorylation, acetylation, methylation,ubiquitinlation, ADP ribosylation, etc), which allow regulatable contacts with the underlyingDNA. Strahl has proposed in 2000 that distinct histone modifications, on one or more tails, actsequentially or in combination to form a 'histone code' that is, read by other proteins to bringabout distinct downstream events. The acetylation status of chromatin that is associated withparticular genes is dependent on both histone acetyltransferases(HATs) and histonedeacetylases(HDACs) activities. HDACs are involved primarily in the repression of genetranscription by virtue of the compaction of chromatin structure that accompanied the removalof charge-neutralizing acetyl groups from the histone lysine tails, while HATs act contrariwise.Histone deacetylase inhibitors (HDAC inhibitors, HDIs) have been tested as antitumordrugs recently, which can induce cell cycle arrest, stimulate differentiation, and provokeapoptosis in tumors as reported. Gene profiling studies indicate that HDIs modulate only4%-12% of genes and surprisingly, a similar proportion of genes are activated and repressed,although often with different kinetics. The naturally occurring anti-fungal antibiotic TSA wasone of the first HDAC inhibitors identified as an antiproliferative agent, and although it hasnever progressed as a clinical candidate, has been an invaluable tool in validating HDACen2ymes as potential anti-cancer targets.P 2|WAFI/CIPI js the grst jdejtfjfied inhibitor of cyclin-CDK complexes, whichis a potent negative regulating factor in cell cycle checkpoint and regulate transitionsbetween different phases of the cell cycle. p21 expression has been shown to beregulated largely at the transcriptional level by both p53 -dependent and-independent mechanisms, and p21 plays a critical role in inducing p53-dependentand p53-independent growth arrsst after DNA damage. Through its directinteraction with proliferating cell nuclear antigen(PCNA), p21 can also block DNAsynthesis.It is known that p21 is non-expressed or low-expressed in most of tumor cells,with abberent checkpoint function. While up to date, almost no mutations or loss ofheterozygosity(LOH) of p21WAF1/cn>1 coding regionhave been found in tumor cells. However, somerecent experiments have suggested that DNA methylation and histone deacetylation, two knownepigenetic modifications, seem to be the best candidate mechanisms for p2iWAF!/CIPIinactivation inthese tumors. We asked weather there are any acetylation modification of p21 in malignanthematological cells, and weather TSA can be used as a universal antitumor drug in leukemiatreatment? Sowa has proved that TSA can induce the expression of p21 through thetypical Spl sites in a p53-independent manner in human osteosarcoma cell lineMG63. So, how does TSA induce growth arrest and apoptosis in human cervixcarcinoma HeLa cells? What is the exact molecular mechanism? As we know thatp300/CBP and PCAF can up-regulation p53 by acetylating its specific residues, so, act asHATs, could acetylation induced by TSA treatment be a universal modification for p53function? Could p53 act synergistically with TSA during the p21 induction? To elucidatethose questions, efforts were made on three aspects: Firstly, examine the reactionsof JurkaU K562N HL60> Namalwa and Molt-4 cell lines by flow cytometry after treatedby TSA, checkpoint-related genes expression were analyzed by Western blot andNorthern bolt respectively. Secondly, to investigate the molecular mechanisms of TSA inhuman neoplasia cells, cervix carcinoma HeLa cells were treated with TSA and assays werecarried out by Flow cytomeo^ Western blot> RT-PCR and Dual-luciferase Reporter Assay.Thirdly, p21 knock down cell line were constructed by antisense-RNA and RNAi technology.The preliminary results as follows:1. TSA can induce G2/M arrest in Jurkat, K562, Namalwa and Molt-4 cell line while Gl arrest in HL-60 cell line, which indicate that TSA is probably versatile as a antitumor drug, at least in malignant hematologic cells.2. TSA can induce the expression of p21 in all the above cells, which may be one of the main molecular mechanisms for proliferation inhibition of neoplasia cells by HDIs.3. Results show that Gl and G2 arrest can be induced by TSA in HeLa cells, and significant apoptosis can be detected with increasing dose and longer time. p21...