Histone Deacetylase Inhibitor Trichostatin A Induced The Molecular Mechanisms Of Lung Adenocarcinoma Cell Apoptosis And Cell Cycle Arrest

Lung cancer is the leading cause of cancer deaths in the world, among both men andwomen. It has been shown that in a variety of tumor cells, HDAC function is inhibited,histones remain acetylated, resulting in a more open chromatin conformation, whichfacilitates the transcription of genes, and these changes resulted in a reduction ofproliferation and metabolic activity and in an induction of apoptosis and differentiationboth in vitro and in vivo. HDAC inhibitors have been shown to induce cell cycle arrest anda differentiated phenotype in a variety of tumor types including leukemia, colon cancer,and breast cancer, thus, histone deacetylase (HDAC) has recently been discovered as apotential target for the development of new therapeutic agents. Thus far, a number ofhistone deacetylase inhibitors (HDACI) have been identified with different anti-tumoractivity, specificity, toxicity, and stability. To date, the underlying mechanism for theseobserved effects is not known. Therefore, we have investigated the effect of TSA on thecell cycle distribution and the expression of cell cycle regulating proteins in a panel ofNSCLC cell lines---A549 and the apoptosis mechanism, chromatin remodeling and thebinding of associated transcription factors to the muc1 and cdc2 promoter wereinvestigated.Chromatin modifying enzymes can modify the chromatin and regulate its structure,which can activate or restrain the binding of transcription factors to the DNA and activatedor repressed the expression of the gene. Chromatin Immunoprecipitation (CHIP) assay wasdeveloped in recent years to detect protein factors that bind to chromatin. The binding ofmSin3A, p53, Ac-p53, BRM, PCAF, p300, H3K9-Ac and H3K14-Ac to the muc1 and cellcycle regulating genes promoter in control or A549 cells induced by TSA was studied bythis method. And the effect of PCAF, p300 and BRM to the promoter activity of muc1 wasinvestigated by transient transfection and RT-PCR method.1. TSA induces cell cycle arrest and apoptosis in NSCLC cell line A549. When the A549 cells were treated with TSA, the percentage of G1-phase as well asG2/M-phase cells increases, especially the G2/M-phase. And mRNA expression ofp21,cdc2, cdc25c and cyclinB2 in A549 cells treaeted by TSA were detected by RT-PCR,the result showed that p21 was increased, on the other hand, transcription of genesinvolved with cell cycle progression such as cdc2, cdc25c and cyclinB2 and muc1 geneswere inhibited. In A549 cells treated with TSA, the percentage of apoptotic cells in FACSand the cleavage of PARP are higher than that of the untreated control.2. Effects of p53 protein on the promoter activity of muc1 gene in A549 cells(1)A549 cells were cotransfected with reporter gene plasmids pREP4m-muc1-2.4k orpREP4m-cdc2-CAT and control plasmids pM-CAT and treated by TSA, the analysis ofpromoter activity show that TSA can inhibit the promoter activity of muc1 and cde2gene.Furthermore, reporter plasmid containing different length of muc1 gene promoter(pREP4m-muc1-2.4k, pREP4m-muc1-1.6k, pREP4m-muc1-724, pREP4m-muc1-298)and transfection control plasmid pM-CAT. Promoter activity of muc1 gene was thencalculated in TSA treated A549 cells. The results showed that TSA could inhibit the muc1gene promoter activity significantly in pREP4m-muc1-2.4k, pREP4m-muc1-1.6k. Theresults indicated that inhibition of muc1 gene promoter activity by TSA probably dependon the cis-element upstream -724 in A549 cells.(2)p53 protein expression plasmid or mutant expression plasmid were cotransfectedinto A549 cells with reporter plasmid pREP4m- muc1-2.4k and transfection controlplasmid pM-CAT. Promoter activity of muc1 gene was then calculated. The results showedthat p53 protein decreased the gene promoter activity. Mutant- p53 can restrain theinhibition effect of TSA on the muc1 promoter activity, moreover, p53 protein wasupregualted upon TSA treated, which suggested TSA may control the expression of muc1though p53 pathway indirectly. For further studying the effects of p53 on muc1 genepromoter activity, site-directed mutation was used to mutate the core sequence of CCAATbox and p53 binding site. The mutated CCAAT box or p53 binding site of muc1 genepromoter sequences are inserted into pREP4m-CAT for constructing reporter plasmids. Promoter activity of muc1 gene was then calculated. The results showed that p53 proteinhas no effect on p53 binding site mutated promoter, which suggested that p53 may inhibitmuc1 gene promoter activity through p53 binding site.3.The effects of chromatin modifiers on the promoter activity of muc1 duringTSA induced A549 cells(1)The expression plasmids of PCAF or p300 were cotransfected with pREP4m-muc1-2.4k and pM-CAT into A549 cells and the promoter activity of muc1 was detected byRT-PCR. The experiment results show that PCAF and p300 both can increase the promoteractivity of muc1.(2)A549 cells express only BRM, but not BRG1. The cotransfection assays show thatBRG1 and BRM both inhibit the muc1 promoter and dominant negative BRG1 and BRMcan reverse the repressive effect. Furthermore, it was also found that dominant negativeBRM could abolish the repressive effect of TSA to the muc1 promoter activity. All theseindicate that BRM is necessary for the TSA induced downregulation of muc1.4. The binding of chromatin modifiers to the promoter of muc1 during A549induced by TSAChIP assays results show that:(1)p300 and PCAF both bound muc1 promoter, however, only PCAF released fromnegative regulative region of muc1 promoter after TSA induced; TSA promoted p53 andacetylated-p53 bind with muc1 promoter, and that, mSin3A and Brm bound with increasedinduced by TSA. But the acetylation of both H3K9 and H3K14 has no change whetherTSA treated or not.(2)On the promoter of cdc2, cdc25c and cyclinB2, p53 binds their CCAAT boxwhether TSA treated or not; acetylated-p53 and mSin3A increased in A549 cells treated byTSA ,but p300 decreased. PCAF binds cdc2 and cdc25c gene both treated or untreatedA549 cells, nevertheless, no PCAF bind to cyclinB2 gene promoter whether TSA treated ornot. Moreover, after TSA treated, acetylation of H3K14 and H3K9 altered in these threegenes differently. Furthermore, BRM and PCAF co-immunoprecipitation results show that:interactionbetween BRM and mSin3A increases, but decreases between PCAF and mSin3A in TSAtreated A549 cells.According to the results above, we present a hypothesis: The treatment of TSA inA549 cells promote PCAF and p300 acetylate p53, the acetylated p53 may recruitSWI/SNF and mSin3A to the muc1 promoter, which remodel the chromatin of muc1promoter, then regulate the expression of muc1 gene.