| Backgroundp300 is a transcription cofactor with histone acetyltransferase(HAT) activity,which is capable of acetylating all the 4 core histones as well as other transcription factors. p300 participates in regulating many cellular processes, including cell growth,cell proliferation as well as cancer development and apoptosis.The histone acetylation of the antioncogene promoter reduced will lead to its downexpression. CBP/p300 is very important to the transcriptional activation of p53, BRCA1 and FOXO3 a all of which are the key tumor suppressors. In such situations CBP / p300 can inhibit the occurrance of tumor. Meanwhile, being coactivators of oncogene c-Myc, c-Myb and the AR, CBP / p300 can also facilitate cell proliferation and cancer development as well. Wild-type p300 contained in Plasmid pc DNA3.1 has the common structure of HAT, mutant-type p300 contained in Plasmid pc DNA3.1 does not have HAT domain.ObjectiveTo observe the effect of p300 on gastric cancer cell proliferation, to observe the effects of wild-type p300 and mutant-type p300 on gastric cancer cells histone acetylation, to explore the mechanism of p300 affecting gastric cancer cell proliferation.Materials and methods1. Experimental group Gastric cancer cells SGC7901 and BGC823 were randomly divided into four groups: the experimental groups were divided into three groups which were transfected with 2μl of plasmid pc DNA3.1containing wild-type p300 at 400ng/μl concentration, plasmid pc DNA3.1containing mutant-type and the empty plasmid pc DNA3.1 and cultured for 24 h, 48 h and72h,respectively; the control group was added with the same volume RPMI1640 medium without serum or antibody.2. MTT After collecting the cells from three experimental groups and one control group, MTT method was used to detect the proliferating capacity of gastric cancer cell SGC7901 and BGC823 at 24 h, 48 h and 72 h, respectively.3. Immunocytochemistry A series of cell drop sheets were made after collecting the cells of three experimental groups and one control group at 24 h, 48 h and 72 h.The Immunofluorescence method was used to observe the immunoreactivity of ac K14-H3 and p300 in different groups for semi-quantitative analysis and to detect the histone acetylation levels of gastric cancer cells containing different types of p300 pc DNA3.1.4. Flow cytometry Flow cytometry was used to detect the cell cycle of gastric cancer cell SGC7901 and BGC823 transfected with different p300 plasmids pc DNA3.1 and to observe whether p300 can induce cell cycle arrest.5. Statistical analysis All data were analyzed with SPSS20.0 statistical software. P<0.05 was considered as statistical significance.Results1. Effects of p300 on the gastric cancer cell proliferation. For gastric cancer cell SGC7901, the proliferating capacity of the wild-type p300 gastric cancer cell group had no significant difference in the group transfected with the mutant-type p300 at 24h(P>0.05); The proliferation of the wild-type p300 gastric cancer cell group was significantly lower than the mutant p300 group at 48 h and 72h(P<0.05). For gastric cancer cell BGC823, The proliferation of the wild-type p300 gastric cancer cell group was significantly lower than the mutant p300 group at24 h and 48h(P <0.05); The proliferation of the wild-type p300 gastric cancer cell group had no significant difference with those transfected with the mutant-type at 72h(P> 0.05). The proliferation of the experimental groups was significantly lower than the control group(P <0.05). It illustrates that different p300 cloned in plasmid pc DNA3.1 can obviously inhibit the proliferation of gastric cancer cell(P<0.05).2. Effects of p300 on the histone acetylation in gastric cancer cell. The ac K14-H3 IR appeared as green fluorescence. For gastric cancer cell SGC7901,the average level of ac K14-H3 IR in positive cells of wild-type p300 group and mutant-type p300 group was significantly higher than that in control group(P<0.01).Moreover, the average level of ac K14-H3 IR in positive cells of wild-type p300 group was significantly higher than that of the mutant-type p300group(P < 0.01). For gastric cancer cell BGC823,the average level of ac K14-H3 IR in positive cells of wild-type p300 group was significantly higher than that of the control group at 24 h and 48h(P<0.01),and the average level of ac K14-H3 IR in positive cells of wild-type p300 group was significantly higher than that of the mutant-type p300 group at 24 h and 48h(P<0.01),but the average level of ac K14-H3 IR for positive cells in wild-type p300 group had no obvious difference with the control group at 72h( P > 0.05).It shows that p300 can obviously increase the average level of ac K14-H3 IR in gastric cancer cell SGC7901 and BGC823.3. The p300 immunoreactivity in gastric cancer cell The p300 IR appeared as green fluorescence in cell nucleus. In gastric cancer cell SGC7901 and BGC823,the average level of p300 IR in positive cells of wild-type p300 group was significantly higher than that in the control group(P<0.01),and the average level of p300 IR in positive cells of wild-type p300 group was significantly higher than that in the mutant-type p300 group(P<0.01). It appears that p300 can obviously increase the average level of p300 IR in gastric cancer cell SGC7901 and BGC823.4. Flow cytometry results Compared with the control group, gastric cancer cell transfected with wild-type p300 and mutant-type p300 had a higher percentage of G0/G1 phase cells and lower percentage of S phase cells(P<0.05),there is no significant difference between the wild-type p300 group and the mutant p300group(P>0.05). It proves that p300 can induce cell cycle arrest.ConclusionsWild-tipe p300 contained in Plasmid pc DNA3.1 and the mutant-type p300 contained in Plasmid pc DNA3.1 without the HAT domain can inhibit gastric cancer cell proliferation and the wild-tipe p300 contained in Plasmid pc DNA3.1 has a stronger inhibitory effect on the grastric cancer cells.This process maybe occur via increasing histone acetylation levels of tumor suppressor gene in gastric cancer cells. p300 can induce cycle arrest. |
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