Effect Of Melanoma Associated Antigen MAGE-A9on The Transcriptional Activity And Stability Of P53

Objective:Performed as a gene guard, wild-type p53plays an important role inmaintaining the genomic integrity. P53protein is normally present atextremely low level in cells. In response to cellular stresses such as DNAdamage, p53is transcriptional activated and promotes the expression of a widerange of downstream target genes, which induce cell cycle arrest and/orapoptosis. Recently, several studies have demonstrated that a few membersfrom melanoma associated antigen-A sub-family is interlinked with p53, thetumor suppressor. MAGE-A inhibits the transcriptional activity of p53, andinteracts with MDM2to promote p53ubiquitination and degradation. ThusMAGE-A represses p53to function as a tumor suppressor. This paper is aimedto study the effect of MAGE-A9on the transcriptional activity and thestability of p53, and to understand the potential signaling pathways andmechanisms of MAGE-A9in tumorigenesis.Methods:1Gene transfection, RT-PCR, Western Blot, luciferase reporter assaywere performed to evaluate the effect of exogenous MAGE-A9on theexpression of p21WAF1in MDA-MB-231cells.2The proliferation activity of MDA-MB-231cells was detected bycolony formation assay and MTT assay.3Gene transfection, RT-PCR, Western Blot were used to analyze theeffect of MAGE-A9on the stability of p53protein in MDA-MB-231cells.4Expressions of p53protein in MDA-MB-231cells were researched indifferent periods and different groups by cycloheximide protein synthesisinhibition assay. Results:1The results of RT-PCR showed that the transfection of p53markedlyincresed the expression of p21WAF1mRNA, compared with the empty vectorsgroup (P<0.05). The mRNA expression levels of p21WAF1were significantlylower after co-transfected with p53and MAGE-A9than those transfeced withp53only (P<0.05). MAGE-A9had no significant effect on the expression ofp21WAF1(P>0.05).2The results of Western Blot revealed that, after transfected with p53gene, the expression of p21WAF1protein increased more obviously than that inthe empty vectors group (P<0.05). The expression of p21WAF1protein waslower after co-transfected with p53and MAGE-A9than that transfeced withp53only (P<0.05).3The luciferase reporter gene assay showed that, In breast cancer cellline MDA-MB-231, the luciferase activity in the control group was1.00±0.00.The luciferase activity induced by p21WAF1promoter in p53-transfected groupwas obviously greater than that in the control group (P<0.05). Afterco-transfected with p53and gradually increased MAGE-A9(100ng,200ng,400ng), the luciferase activity induced by p21WAF1promoter were49.40±5.74,35.93±2.27,22.48±4.98, respectively. They were fewer than that in thep53-transfected group (P<0.05).4The results of colony formation assay discovered that, the numbers ofcell colony with G418resistance in empty vector group and p53-transfectedgroup were143.50±11.39,53.25±6.08, respectively. The latter was lower thanthe empty vector group (P<0.05). Compared with the p53-transfected group,the colony number raised significantly when co-transfected with p53andMAGE-A9gene (P<0.05). There was no significant effect on the colonynumber in MAGE-A9group as compared with the empty vectors group(P>0.05).5MTT assay revealed that, transfected cells were incubate in96-wellplate for24hours or48hours, the relative proliferation rates in differentgroups were135.18%and337.23%,138.27%and228.89%,139.44%and291.51%,142.62%and330.71%, respectively. There was no significant difference in the relative proliferation rate after incubating for24hours(P>0.05). Incubating for48hours later, the relative proliferation rates inp53-transfected group and co-transfected group reduced markedly (P<0.05).The relative proliferation rate in p53-transfected cells was lower than that inco-transfected group (P<0.05).6The data from the effect of MAGE-A9on p53stability detected byRT-PCR showed that, the ratios of grey level between p53and GAPDH inempty vector group,0.5g p53-transfected group, co-transfected with0.5gp53and0.5g MAGE-A9group, co-transfected with0.5g p53and1.0gMAGE-A9group were0.492±0.003,0.723±0.026,0.709±0.014,0.712±0.014,respectively. There was no significant differences between any two groupsexcept for the control group (P>0.05).7The effect of MAGE-A9on p53stability detected by Western Blotdiscovered that, the ratios of p53protein and GAPDH in empty vector group,0.5g p53-transfected group, co-transfected with0.5g p53and0.5gMAGE-A9group, co-transfected with0.5g p53and1.0g MAGE-A9groupwere0.747±0.011,0.961±0.027,0.677±0.021,0.607±0.023, respectively.Significant differences were found between every two groups (P<0.05).8Cycloheximide (CHX) protein suppression analysis revealed that, aftertransfected with p53and treated with CHX for0h,1h,2h,4h,6h, the ratios ofp53protein and GAPDH were0.918±0.014,0.777±0.032,0.607±0.059,0.383±0.032,0.208±0.017, respectively. While after co-transfected with p53and MAGE-A9and treated with CHX for0h,1h,2h,4h,6h, the ratios of p53protein and GAPDH were0.890±0.070,0.495±0.039,0.268±0.032,0.165±0.017,0.084±0.018, respectively. There was significant differencebetween two groups at every indicated time (P<0.05).Conclusion:Melanoma associated antigen-A9inhibits the transcriptional activity ofp53, suppresses its stability, and affects its biological functions.