摘要
目的研究异常转录因子PML/RARα对GADD45A的转录调控机制,为进一步阐释PML/RARα在急性早幼粒细胞白血病(APL)发病中的作用机制提供线索。方法运用ChIP-PCR技术研究PML/RARα与GADD45A的结合情况;采用Real-TimePCR技术检测PR9细胞中GADD45A mRNA的表达水平,并分析其与PML/RARα融合蛋白表达的相关性;利用真核细胞表达载体在H1299细胞中过表达PML/RARα和p53蛋白,研究PML/RARα与p53对GADD45A的共调控关系。结果 PML/RARαChIP-qPCR结果显示:PML/RARα特异性抗体能够富集GADD45A基因功能区第3个内含子区的DNA片段,而非特异性IgG不能够富集;在PR9细胞中发现GADD45A mRNA表达水平与PML/RARα蛋白的表达呈负相关,说明在PR9细胞中PML/RARα融合蛋白能够抑制GADD45A的表达;在H1299细胞中单独转染野生型p53表达质粒后,GADD45A在mRNA水平显著上调,而共转染PML/RARα和野生型p53表达质粒后,PML/RARα能够显著抑制p53对GADD45A在mRNA水平的上调作用(P<0.01)。结论 PML/RARα能够结合在GADD45A第3个内含子区域并抑制其转录表达。GADD45A同时也是p53的靶基因,PML/RARα与p53对GADD45A有共调控关系。PML/RARα能够抑制p53对GADD45A的转录激活效应。
Objective To investigate the mechanism by which the aberrant fusion protein,PML/RARα,transcriptionally regulates GADD45A in acute promyelocytic leukemia(APL) cells.Methods ChIP-PCR was employed to study the enrichment of PML/RARα at the intron 3 of GADD45A.Real-Time PCR was conducted to investigate whether the ectopic expression of PML/RARα could impact the mRNA level of GADD45A gene in PR9 cell line.Eucaryotic expression vector was utilized to overexpress PML/RARα and p53 protein in H1299 cells,in order to investigate the co-regulation relationship between PML/RARα and p53 on GADD45A gene.Results ChIP-PCR revealed that the specific PML/RARα antibody could enrich the DNA segment of intron 3 of GADD45A gene compared to the negtive results of non-specific normal IgG,which indicated that PML/RARα could bind to the intron 3 of GADD45A.In PR9 cells,the expression of PML/RARα exhibited significant negtive correlation with GADD45A mRNA expression.PML/RARα could inhibit the transcriptional activity of p53 on GADD45A in H1299 cells(P<0.01).Conclusion PML/RARα can bind to the intron 3 of GADD45A gene.GADD45A is also the target of p53 protein,and PML/RARα can inhibit GADD45A expression and interrupt p53 dependent transcriptional effect of GADD45A gene.
引文
[1]De ThéH,Chen Z.Acute promyelocytic leukaemia:novel insightsinto the mechanisms of cure[J].Nat Rev Cancer,2010,10(11):775-783.
[2]Grignani F,Fagioli M,Alcalay M,et al.Acute promyelocyticleukemia:from genetics to treatment[J].Blood,1994,83(1):10-25.
[3]Wang K,Wang P,Shi J,et al.PML/RARalpha targets promoterregions containing PU.1 consensus and RARE half sites in acutepromyelocytic leukemia[J].Cancer Cell,2010,17(2):186-197.
[4]Levine AJ,Momand J,Finlay CA.The p53 tumour suppressor gene[J].Nature,1991,351(6326):453-456.
[5]Petitjean A,Achatz MI,Borresen-Dale AL,et al.TP53 mutationsin human cancers:functional selection and impact on cancer progno-sis and outcomes[J].Oncogene,2007,26(15):2157-2165.
[6]Longo L,Trecca D,Biondi A,et al.Frequency of RAS and p53mutations in acute promyelocytic leukemias[J].Leuk Lymphoma,1993,11(5-6):405-410.
[7]Trecca D,Longo L,Biondi A,et al.Analysis of p53 gene mutationsin acute myeloid leukemia[J].Am J Hematol,1994,46(4):304-309.
[8]Zhou GB,Zhang J,Wang ZY,et al.Treatment of acute promyelo-cytic leukaemia with all-trans retinoic acid and arsenic trioxide:aparadigm of synergistic molecular targeting therapy[J].Philos TransR Soc Lond B Biol Sci,2007,362(1482):959-971.
[9]Liu TX,Zhang JW,Tao J,et al.Gene expression networks under-lying retinoic acid-induced differentiation of acute promyelocytic leu-kemia cells[J].Blood,2000,96(4):1496-1504.
[10]Riley T,Sontag E,Chen P,et al.Transcriptional control of humanp53-regulated genes[J].Nat Rev Mol Cell Biol,2008,9(5):402-412.
[11]Insinga A,Monestiroli S,Ronzoni S,et al.Impairment of p53acetylation,stability and function by an oncogenic transcriptionfactor[J].EMBO J,2004,23(5):1144-1154.
[12]Wang XW,Zhan Q,Coursen JD,et al.GADD45 induction of aG2/M cell cycle checkpoint[J].Proc Natl Acad Sci U S A,1999,96(7):3706-3711.
[13]Hollander MC,Sheikh MS,Bulavin DV,et al.Genomic instabilityin Gadd45a-deficient mice[J].Nat Genet,1999,23(2):176-184.
[14]Hollander MC,Jr Fornace AJ.Genomic instability,centrosomeamplification,cell cycle checkpoints and Gadd45a[J].Oncogene,2002,21(40):6228-6233.
[15]Mak SK,Kultz D.Gadd45 proteins induce G2/M arrest and modu-late apoptosis in kidney cells exposed to hyperosmotic stress[J].JBiol Chem,2004,279(37):39075-39084.
[16]Takekawa M,Saito H.A family of stress-inducible GADD45-likeproteins mediate activation of the stress-responsive MTK1/MEKK4MAPKKK[J].Cell,1998,95(4):521-530.
[17]Yi YW,Kim D,Jung N,et al.Gadd45 family proteins are coacti-vators of nuclear hormone receptors[J].Biochem Biophys ResCommun,2000,272(1):193-198.
[18]Wang ZY,Chen Z.Acute promyelocytic leukemia:from highly fatalto highly curable[J].Blood,2008,111(5):2505-2515.
[19]Duprez E,Wagner K,Koch H,et al.C/EBPbeta:a major PML-RARA-responsive gene in retinoic acid-induced differentiation ofAPL cells[J].EMBO J,2003,22(21):5806-5816.
[20]Grandér D.How do mutated oncogenes and tumor suppressor genescause cancer[J].Medical Oncology,1998,15(1):20-26.
[21]Rosemary SA,Richardson DR.Growth arrest and DNA damage-45alpha(GADD45alpha)[J].Int J Biochem Cell Biol,2009,41(5):986-989.
[22]Hollander MC,Philburn RT,Patterson AD,et al.Genomic insta-bility in Gadd45a-/-cells is coupled with S-phase checkpoint de-fects[J].Cell Cycle,2005,4(5):704-709.
[23]Valk PJ,Verhaak RG,Beijen MA,et al.Prognostically usefulgene-expression profiles in acute myeloid leukemia[J].J N Engl JMed,2004,350(16):1617-1628.
[24]Reddy SP,Britto R,Vinnakota K,et al.Novel glioblastoma markerswith diagnostic and prognostic value identified through transcriptomeanalysis[J].Clin Cancer Res,2008,14(10):2978-2987.
[25]Cretu A,Sha X,Tront J,et al.Stress sensor Gadd45 genes as thera-peutic targets in cancer[J].Cancer Ther,2009,7(A):268-276.
[26]Liebermann DA,Hoffman B.Prostate cancer:JunD,Gadd45a and Gadd45g as therapeutic targets[J].Cell Cycle,2011,10(20):3428.
