MDM2 SNP309 Polymorphisms (SNPs) In The Role Of The Risk Of The Occurrence Of Acute Myeloid Leukemia And Sensitivity To Chemotherapeutic Drugs

Part one.Risk of MDM2 SNP309 alone or in combination with p53 codon 72 polymorphism in acute leukemiaObjectives:The p53 tumor suppressor pathway is aberrant in most human tumors with over 50%expressing mutant p53 protein.The pathway is critically controlled by the protein mouse double minute 2(MDM2),which is a key negative regulator of p53.A single nucleotide polymorphism(SNP) in the promoter of MDM2 gene,SNP309 T＞G(a T to G exchange at nucleotide 309 in the first intron),can increase the expression level of MDM2,thereby causing an impairment of p53 tumor suppressor activity.A G to C exchange at p53 codon 72 polymorphism results in a substitution of proline(Pro) for arginine(Arg) in the transactivation domain,which was shown to alter the primary structure of the p53 protein.Both polymorphisms have been implicated in cancer.The purpose of this study is to investigate whether that MDM2 SNP309 and p53 codon 72 polymorphism should be at least partially responsible for genetic susceptibility to acute myeloid leukemia(AML).Methods:Genotyping of MDM2 SNP309 and p53 codon 72 polymorphism were determined in 231 patients with AML and 128 healthy individuals by allele-specific polymerase chain reaction(AS-PCR) and PCR-based restriction fragment length polymorphism(RFLP) methods.Genotype frequencies were performed for Hardy-Weinberg equilibrium in controls using the Pearson's x² test.Logistic regression models were used to examine whether the MDM2 SNP309 and p53 codon 72 polymorphisms were associated with AML.Odds ratios(OR) with the corresponding 95%Confidence intervals(95%CI) and P-values were calculated using unconditional logistic regression with age adjustment.The interaction between the MDM2 SNP309 and p53 codon 72 polymorphisms was performed by adding an interaction term to the logistic model and computing the likelihood ratio statistic.A more-than-additive interaction was suggested when OR₁₁＞OR₁₀+OR₀₁—1,for which OR₁₁=OR when both factors were present,OR₁₀=OR when only factor 1 was present and OR₀₁=OR when only factor 2 was present.A more-than-multiplicative interaction was suggested when OR₁₁＞OR₁₀×OR₀₁.Genotype-specific distributions of age of disease onset were compared via Wilcoxon Mann-Whitney test or Kruskal-Wallis H-test.The correlation of genotypes and any other clinical parameters(gender,cytogenetic risk,initial WBC count and so on) was analyzed via the Fisher's exact test or x² test as appropriate.All P-values were two-sided with a P-value＜0.05 considered to be statistically significant.All statistical analyses were conducted using SPSS 13.0 software(SPSS Science).Results:The genotype distribution of the MDM2 SNP309 and p53 codon 72 polymorphisms in controls did not deviate from the Hardy-Weinberg equilibrium. Frequencies of MDM2 309TT,TG,and GG genotypes among patients were significantly different from those among controls(x²=9.75;p=0.002;df=2),with the GG homozygotes being significantly over-represented among patients than among controls(32.9%verus 19.5%;p=0.003).The MDM2 SNP309G allele was associated with increased risk of AML[GG versus TT:odds ratio(OR)=3.52,95%confidence interval(95%CI) =1.80-6.87].Furthermore,the p53 codon 72 and MDM2 SNP309 polymorphisms did not associated with age of onset and any other clinical parameters studied.When the p53 and MDM2 polymorphisms were combined,no multiplicative joint effect between the MDM2 GG and p53 Pro/Pro genotypes exists in the risk of developing AML.Conclusions:The MDM2 SNP309 homozygous GG genotype may be a genetic susceptibility factor in the pathogenesis of AML. Part two.Effect of MDM2 gene polymorphism and chemotherapy drugs in leukemia cell linesObjectives:DNR and etopside play key role in leukemia therapy.Many cell cyie regulatory proteins are involved in the etopside-induced apoptosis.Among those,p53, c-Myc and BAFF have been identified as pathway utilized to arrest cell cycle progress and induce apoptosis in certain cell lines exposed to etopside.Etoposide activated two pathways which lead to G2M arrest,one of which depends on the presence of p53 wheras the other is p53 independent.DNR triggers both positive and negative regulatory pathways of apoptosis.The purpose of this investigation is to clarify the effect of MDM SNP309 on chemotherapeutic drugs.Methods:We detected MDM2 SNP309 genotype and p53 mutation of leukemia cell lines by PCR amplification and sequencing methods.Leukemia cell lines were treated with DNR and etopside.Cell's ability of prolification were analyzed by MTT methods.At the same time,cell apoptosis were detected by DNA ladder analysis and Annexin V method.Results:The MDM2 SNP309 polymorphism has no effect on the proliferation ability of leukemia cells lines.However,DNA ladder analysis suggests that the leukemia cell lines with the homozygous TT genotype is more sensitive to apoptosis induced by DNR and VP-16.Conclusions:MDM2 SNP309 genotypes could affect the sensitivity to chemotherapeutic drugs in leukemia cell lines. Part three.AML1 and AML1-ETO on the regulation of MDM2 expressionObjectives:The aims of this study are to observe the impact of AML1 and AML1-ETO fusion gene on the transcription activity of different SNP309 MDM2 P2 promoter,and to explore the mechanism of induction of leukemia pathogenesis by AML1-ETO.Methods:The luciferase reporter plasmids of MDM2 P2 promoter containing different SNP309 were contructed,and cotransfected into CV-1 cells with AML1-ETO,AML1 and SP1 expression plasmid.The transactivities of luciferase were assayed by luminometer to determine the impact of AML1,AML1-ETO,and SP1 on MDM2 P2 promoter.Results:In CV-1 cells,if AML1-ETO was set within a certain concentration range (50-500ng),pGL3-MDM2 G-Basic vector relative to pGL3-MDM2 T-Basic vector, luciferase expression decrease 1.2-4 folds.However,no significant variation demonstrates for the transcriptional regulation effect of AML1 on different SNP309 genotype of the MDM2 P2 promoter.The AML1-ETO with the fixed-dose of SP1 were transfected with MDM2 P2 promoter reporter gene vector containing different genotype of the SNP309,and we found that the luciferase reporter gene expression of SNP309 GG promoter was significantly reduced in a dose-independent manner,whereas the luciferase reporter gene expression of SNP309 TT promoter increase.With the increased AML1-ETO dose,luciferase expression decreased.Conclusions:AML1-ETO can impact transcriptional regulation of the MDM2 P2 promoter via the locus of SNP309.