Abstract
Abstract Background. The ubiquitin ligase protein MDM2 plays an important role in cellular processes like growth arrest, senescence and apoptosis, and one of its main targets is the key cell cycle regulator p53. MDM2 is found overexpressed in several solid tumors, and its dysregulation is caused by gene amplification or by enhanced transcription or translation. The MDM2 gene has two main promoters; promoter P1 is constitutive while P2 is considered to be inducible in response to cellular stress. One polymorphism located in the MDM2 promoter P2 region, SNP309T>G (rs2279744), has been shown to enhance Sp1 transcription factor binding and is associated with increased cancer risk. Recently we reported a second polymorphism SNP285G>C (rs117039649) located 24 bp upstream of rs2279744. SNP285C resides on the SNP309G allele and was found to reduce Sp1 binding and lower the risk of ovarian, endometrial and breast cancer. A third SNP located in the same region, SNP344T>A (rs1196333) has been found not to be associated with cancer risk. In this study we examined the expression levels from the two MDM2 promoters among individuals harboring the different SNP variants (SNP309T/G, SNP285G/C and SNP344T/A) before and after irradiation. Materials and methods. Blood samples were collected from 219 healthy individuals. From this blood the buffy coat fraction was separated, divided into two and cultured. Prior to RNA extraction and subsequent cDNA synthesis, one half of the cells were irradiated with a dose equivalent to 3 Grey. Each sample was investigated for SNP285, SNP309 and SNP44 status by sequencing, and MDM2 expression was determined by qPCR using hydrolysis probes. All statistical analyses were calculated using SPSS and GraphPad Prism 5. Results. After stress (ionizing radiation) we observed, on average, a 8 and 60 fold increase in mRNA levels from promoter P1 and P2, respectively (p<0,0001). Statistical analysis of MDM2 mRNA expression from promoter P1 and P2, showed no significant difference between individuals harboring the different genotypes of SNP309 (TT/TG/GG), SNP285 (GG/GC/CC) or SNP344 (TT/TA/AA) before or after stress. Since the SNP285C allele counteracts the effect of SNP309G (reduces Sp1 binding), we analyzed the impact of SNP309G in the subgroup of individuals harboring SNP285 GG, but found no correlation to higher expression levels in this subgroup either. Conclusions. Despite their association with risk of several major solid tumor forms, we found no correlation between MDM2 promoter SNP285 or SNP309 with mRNA expression levels in WBC. Citation Format: Liv B. Gansmo, Stian Knappskog, Per E. Lonning. MDM2 promoter variants and mRNA expression in irradiated lymphocytes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1792. doi:10.1158/1538-7445.AM2013-1792
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