Abstract:
Exposure to methylmercury (MeHg) in the environment poses a risk of impaired function and death to neurons, with the developing brain being more susceptible. Our lab has hypothesized that MeHg damage can be characterized by changes in cell cycle progressionânamely G2M arrest. The tumor suppressor gene p53, acting as a transcription factor in response to genotoxic stress, serves as a pivotal cell cycle checkpoint and thus may play a role in modulating MeHg toxicity even in the absence of direct DNA damage. We have employed cell cultures of p53 transgenic mouse embryonal fibroblasts to examine differential sensitivity to MeHg, and specifically the gene expression pathways linked to MeHg-induced cytotoxicity. Cyclin B1 and p21Cip1/WAF1 were of particular interest for their respective promotion and inhibition of cyclin dependent kinases. The p53-dependent and independent growth arrest genes GADD45 and GADD153 were also assayed by semi-quantitative RT-PCR. A 2.5mM MeHg dose causing G2M arrest without overt cytotoxicity was chosen to highlight the changes in transcriptional abundance. To expand the scope of genes impacted by MeHg, two types of arrays were hybridized with total RNA isolated from untreated and treated cells. GEArraysâa nylon membrane-based, cDNA platform comprised of 96 genes specific to the cell cycle or p53 pathway, were compared to the CodeLink glass slide-based 10K oligonucleotide array system. Differences in probe design, labeling technique, and quantification of positive signals between the GEArray and CodeLink technologies were also considered for their effect on sensitivity of detection. After 24 hours, p53 genotype did contribute to differential gene expression as assessed by RT-PCR and both array methods. P53+/+ fibroblasts up-regulated p21 transcript in response to MeHg treatment while p53-/- cells did not. Conversely, from RT-PCR and only the CodeLink arrays, cyclin B1 mRNA expression was suppressed in treated p53+/+ cultures but marginally up regulated in the p53-/-. In addition to the expected changes in gene expression, several unknown effectors downstream of p53 were further identified for a possible role in MeHg-induced cell cycle arrest. For example, the expression levels of G2 S phase expressed protein 1 (Gtse1), which causes G2 arrest independent of p53 status by interfering with microtubule rearrangements necessary for mitosis, was found to increase on both GEArrays and CodeLink slides. Identification of multiple pathways influenced by MeHg treatment in relation to p53 status was possible using cDNA and oligo arrays as a screening tool.