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Biomarkers and Early Detection: Novel Assay Technologies

Detection of DNA strand breaks in the p53 gene: Assay validation and effect of folate depletion.

Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA

Abstract

B23

Low dietary folate intake is associated with an elevated risk for colorectal carcinogenesis. One putative mechanism by which folate depletion promotes carcinogenesis is by inducing gene-specific strand breakage and impaired expression of affected genes. We designed and validated two assays to study the integrity of the hypermutable region of the human p53 tumor suppressor gene. A conventional PCR-based assay targeted a 1547 bp region spanning exons 5 - 8 and a real time PCR-based assay targeted a 615 bp region spanning exons 7 - 8 of the p53 gene were tested. Primary human lymphocytes were then cultured in RPMI-1640 media containing 15 (deplete), 30 (low) or 120 nM (replete) folic acid for nine days. p53 strand breaks, gene and protein expression, and p21 transcript were determined. Cells grown in 15 nM folate developed significantly elevated levels of p53 strand breaks, reflected by reductions in amplifiable DNA from p53 exons 5-8 (~40% loss, P< 0.0001) and exons 7-8 (~26% loss, P< 0.0001) compared to 30 and 120 nM. Nevertheless, RT-PCR revealed that the steady-state abundance of p53 transcript was elevated approximately 2-fold in 15 and 30 compared to 120 nM (P< 0.001). p53 protein abundance increased with decreasing media folate, as did p21 transcript. The Cytokinesis-Block Micronucleus assay demonstrated a 3-fold increase in chromosomal damage (micronuclei, nucleoplasmic bridges and nuclear budding) at the two lower folate concentrations (P< 0.01). In primary human lymphocytes, folate depletion induces a marked increase in p53 exon 5 - 8 breaks, but does not reduce steady state levels of p53 mRNA, protein, or impair downstream signaling. We speculate that under these conditions p53 expression may be maintained, despite elevated strand breakage, by increased transcription and transcript half life. The induction of p53 strand breaks by folate depletion does not impair p53 expression or action within all human cells.