Carcinogenesis, Vol 18, 1709-1714, Copyright © 1997 by Oxford University Press
SJ Duthie and P McMillan
Poor folate status may be important in the aetiology of several epithelial
cell malignancies including cancer of the uterine cervix. Folic acid is
essential in the synthesis of purine nucleotides and the pyrimidine
nucleoside thymidine and it is probable that imbalances in these DNA
precursors negatively effect DNA stability and may ultimately lead to
malignant transformation. The development of a modified 'comet assay' using
the bacterial DNA repair enzyme uracil DNA glycosylase, to detect
misincorporated uracil in human DNA is reported here. The effect of
perturbing folic acid and deoxyuridine levels on uracil misincorporation in
normal human lymphocytes and cultured human tumour cells was investigated
using this assay. HeLa cells and peripheral human lymphocytes incubated as
agarose-embedded nucleoids, with 1 unit of uracil DNA glycosylase per
microg of DNA, contained low levels of uracil in their DNA. Both HeLa cells
and stimulated human lymphocytes cultured in folate-deficient medium were
growth arrested. Incubating human lymphocytes in folate-deficient medium
significantly increased the level of uracil detected compared with control
cells. HeLa cells showed an increase in non-specific DNA damage (strand
breaks). Deoxyuridine (100 microM) significantly increased the level of
uracil detected in the DNA of both folate-deficient and control HeLa cells.
It appears that this modified comet assay specifically detects
misincorporated uracil in single human cells. It should, therefore, prove
valuable in determining the role of folic acid status in DNA instability
and cancer.
ARTICLES
Uracil misincorporation in human DNA detected using single cell gel electrophoresis
Rowett Research Institute, Bucksburn, Aberdeen, Scotland, UK.
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