Nucleic Acids Research, Vol 26, Issue 13 3194-3201, Copyright © 1998 by Oxford University Press
K Bialkowski and KS Kasprzak
8-Oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is a product of
oxidative modification of dGTP, thatcan be misincorporated into DNA,
causing AT-->CG mutations. Cells are protected against 8-oxo-dGTP by 8-
oxo-dGTP 5'-pyrophosphohydrolases (8-oxo-dGTP-ases) that convert it to
8-oxo-dGMP. Thus, inhibition of 8-oxo-dGTPases may lead to cancer. To
elucidate the involvement of 8-oxo-dGTPases in carcinogenesis, an assay of
the 8-oxo-dGTPase activity is required. This paper presents such an assay
developed for Chinese hamster ovary (CHO) cells that can be applied to any
biological material. It includes: (i) a convenient method for preparing
8-oxo-2'-deoxyguanosine 5'-phosphates; (ii) an HPLC/UV quantification of
8-oxo-dGTP hydrolysis products and (iii) separation of 8-oxo-dGTPase
activity from interfering 8-oxo-dGTP phosphatase(s). The 8-oxo-dGTPase
activity of CHO cells depends on magnesium, has a pH optimum of 8.5, Km for
8-oxo-dGTP of 9.3 microM, and is inhibited by 8-oxo-dGDP, the product of
interfering 8-oxo-dGTP phosphatases. The latter must be removed from the
assayed samples by ultrafiltration through 30 kDa cut-off membranes. The
method was used to test the inhibition by cadmium ions of the activity of
8-oxo-dGTPase in CHO cells. The cells cultured with 0.3-3 microM
cadmium(II) acetate for up to 24 h had their 8-oxo-dGTPase activity
suppressed in a Cd(II) concentration-dependent manner, down to 70% of the
control value.
ARTICLES
A novel assay of 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) activity in cultured cells and its use for evaluation of cadmium(II) inhibition of this activity
Laboratory of Comparative Carcinogenesis, National Cancer Institute- FCRDC, Building 538, Room 205E, Frederick, MD 21702, USA. karolb@mail.ncifcrf.gov
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