Depletion of O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine in three-dimensional collagen cultures of normal human breast epithelial cells.
O6-Alkylguanine--DNA alkyltransferase (AGT) is a protein which removes the promutagenic O6-alkylguanine lesion induced in DNA by alkylating agents. Our results demonstrate that freshly isolated organoids from reduction mammoplasty specimens contain significant levels of AGT activity. AGT activity in breast epithelial cells shows interindividual variation. Constitutive levels of AGT activity remain unchanged during short-term serum-free culture of breast epithelial cells inside three-dimensional rat-tail collagen gel matrix. In the present study, we optimized conditions for depleting AGT activity in human breast epithelial cells cultured in three-dimensional collagen gel matrix using O6-methylguanine and O6-benzylguanine which are substrates for AGT. AGT activity was efficiently inactivated by exposure of cells to O6-methylguanine or O6-benzylguanine. Inactivation with O6-benzylguanine was more rapid, of greater magnitude and consistency and occurred at lower concentrations than with O6-methylguanine. Near-complete inactivation (> 99.5%) of AGT activity was reproducibly achieved with 50 microM O6-benzylguanine. In contrast, 500 microM O6-methylguanine was needed to obtain a maximal effect and this reduced AGT activity by only 53-93% of control. Within 30 min of adding the free base, 50 microM O6-benzylguanine depleted 95% of the levels of AGT compared to 30% inhibition with 500 microM O6-methylguanine. The profile for restoration of AGT activity was different following a 24 h incubation and subsequent removal of each of the guanine derivatives. AGT activity levels remained undetectable for at least 2 days after removal of 50 microM O6-benzylguanine from the medium and recovered to only 53% of control values after an additional 3 days. AGT activity levels remained undetectable for at least 2 days after removal of 50 microM O6-benzylguanine from the medium and recovered to only 53% of control values after an additional 3 days. In contrast, following removal of 500 microM O6-methylguanine, the activity was restored from its nadir of 16% of control values reaching pretreatment levels after 5 days. These results suggest that treatment with O6-benzylguanine may be used to modulate the incidence of transforming mutations in cultured human breast epithelial cells treated with chemical carcinogens which give rise to O6-alkylguanine adducts.
Hamdan
M A
MA
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Guzman
R C
RC
Yang
J
J
Beattie
C
C
Mascharack
P
P
Nandi
S
S
D000818:Animals; D001483:Base Sequence; D001940:Breast / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D002471:Cell Transformation, Neoplastic / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen; D003470:Culture Media; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology; D005260:Female; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D008593:Menopause / Q000502:physiology; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000172:deficiency* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase; D051381:Rats
D003470:Culture Media; C064976:O(6)-benzylguanine; D006147:Guanine; D003094:Collagen; C008449:O-(6)-methylguanine; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase