Increasing the DNA damage threshold in breast cancer cells.
The biochemical role of estrogens in the development of estrogen-dependent breast cancer remains to be elucidated, and the involvement of estrogens in tumor initiation remains controversial. Reports regarding estrogen-mediated DNA damage include the induction of 8-oxo-2'-deoxyguanosine (8-oxo-dG) in vitro and in vivo, indicating a role for oxidative stress in tumor initiation and/or progression. However, DNA isolation, cellular DNA repair, and high antioxidant status have made the measurement of 8-oxo-dG in vivo and in cell culture somewhat challenging. In this regard, a potentiation in DNA damage can be achieved by depleting cellular stores of glutathione. We chose to deplete glutathione in the estrogen receptor (ER)-positive MCF-7 breast cancer cell line with a gamma-glutamylcysteine transpeptidase enzyme inhibitor buthionine sulphoximine (BSO) for the purpose of studying estrogen-induced DNA damage. Treatment of GSH-depleted MCF-7 cells with 10 microM 2-OH-E2 or 4-OH-E2 for 30 min resulted in a statistically significant increase in 8-oxo-dG/10(5) dG of 127 and 160%, respectively. A potentiation in catechol estrogen-induced DNA damage was observed with the addition of copper(II) chloride for both 2-OH-E2 and 4-OH-E2 by 165 and 200%, respectively. In addition, 100 nM and 1.0 microM estradiol increased DNA damage in a dose-response-like fashion by 145 and 189%, respectively. The depletion of GSH by BSO may prove to be an advantageous technique for the study of DNA damage in cells otherwise resistant to oxidative stress and/or alkylating agents and has proven useful in the study of estrogen-induced oxidative DNA damage in a highly reproducible and sensitive manner.
Mobley
James A
JA
Division of Medicinal Chemistry and Pharmacognosy, The Ohio State University, Columbus, Ohio 43210, USA.
Brueggemeier
Robert W
RW
D000080242:8-Hydroxy-2'-Deoxyguanosine; D019328:Buthionine Sulfoximine / Q000494:pharmacology*; D004249:DNA Damage / Q000187:drug effects*; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004958:Estradiol / Q000378:metabolism / Q000494:pharmacology*; D005978:Glutathione / Q000172:deficiency* / Q000502:physiology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects*; D011960:Receptors, Estrogen / Q000187:drug effects / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D011960:Receptors, Estrogen; D004958:Estradiol; D019328:Buthionine Sulfoximine; D000080242:8-Hydroxy-2'-Deoxyguanosine; D007770:L-Lactate Dehydrogenase; D003849:Deoxyguanosine; D005978:Glutathione