Increased oxidative DNA damage in mammary tumor cells by continuous epidermal growth factor stimulation.
BACKGROUND
Growth factors can enhance the malignant potential of tumor cells. To examine the relationship between growth factors and tumor progression, we previously established a weakly malignant cell line, ER-1. We found that a 24-hour exposure of ER-1 cells to epidermal growth factor (EGF) induced malignant properties (tumor progression) that were reversible but that, after a 1-month exposure, these changes were irreversible. In this study, we investigated the irreversible changes induced in ER-1 cells by a 1-month exposure to EGF and the possible involvement of oxidative stress.


METHODS
ER-1 cells were treated with EGF (100 ng/mL) for 1 month in the presence or absence of an antioxidant, N-acetylcysteine or selenium, and compared with untreated control ER-1 cells. We assessed tumor progression by measuring intracellular peroxide levels, 8-hydroxydeoxyguanosine (a marker for oxidative DNA damage) levels, in vitro invasiveness, and in vivo tumorigenicity and metastatic ability. All statistical tests are two-sided.


RESULTS
After ER-1 cells were treated for 1 month with EGF, levels of intracellular peroxide and 8-hydroxyguanosine in the DNA of treated cells were higher than those in the DNA of control cells, and treated ER-1 cells were more tumorigenic and metastatic in vivo and more invasive in vitro than untreated control cells (all P<.001). Levels of 8-hydroxyguanosine in DNA increased as the length of the EGF treatment increased (P<.001). However, when N-acetylcysteine or selenium was added with EGF for 1 month, levels of intracellular peroxide and 8-hydroxyguanosine in DNA were comparable to those in control cells (r =.795). Both tumorigenicity (P =.008) and metastatic ability (P<.001) decreased after addition of N-acetylcysteine or selenium.


CONCLUSIONS
The irreversible changes caused by continuous EGF stimulation of ER-1 cells result from increased oxidative damage in the DNA, which generates tumor cells with more malignant characteristics.
Hamada
J
J
Division of Cancer-Related Genes, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan. jhamada@med.hokudai.ac.jp
Nakata
D
D
Nakae
D
D
Kobayashi
Y
Y
Akai
H
H
Konishi
Y
Y
Okada
F
F
Shibata
T
T
Hosokawa
M
M
Moriuchi
T
T
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000111:Acetylcysteine / Q000494:pharmacology; D000230:Adenocarcinoma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D018450:Disease Progression; D004815:Epidermal Growth Factor / Q000009:adverse effects*; D005260:Female; D016166:Free Radical Scavengers / Q000494:pharmacology*; D005979:Glutathione Peroxidase / Q000378:metabolism; D008325:Mammary Neoplasms, Experimental / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D018384:Oxidative Stress / Q000187:drug effects; D051381:Rats; D011918:Rats, Inbred SHR; D012643:Selenium / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000975:Antioxidants; D016166:Free Radical Scavengers; D004815:Epidermal Growth Factor; D000080242:8-Hydroxy-2'-Deoxyguanosine; D005979:Glutathione Peroxidase; D003849:Deoxyguanosine; D012643:Selenium; D000111:Acetylcysteine