Affiliations of authors: J. Hamada, D. Nakata, T. Moriuchi (Division of Cancer-Related Genes), F. Okada, M. Hosokawa (Division of Cancer Pathobiology), Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan; D. Nakae, Y. Kobayashi, H. Akai, Y. Konishi, Department of Oncological Pathology, Cancer Center, Nara Medical University, Kashihara, Japan; T. Shibata, Second Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Health Science University of Hokkaido, Tobetsu, Ishikari, Japan.
Correspondence to: Jun-ichi Hamada, Ph.D., Division of Cancer-Related Genes, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 0608638, Japan (e-mail: jhamada{at}med.hokudai.ac.jp).
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We isolated (4) a weakly malignant cell line, ER-1, from the rat mammary carcinoma cell line c-SST-2 to identify factors involved in tumor progression and found (5) that epidermal growth factor (EGF) or transforming growth factor- enhanced the tumorigenic, metastatic, and in vitro invasive capacities of these cells. We observed (6) that a 1-month exposure to EGF induced irreversible changes (tumor progression) in ER-1 cells but that a 24-hour exposure did not. We also hypothesized (6) that the increased intracellular oxidative stress induced by EGF might play an important role in both irreversible and reversible tumor progression. Thus, the ER-1 cell line appears to be a good model for investigations of tumor progression induced by continuous growth factor stimulation.
In this report, we investigate the mechanisms of irreversible tumor progression by using ER-1 cells treated with EGF in vitro for 1 month.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SHR rats were purchased from Charles River Japan (Yokohama, Japan). Female SHR rats aged 710 weeks were used for all experiments.
Cell Culture
The ER-1 clonal cell line was derived from a mammary adenocarcinoma that developed spontaneously in a female SHR rat (4). This cell line was grown on tissue culture dishes in Dulbecco's modified Eagle medium (DMEM) supplemented with 7% fetal bovine serum (FBS). The rat lung endothelial (RLE) cell line was provided by Dr. G. L. Nicolson (The University of Texas M. D. Anderson Cancer Center, Houston) (7). Mesothelial cells were isolated from transparent sheets of mesentery in SHR rats by the method of Akedo et al. (8). RLE and mesothelial cells were grown on gelatin-coated tissue culture dishes in DMEM supplemented with 10% FBS. All cell lines were maintained in a CO2 incubator (5% CO2-95% air).
Treatment of ER-1 Cells With EGF
ER-1 cells were seeded on tissue culture plates or dishes in DMEM containing 3% FBS and human recombinant EGF at 100 ng/mL (Wakunaga Pharmaceutical, Hiroshima, Japan). During the 4-week (1-month) treatment with EGF, culture medium containing EGF was replaced every day. One week before assays for tumorigenicity, metastasis, and in vitro invasion, EGF supplementation was stopped and EGF-treated ER-1 cells were cultured in the absence of EGF.
In some experiments, ER-1 cells were treated with 5 or 10 mM N-acetylcysteine (Wako Pure Chemical, Tokyo, Japan) or with sodium selenite at a dose of 1, 10, or 100 ng/mL (Sigma Chemical Co., St. Louis, MO) in the presence or absence of EGF for 4 weeks (1 month).
Assay for Tumorigenicity and Metastasis
After ER-1 cells were treated with EGF as described above, they were washed in phosphate-buffered saline (PBS) and detached from culture dishes with PBS containing 2 mM EDTA. Recovered cells were washed twice in PBS followed by centrifugation at 600g for 5 minutes at 4 °C. To evaluate the tumorigenicity of the cells, we injected 1 x 105 cells resuspended in PBS intraperitoneally into an SHR rat (five or six rats per treatment group). Four weeks after the injection, the rats were killed under ether anesthesia by cervical dislocation, and disseminated tumor nodules in the peritoneum were examined. To evaluate the metastatic capacity of the cells, we injected 1 x 104 cells resuspended in PBS intravenously into the tail vein of an SHR rat (five rats per treatment group). Five weeks after the injection, the rats were killed under ether anesthesia by cervical dislocation and examined for pulmonary metastases. The experiments were approved by the Animal Care and Use Committee of Hokkaido University School of Medicine.
Assay for In Vitro Invasion of Mesothelial or Endothelial Cell Monolayer by ER-1 Cells
The in vitro invasion of rat mesenteric mesothelial cell monolayers and RLE cell monolayers by ER-1 cells was assayed as described previously by Akedo et al. (8) and Ohigashi et al. (9). Briefly, when the RLE or mesothelial cells reached confluence in 60-mm tissue culture dishes with grids, 2 x 104 ER-1 cells were layered over the RLE or mesothelial cell monolayer. The invasive capacity of ER-1 cells was measured 1 week after the tumor cell seeding by counting the number of colonies per square centimeter formed under the RLE cell monolayer by use of a phase-contrast microscope.
Assay for In Vitro Invasion of a Matrigel Reconstituted Basement Membrane by ER-1 Cells
Chemoinvasion of basement membrane-like matrices by ER-1 cells was assayed by use of Matrigel (Collaborative Research, Inc., Bedford, MA) and Transwell chambers (Costar, Cambridge, MA) as described previously (10). Briefly, 2 x 104 ER-1 cells suspended in DMEM supplemented with 0.1% bovine serum albumin were placed in the upper compartment of a Transwell chamber that was separated from the lower chamber by a Matrigel-coated filter with 8-µm pores. The lower chamber contained conditioned medium from newborn SHR rat skin fibroblasts as the chemoattractant. After a 3-day incubation, the cells that had invaded the Matrigel and attached to the lower surface of the filter were fixed with 10% formalin, stained with a 5% Giemsa solution, and counted under a microscope. The invasive capacity was calculated from the number of cells that invaded the Matrigel per microscopic field (x200 magnification).
Measurement of Intracellular Peroxides by Confocal Laser Scanning Microscopy
Intracellular peroxide levels were measured by use of 2',7'-dichlorofluorescin diacetate (Eastman Kodak, Rochester, NY) as reported previously by Ohba et al. (11). 2',7'-Dichlorofluorescin diacetate is a membrane-permeable nonfluorescent compound that is converted into membrane-impermeable nonfluorescent 2',7'-dichlorofluorescin by alkaline hydrolysis. 2',7'-Dichlorofluorescin is rapidly oxidized to fluorescent 2',7'-dichlorofluorescein in the presence of hydrogen peroxide and peroxidases (12). We also used an oxidized probe, 5- (and 6-) carboxy-2',7'-dichlorofluorescein diacetate (carboxy-DCFDA) (Molecular Probes, Inc., Eugene, OR), to check for differences in esterase activity and efflux of the probe. Briefly, ER-1 cells that had been treated with or without EGF and/or N-acetylcysteine or sodium selenite for 1 month were cultured on an eight-chamber Chamber SlideTM (Nunc, Naperville, IL) in DMEM containing 3% FBS in the presence or absence of N-acetylcysteine (5 or 10 mM) or sodium selenite (1, 10, or 100 ng/mL) for 13 days. EGF at a dose of 100 ng/mL was then added to some cultures; 30 minutes later, the medium in all cultures was replaced with DMEM including 5 µM 2',7'-dichlorofluorescin diacetate or carboxy-DCFDA for 5 minutes, and the cellular fluorescence intensity was measured under a confocal laser scanning microscope. Intracellular peroxide levels were shown by the relative fluorescence intensity, i.e., the fluorescence intensity of a cell treated with 2',7'-dichlorofluorescin diacetate divided by the fluorescence intensity of a cell treated with carboxy-DCFDA.
Determination of 8-Hydroxydeoxyguanosine Levels in DNA of ER-1 Cells
Genomic DNA was extracted from ER-1 cells by use of a SepaGene® kit (Sanko Junyaku, Tokyo, Japan) as described previously (13). In a Eppendorf tube, the extracted DNA was resolved with 250 µL of distilled deionized water, incubated at 95 °C for 2 minutes, and immediately placed on ice for 3 minutes. Six microliters of 1 M sodium citrate was then added, and the mixture was vortex mixed. Fifteen microliters of nuclease P1 (EC 3.1.30.1; Yamasa Shoyu, Chohshi, Japan) at 2000 U/mL was added, and the mixture was mixed well and incubated at 60 °C for 1 hour. After the incubation, 20 µL of 1 M TrisHCl (pH 7.5) was added, the mixture was mixed well, 15 µL of alkaline phosphatase at 1000 U/mL (EC 3.1.3.1; Sigma Chemical Co.) was added, and the mixture was mixed again. After a 1-hour incubation at 37 °C, enzyme-digested DNA was stored at -20 °C until the levels of 8-hydroxydeoxyguanosine were measured.
8-Hydroxydeoxyguanosine levels in these DNA samples were measured by the use of high-pressure liquid chromatography equipped with an electrochemical detector (HPLC/ECD) (Coulochem II; ESA, Bedford, MA) system, as reported previously by Nakae et al. (13), and standard curves made with authentic 8-hydroxyguanosine and deoxyguanosine. Data are expressed as the number of 8-hydroxyguanosine nucleosides per 105 deoxyguanosine nucleosides.
Assay for Glutathione Peroxidase
Glutathione peroxidase activity was determined by the method of Beutler et al. (14), as modified by Yoshimura et al. (15). The amount of reduced nicotinamide dinucleotide phosphate (NADPH) was followed at 340 nm, with cumene hydroperoxide (0.23 mM) as substrate. One unit of enzyme activity was defined as 1 µmol of NADPH oxidized per minute at 37 °C. The enzyme activity was expressed as milliunits per milligram of protein.
Statistical Analysis
Statistical significance was determined by one-way analysis of variance followed by Fisher's probable least-squares difference analysis as a post hoc test. In all statistical comparisons, P<.01 was used to indicate a statistically significant difference. The incidence of tumorigenicity and of metastasis in Table 1 was analyzed by Fisher's exact test. Pearson's correlation analysis was done to determine statistical correlation. All statistical tests are two-sided.
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Phagocytes, such as macrophages and neutrophils, are known as a source of ROS in tumor microenvironment (1721). The large amount of ROS produced extracellularly by activated phagocytes can cause cell death or injury, including mutations to target cells that contribute to carcinogenesis and subsequent tumor progression (1721). The amount of intracellular ROS generated in growth factor-stimulated cells is not high enough to be cytotoxic because it did not reduce the viability and growth rate of the ER-1 cells (data not shown). The 8-hydroxyguanosine levels in DNA from ER-1 cells treated with EGF for 1 week, 2 weeks, 3 weeks, and 4 weeks were 1.5-fold, 1.7-fold, 2.2-fold, and 3.6-fold, respectively, greater than those in the DNA from untreated cells. Such a time-dependent increase in the amount of 8-hydroxyguanosine leads us to speculate that the antioxidant system in EGF-treated ER-1 cells is less active. Actually, several reports (2124) show that some tumor cells have defective antioxidant systems with decreased levels of antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione peroxidases, as well as dysfunctional 8-hydroxyguanosine repair enzymes. Therefore, tumor cells with defective antioxidant systems may be prone to oxidative DNA damage even if a relatively low amount of ROS is generated. 8-Hydroxyguanosine mispairs preferentially with adenosine during replication and thereby causes G : C to T : A transversion mutations (25,26). Therefore, EGF-treated ER-1 cells with the higher levels of 8-hydroxyguanosine in their DNA may be in a mutation-prone state.
The generation of ROS has been detected in various cells stimulated with growth factors and cytokines, including transforming growth factor-1 (11,27,28), platelet-derived growth factor (29,30), basic fibroblast growth factor (30,31), EGF (5,32), interleukin 1 (33), and tumor necrosis factor-
(31,33). Consequently, other growth factors that are found in the tumor microenvironment may also stimulate the production of ROS, resulting in oxidative DNA damage, genetic alteration, and cellular diversification.
![]() |
NOTES |
---|
We dedicate this work to the memory of Dr. Noritoshi Takeichi, whose advice and encouragement were invaluable to us. We thank Miss Masako Yanome for assistance in preparing the manuscript. We also thank Drs. Charles W. Boone and Silvio De Flora for critical reading of the manuscript.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1 Nowell PC. Mechanisms of tumor progression. Cancer Res 1986;46:22037.[Medline]
2 Miller FR, Heppner GH. Cellular interactions in metastasis. Cancer Metastasis Rev 1990;9:2134.[Medline]
3 Nicolson GL. Tumor cell instability, diversification, and progression to the metastatic phenotype: from oncogene to oncofetal expression. Cancer Res 1987;47:147387.[Abstract]
4 Hamada J, Takeichi N, Okada F, Ren J, Li X, Hosokawa M, et al. Progression of weakly malignant clone cells derived from rat mammary carcinoma by host cells reactive to plastic plates. Jpn J Cancer Res 1992;83:48390.[Medline]
5
Li X, Nagayasu H, Hamada J, Hosokawa M, Takeichi N. Enhancement of tumorigenicity and invasion capacity of rat mammary adenocarcinoma cells by epidermal growth factor and transforming growth factor-. Jpn J Cancer Res 1993;84:11459.[Medline]
6 Nagayasu H, Hamada J, Nakata D, Shibata T, Kobayashi M, Hosokawa M, et al. Reversible and irreversible tumor progression of a weakly malignant rat mammary carcinoma cell line by in vitro exposure to epidermal growth factor. Int J Oncol 1998;12:197202.[Medline]
7 Nakajima M, Welch DR, Belloni PN, Nicolson GL. Degradation of basement membrane type IV collagen and lung subendothelial matrix by rat mammary adenocarcinoma cell clones of differing metastatic potentials. Cancer Res 1987;47:486976.[Abstract]
8 Akedo H, Shinkai K, Mukai M, Mori Y, Tateishi R, Tanaka K, et al. Interaction of rat ascites hepatoma cells with cultured mesothelial cell layers: a model for tumor invasion. Cancer Res 1986;46:241622.[Abstract]
9 Ohigashi H, Shinkai K, Mukai M, Ishikawa O, Imaoka S, Iwanaga T, et al. In vitro invasion of endothelial cell monolayer by rat ascites hepatoma cells. Jpn J Cancer Res 1989;80:81821.[Medline]
10 Hamada J, Nagayasu H, Takayama M, Kawano T, Hosokawa M, Takeichi N. Enhanced effect of epidermal growth factor on pulmonary metastasis and in vitro invasion of rat mammary carcinoma cells. Cancer Lett 1995;89:1617.[Medline]
11
Ohba M, Shibanuma M, Kuroki T, Nose K. Production of hydrogen peroxide by transforming growth factor-1 and its involvement in induction of egr-1 in mouse osteoblastic cells. J Cell Biol 1994:126:107988.[Abstract]
12
Bass DA, Parce JW, Dechatelet LR, Szejda P, Seeds MC, Thomas M. Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J Immunol 1983;130:19107.
13 Nakae D, Mizumoto Y, Kobayashi E, Noguchi O, Konishi Y. Improved genomic/nuclear DNA extraction for 8-hydroxydeoxyguanosine analysis of small amounts of rat liver tissue. Cancer Lett 1995;97:2339.[Medline]
14 Beutler E, Blume KG, Kaplan JC, Lohr GW, Ramot B, Valentine WN. International Committee for Standardization in Haematology: recommended methods for red-cell enzyme analysis. Br J Haematol 1977;35:33140.[Medline]
15 Yoshimura S, Komatsu N, Watanabe K. Purification and immunohistochemical localization of rat liver glutathione peroxidase. Biochim Biophys Acta 1980;621:1307.[Medline]
16 Sunde RA. Molecular biology of selenoproteins. Annu Rev Nutr 1990;10:45174.[Medline]
17 Yamashina K, Miller BE, Heppner GH. Macrophage-mediated induction of drug-resistant variants in a mouse mammary tumor cell line. Cancer Res 1986;46:2396401.[Abstract]
18 Weitzman SA, Weitberg AB, Clark EP, Stossel TP. Phagocytes as carcinogens: malignant transformation produced by human neutrophils. Science 1985;227:12313.[Medline]
19 Cerutti PA, Trump BF. Inflammation and oxidative stress in carcinogenesis. Cancer Cells 1991;3:17.[Medline]
20 Weitzman SA, Gordon LI. Inflammation and cancer: role of phagocyte-generated oxidants in carcinogenesis. Blood 1990;76:65563.[Abstract]
21 Okada F, Nakai K, Kobayashi T, Shibata T, Tagami S, Kawakami Y, et al. Inflammatory cell-mediated tumour progression and minisatellite mutation correlate with the decrease of antioxidative enzymes in murine fibrosarcoma cells. Br J Cancer 1999;79:37785.[Medline]
22 Oberley TD, Oberley LW. Antioxidant enzyme levels in cancer. Histol Histopathol 1997;12:52535.[Medline]
23 Sun Y. Free radicals, antioxidant enzymes, and carcinogenesis. Free Radic Biol Med 1990;8:58399.[Medline]
24 Lu R, Nash HM, Verdine GL. A mammalian DNA repair enzyme that excises oxidatively damaged guanines maps to a locus frequently lost in lung cancer. Cur Biol 1997;7:397407.[Medline]
25
Cheng KC, Cahill DS, Kasai H, Nishimura S, Loeb LA. 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes GT and A
C substitutions. J Biol Chem 1992;267:16672.
26 Grollman AP, Moriya M. Mutagenesis by 8-oxoguanine: an enemy within. Trends Genet 1993;9:2469.[Medline]
27
Kayanoki Y, Fujii J, Suzuki K, Kawata S, Matsuzawa Y, Taniguchi N. Suppression of antioxidative enzyme expression by transforming growth factor-1 in rat hepatocytes. J Biol Chem 1994;269:1548892.
28
Thannickal VJ, Fanburg BL. Activation of an H2O2-generating NADH oxidase in human lung fibroblasts by transforming growth factor beta 1. J Biol Chem 1995;270:303348.
29 Sundaresan M, Yu ZX, Ferrans VJ, Irani K, Finkel T. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science 1995;270:2969.[Abstract]
30 Krieger-Brauer HI, Kather H. Antagonistic effects of different members of the fibroblast and platelet-derived growth factor families on adipose conversion and NADPH-dependent H2O2 generation in 3T3 L1-cells. Biochem J 1995;307:54956.[Medline]
31
Lo YY, Cruz TF. Involvement of reactive oxygen species in cytokine and growth factor induction of c-fos expression in chondrocytes. J Biol Chem 1995;270:1172730.
32
Bae YS, Kang SW, Seo MS, Baines IC, Tekle E, Chock PB, et al. Epidermal growth factor (EGF)-induced generation of hydrogen peroxide. Role in EGF receptor-mediated tyrosine phosphorylation. J Biol Chem 1997;272:21721.
33 Meier B, Radeke HH, Selle S, Younes M, Sies H, Resch K, et al. Human fibroblasts release reactive oxygen species in response to interleukin-1 or tumour necrosis factor-alpha. Biochem J 1989;263:53945.[Medline]
Manuscript received July 13, 2000; revised November 24, 2000; accepted November 30, 2000.
This article has been cited by other articles in HighWire Press-hosted journals:
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |