Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan 48201
Received April 1, 2003; accepted May 10, 2003
ABSTRACT
Oxidative damage to DNA is thought to play a significant role in mutagenesis, aging, and cancer. Sensitivity to oxidative DNA damage and DNA repair efficiency were examined using a series of human breast epithelial cell linesMCF-10A, MCF-10AT, and MCF-10ATG3Bwith progressively elevated Ras protein. Breast epithelial cells were treated with H2O2, in the absence and presence of the DNA-repair inhibitors hydroxyurea (HU) and cytosine arabinoside (Ara-C). DNA strand breaks were assessed by the mean olive tail moment (µm) using the alkaline single-cell gel electrophoresis (Comet) assay. In untreated cells, the mean olive tail moment values were 4.3 ± 0.7, 8.3 ± 1.1, and 7.1 ± 0.6 µm in the MCF-10A, MCF-10AT, and MCF-10ATG3B cells, respectively. Five min H2O2 treatment produced concentration-dependent DNA damage, with the MCF-10A cells most susceptible and the tumorigenic MCF-10ATG3B cells the least susceptible. Treatment with 100 µM H2O2 resulted in ~17-, 6-, and 4.5-fold increases in mean olive tail moment values in the MCF-10A, MCF-10AT, and MCF-10ATG3B cells, respectively, compared to untreated cells. The HCC1937 tumor cell line responded in a manner comparable to the MCF-10ATG3B cells treated with H2O2, HU/Ara-C pre-treatment resulted in a ~1.5-fold increase in olive tail moment values in all three cell lines. Protein levels of antioxidant enzymes, including catalase, copper/zinc superoxide dismutase (Cu/Zn SOD), and manganese SOD (MnSOD) were determined in order to examine a potential mechanism for increased resistance to H2O2-mediated DNA damage. Levels of these enzymes increased progressively, with highest expression in MCF-10ATG3B cells. Increased cellular resistance also coincided with marked decreases in p53 protein levels. These results demonstrate that, in this cell lineage, sensitivity to oxidative DNA damage by H2O2 decreases with tumorigenicity (i.e., MCF-10A vs. MCF-10ATG3B), and show that DNA repair, altered Ras, and p53 expression, or compensatory mechanisms involving elevated antioxidant enzymes are involved in mediating these effects.
Key Words: oxidative stress; breast epithelial cells; Ha-Ras protein; antioxidant enzymes; p53 protein.
Oxidative stress resulting from oxygen and reactive oxygen species (ROS) is associated with the induction of single- and double-stranded deoxynucleic acid (DNA) breaks and is considered to be the first step in several human degenerative diseases such as cancer and in aging. ROS are generated during many physiological processes including defense against pathogenic microorganisms, cellular metabolism, cell proliferation, induction of cell death, and response to selected environmental agents.
Since human mammary epithelial cells constitute the target cells in the development of breast cancer (Martin et al., 1999b; Thompson et al., 1998
), we were interested in the susceptibility of these cells to oxidative DNA damage, and in whether sensitivity to DNA damaging agents is altered with progression towards tumorigenesis/carcinogenesis. We utilized a series of related human breast epithelial cell lines that represent a progressive model of human proliferative breast disease (PBD) development. These cell lines include the parental MCF-10A cells, the MCF-10AT cells that are T24 Ha-ras-transformed MCF-10A cells, and the third generation MCF-10AT descendant, the MCF-10ATG3B cells. When implanted in nude/beige mice, these three cell lines represent a progressive model of human PBD, with the MCF-10A breast epithelial cells being nontumorigenic and the MCF-10AT and MCF-10ATG3B cells tumorigenic. In mice, the MCF-10AT cells form small nodules that sporadically progress to carcinomas over the course of one year, while the MCF-10ATG3B cells develop tumors within one month and progress to ductal carcinoma in situ at a frequency of 25 to 30%, and ultimately to fully invasive malignant carcinoma (Dawson et al., 1996
).
We have previously shown that the MCF-10A cells are much more susceptible to Fas-mediated apoptosis than either the MCF-10AT or the MCF-10ATG3B cells, and that this resistance is associated with a progressive elevation in Ras expression (Starcevic et al., 2001). Increased Ras protein levels have been shown to accompany chromosomal damage (Anderson et al., 1997
). In view of the association of elevated Ras with DNA damage, we were interested in whether sensitivity to oxidative DNA damage and DNA repair efficiency was altered in this cell lineage. We utilized hydrogen peroxide (H2O2) to produce oxidative stress and damage DNA. DNA damage was assessed using the comet assay, which is a rapid, visual method for quantitatively assessing DNA damage in single cells and is an established technique in DNA damage and repair studies. Our results demonstrate that H2O2 induces substantial DNA damage that is concentration-dependent. Sensitivity to H2O2-mediated DNA damage decreases with progression towards an increasing rate of tumorigenicity. The MCF-10ATG3B cells, which have the most rapid rate of tumorigenesis in the mouse explant model, are the least sensitive to DNA damage. This decreased sensitivity may be attributed to increased expression of the antioxidant enzymes catalase, copper/zinc superoxide dismutase (Cu/Zn SOD), and manganese SOD (MnSOD) expressed in these cells relative to the parental, nontumorigenic MCF-10A cells, as shown by immunoblot analysis. We also show that this increased cellular resistance coincides with marked decreases in p53 protein levels.
MATERIALS AND METHODS
Cell lines.
The MCF-10AT cell series (MCF-10A, MCF-10AT, and MCF-10ATG3B cells) was obtained from Dr. F. Miller (Karmanos Cancer Institute, formerly the Michigan Cancer Foundation, Detroit, MI). The HCC 1937 cells were obtained from Dr. Adi Gazdar (University of Texas Southwestern Medical Center, Dallas, TX). MCF-10A cells, the progenitor line of the MCF-10AT series, are spontaneously immortalized breast epithelial cells obtained from a woman with fibrocystic breast disease (Soule et al., 1990). MCF-10A cells were transfected with a mutated T24 Ha-ras gene to generate MCF-10AT cells (Basolo et al., 1991
; Russo et al., 1991
). Unlike the MCF-10A cells, MCF-10AT cells persist as xenografts in nude/beige mice and will develop into carcinomas in about 25% of the animals (Miller et al., 1993
). A family of MCF-10AT cell lines was generated by re-establishing cells isolated from the carcinomas in culture and subsequently reinjecting these cells into nude/beige mice (Dawson et al., 1996
). With each subsequent generation, the onset of PBD and the development of invasive cancer appeared more quickly after implantation. The MCF-10ATG3B cell line was generated from cells that have been through this process of transplantation in nude/beige mice and re-establishment in culture three times. These cells form focal cribiforming ducts within 1 month, progress to ductal carcinoma in situ at a frequency of 2530%, and ultimately progress to invasive carcinoma when implanted in nude/beige mice (Dawson et al., 1996
).
Cell culture and treatment.
Cells were maintained in a humidified environment of 5% CO2/95% air at 37°C. The MCF-10AT series of cells was cultured in Dulbeccos modified Eagle medium/F-12 medium supplemented with 10 mg/ml human insulin, 20 ng/ml of epidermal growth factor, 0.5 mg/ml of hydrocortisone, 5% horse serum, 100 U/ml of penicillin, and 100 mg/ml of streptomycin. HCC1937 cells were maintained in RPMI 1640 medium with 10% fetal bovine serum, 2.5 g/l D-glucose, 1 mM sodium pyruvate, 100 U/ml of penicillin, and 100 mg/ml of streptomycin. All cell culture reagents were purchased from Invitrogen (Carlsbad, CA).
The cell lines were passed in 60 cm2 tissue culture dishes. Cells were treated, when they were 100% confluent, with fresh H2O2 (CAS no. 722-84-1; Sigma Chemical Co., St Louis, MO) in Hanks Balanced Salt Solution (HBSS; Invitrogen) for 5 min on ice. H2O2 was prepared immediately prior to use and a new bottle of H2O2 was used in all experiments. For the DNA repair studies, cells were pretreated with the DNA repair inhibitors hydroxyurea (HU; 10 mM; CAS no. 127071; Sigma) and cytosine arabinoside (Ara-C; 1.8 mM; CAS no. 69-74-9; Sigma) at 37°C for 30 min prior to H2O2 treatment (Martin et al., 1999a).
Immunoblot analysis.
Catalase (Sigma), Cu/Zn SOD (BD PharMingen, San Diego, CA), MnSOD (BD Transduction Laboratories, Lexington, KY), p53 (Santa Cruz Biotechnology, Santa Cruz, CA), and Ras (Santa Cruz Biotech.) protein levels were examined by immunoblot analysis of whole-cell lysates. Cells cultured on plastic were washed with HBSS and harvested with lysis buffer (50 mM HEPES, pH 7.2, 150 mM NaCl, 1.5 mM MgCl2, 1.5 mM EGTA, 10% glycerol, 1% Triton X-100, 1 mM MnCl2) containing protease inhibitors (1 mM sodium orthovanadate, 10 mg/ml leupeptin, 2 mM phenylmethylsulfonyl fluoride, 200 units aprotinin). The lysates were passed several times through a 21-gauge needle and incubated on ice for 1 h, after which the lysates were clarified by centrifugation at 16,000 x g for 20 min, 4°C. The resulting supernatant represents the whole cell lysate. Protein concentrations were determined using the BCA protein assay (Sigma). Proteins (25 mg) were resolved on a 7.5% SDSPAGE gel and then transferred to Immobilon-P (Millipore Corp., Bedford, MA). After blocking the Immobilon-P for 1 h in 5% milk in TBST (20 mM TrisHCl, 500 mM NaCl, pH 7.5; 0.05% Tween-20), membranes were incubated with the respective primary antibody overnight in TBST with 5% milk, and then incubated with a horseradish peroxidase-conjugated secondary antibody (BioRad, Hercules, CA) for 1 h in TBST containing 5% milk powder. The membranes were rinsed with TBS. Proteins were detected by enhanced chemiluminescence (Amersham Life Science, Piscataway, NJ) on Kodak X-OMAT film (Sigma). For immunoblot analysis, densitometry was performed on three separate protein preparations using a Molecular Dynamics scanning laser densitometer, and the band density was quantified using the ImageQuant analysis program (Amersham Life Science).
Cell doubling times.
For the MCF-10AT series of cell lines, the cell population doubling time (Td) was calculated from the growth rate by the formula: td = 0.693t/ln(Nt/N0), where t is the time in h, Nt is the cell number at time t, and N0 is the cell number at the initial time. The cell doubling time was determined at 48 h, at which point the cells were at the logarithmic phase of their growth cycle and were ~80% confluent.
Comet assay.
The comet assay, also referred to as the alkaline single-cell gel electrophoresis assay, utilizes single cells that are embedded in an agarose layer on microscope slides, followed by lysis, electrophoresis, and staining with a fluorescent DNA-binding dye. The resulting image, when viewed microscopically, is in the shape of a comet whose tail length and fluorescent intensity are related to the number of DNA strand breaks induced by DNA-damaging agents. The head of the comet represents the long-stranded DNA nucleus of a single-cell, while the comet tail is composed of the smaller pieces of fragmented DNA that have migrated down the gel.
The comet assay was performed under alkaline conditions as described by Singh et al., (1988), with some modifications. Briefly, regular microscope slides were coated with 1% normal-melting-point agarose and allowed to dry overnight at room temperature. Freshly prepared cell suspensions (1 ml; 106 cells/ml) were mixed with 9 ml 0.75% low-melting-point agarose. Samples of 100 ml of this suspension were layered on an agarose precoated slide, covered with a coverslip, and placed on ice for 5 min. The slides were overlaid with low-melting-point agarose a second time and then immersed in freshly prepared cold lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM TrisHCl, 1% Triton X-100, pH 10) for 24 h at 4°C. Following lysis, the slides were placed in a horizontal gel electrophoresis unit with freshly prepared cold electrophoresis solution (300 mM NaOH, 1 mM Na2EDTA, pH > 13, 4°C). After incubating the slides for 30 min to allow the DNA to unwind and to resolve alkali-labile sites, they were electrophoresed at 0.7 V/cm (25 V, 300 mA) for 25 min at 4°C. Following electrophoresis, the slides were neutralized three times (5 min each) with 400 mM TrisHCl (pH 7.5), fixed with 100% ethanol for 2 min, and dried at room temperature. All steps were conducted under dim light to prevent the occurrence of additional DNA damage.
Evaluation of DNA damage.
DNA was stained with propidium iodide (0.1 mg/ml; 0.1 ml/slide), cover-slipped, and analyzed by fluorescence microscopy (excitation filter 515560 nm, barrier filter 590 nm). A computer-based image analysis system (Kinetic Imaging, Ltd., Bromborough, Wirral, U.K.) was used to measure the tail moment values. A total of 100 digitized images, 50 random nuclei from two duplicate slides were scored. The median tail moment of 50 cells/slide was determined from duplicate slides (i.e., 100 cells/concentration were analyzed). Each experiment was repeated three times. The mean olive tail moment value was used as the indicator of DNA damage (Olive, 1989). This parameter includes both the migration of the various DNA fragments forming the tail and their relative amounts of DNA (Hellman et al., 1995
).
Statistical analysis.
Significant differences between groups were determined using the Tukey-Kramer test, with p < 0.05 indicating significant differences.
RESULTS
H2O2 Dose Response
In general, susceptibility to cancer is characterized by high DNA damage, which is the result of low repair capacity. The MCF-10AT series of cell lines, when implanted in nude/beige mice, serve as models for development of human PBD, which is associated with a 45-fold risk factor for breast cancer development. We have used this cell model to determine whether sensitivity to DNA damage is altered at early stages in the progression towards tumorigenesis. Since the MCF-10AT cell model demonstrates increasing propensity for tumorigenicity, we also compared the sensitivity of the MCF-10AT cell model to DNA damage that occurs in HCC1937 cells that contain a mutant BRCA1 (Tomlinson et al., 1998). BRCA1 participates in the regulation of cell cycle progression, apoptosis, and DNA repair pathways, and decreased BRCA1 expression is linked to cell proliferation and transformation (reviewed in Scully and Puget, 2002
).
The ability of H2O2 to induce DNA strand breaks in these human breast epithelial cell lines was examined using the comet assay. In untreated cells, DNA does not migrate far from the origin (Fig. 1A). Following H2O2 treatment, breast epithelial cells with damaged DNA have the shape of a comet, whose tail length and fluorescent intensity are related to the number of DNA strand breaks induced by the DNA-damaging agent (Fig. 1B
). Breast epithelial cells were treated with 0100 µM H2O2 and the mean olive tail moment was determined by the comet assay (Fig. 2A
). In untreated cells, the mean olive tail moments were not significantly different between the three cell lines, with mean olive tail moment values of 4.3 ± 0.7, 8.3.0 ± 1.1, and 7.1 ± 0.6 µm in the MCF-10A, MCF-10AT, and MCF-10ATG3B cells, respectively. Following 110 µM H2O2 treatment, minimal DNA damage was detected in the three cell lines; however, the mean olive tail moment value was generally slightly larger in the MCF-10ATG3B cells than in the MCF-10A and MCF-10AT cells, but the difference was not statistically significant. Treatment with 50 µM H2O2, however, resulted in significant increases, 4- and 2.5-fold, in mean olive tail moment values in the MCF-10A cells (18.8 ± 1.5 µm) and MCF-10AT cells (21.4 ± 1.4 µm) cells compared to cells treated with medium alone. The mean olive tail moment in the MCF-10ATG3B cells was only slightly higher following 50 µM H2O2 treatment, and the difference was not statistically significant compared to untreated cells. A significant increase in DNA damage was observed in all the cell lines following treatment with 100 µM H2O2, with the MCF-10A cells more susceptible than the MCF-10AT and MCF-10ATG3B cells. The MCF-10A cells demonstrated an ~17-fold increase in mean olive tail moment (73.0 ± 1.8 µm) following 100 µM H2O2 treatment compared to untreated cells, while olive tail moment values increased by ~10- and 6-fold in the MCF-10AT (51.1 ± 0.1 µm) and MCF-10ATG3B cells (33.4 ± 1.6 µm), respectively.
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DNA Repair Efficiency
The ability of these cells to repair H2O2-mediated DNA damage in the absence and presence of two DNA repair inhibitors, HU and Ara-C, was examined to determine whether DNA repair capacity changed with progression of the cell series. Ara-C is a potent inhibitor of DNA (,
, and
) polymerases associated with DNA repair (Gedik and Collins, 1991
); HU is a ribonucleotide reductase inhibitor that depletes intracellular deoxribonucleotides levels (Gedik and Collins, 1991
; Mirzayans et al., 1993
; Yarbro, 1992
). Breast epithelial cells were pretreated with HU/Ara-C for 25 min, the medium removed, and the cells were then treated with 100 µM H2O2 (±HU/Ara-C) for 5 min on ice. This concentration was chosen because it produced the greatest extent of DNA damage in the three cell lines. H2O2-treatment was performed on ice because of the rapid repair capacity of these cells. Cells were harvested 5 min following H2O2 treatment or 1 h post-treatment to assess DNA repair efficiency over that time period. Treatment with the DNA repair inhibitors alone (Inhib and Inhib + 1 h) did not significantly increase mean olive tail moment values compared to cells in media alone (Un). HU/Ara-C pretreatment significantly exacerbated the damage induced by 100 µM H2O2 in all three cell lines (Fig. 3
). Mean olive tail moment values rose ~1.5-fold in all the cell lines pretreated with HU/Ara-C and exposed to 100 µM H2O2 (H2O2 + Inhib) when compared to cells treated with 100 µM H2O2 alone. This increased DNA damage did not result in cell death based on trypan blue exclusion (data not shown). The extent of DNA repair at 1 h following removal of H2O2 was also determined in these cell lines. The three cell lines were all able to significantly repair (~49%) DNA strand breaks 1 h following oxidant treatment. All three cell lines, MCF-10A, MCF-10AT, and MCF-10ATG3B, treated with the DNA repair inhibitors were able to repair DNA damage induced by H2O2 after 1 h. Mean olive tail moment values of cells pretreated with HU/Ara-C and treated with H2O2 (H2O2 + Inhib + 1 h) were significantly smaller than cells that were not allowed time for DNA repair (H2O2 + Inhib), indicating that the inhibitory action of HU/Ara-c declines over 1 h or that cells were able to compensate for this inhibition. Complete repair was observed by 4 h following H2O2 removal (data not shown).
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SOD and Catalase Protein Levels
Decreased sensitivity to oxidative DNA strand breaks in the tumorigenic MCF-10ATG3B cells and in the HCC 1937 cells suggests that these cells may have developed mechanisms to minimize DNA damage. Since ROS can potentially damage a wide range of cellular macromolecules including DNA, cells have evolved many mechanisms to combat DNA damage from ROS. Using immunoblot analysis, we examined the status of the antioxidants catalase and SOD. Protein levels of catalase (Fig. 6, top) progressively increased in the MCF-10AT cell series, with the MCF-10AT and MCF-10ATG3B cells containing ~1.7- and 2.6-fold higher levels of catalase than the parent MCF-10A cells, which were the most sensitive to DNA damage by H2O2. Catalase protein levels in the HCC1937 cells were significantly higher than that observed in the MCF-10A cells, and similar to levels detected in MCF-10AT cells.
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DISCUSSION
The natural history of breast cancer suggests that it develops slowly over years, even decades. Thus, we have utilized a cell model of human PBD, a risk factor for breast cancer development, to examine whether sensitivity to oxidative DNA damage by H2O2 is altered with progression towards tumorigenesis and in tumor cells. Our comet analysis results demonstrate that sensitivity to H2O2-mediated DNA damage decreases with progression of this cell lineage. Why such progressive resistance should exist or develop is not clear. Several plausible mechanisms can be proposed. These differences include, but are not limited to, DNA repair status, altered Ras or oncogene expression, and/or compensatory mechanisms that involve elevated expression of antioxidant enzymes.
Extensive DNA damage was observed with the MCF-10A cells following treatment with 100 µM H2O2, with less damage detected in the MCF-10ATG3B cells. The HCC1937 cancer-cell line had comparable mean olive tail moment values to the MCF-10ATG3B cells at 50 and 100 µM H2O2, showing that as the tumorigenicity of the cells increases they become more refractory to oxidative DNA damage by H2O2. Inclusion of the DNA repair inhibitors HU and Ara-C in H2O2 exposure resulted in inhibition of DNA repair and a corresponding increase in the number of DNA strand breaks, causing increased mean olive tail moment values. The three cell lines were all able to significantly repair DNA strand breaks 1 h following H2O2 removal. The fact that pretreatment with the repair inhibitors exacerbates DNA damage by H2O2 indicates that DNA repair pathways provide some degree of rapid protection from H2O2-mediated DNA damage. Taken together, these results show that sensitivity to DNA damage by H2O2 decreases with progression towards tumorigenesis, and that oxidative damage to DNA is repaired rapidly in these human breast epithelial cell lines.
Cells use numerous DNA repair processes to counteract the deleterious effects of DNA damage. The tumor suppressor effects of p53 protein partially rely on its ability to induce apoptotic pathways preventing the replication of cells with genetic errors. p53 also directly participates in DNA repair via the base excision repair and nucleotide excision repair pathways. All of these mechanisms work in concert with the cell cycle and apoptosis to maintain genomic fidelity and integrity. Interestingly, the MCF-10ATG3B cells that contain significantly less p53 protein than its predecessor cell lines, were the least susceptible to DNA damage by H2O2. The fact that repair is rapid in MCF-10ATG3B cells suggests that repair enzymes other than those mediated by p53 are critical in these cells. In agreement with our results, Pani et al. (2000) demonstrated that mouse transformed fibroblasts lacking p53 are significantly less susceptible, than wild-type controls to the cytotoxic effect of a number of pro-oxidant treatments. These researchers have correlated this resistance in part to increased expression of MnSOD, which is negatively regulated at a transcriptional level by p53. MnSOD has been proposed to function as a tumor suppressor gene (Bravard et al., 1992
). MnSOD overexpression in HeLa cells confers resistance to cell death induced by serum deprivation (Palazzotti et al., 1999
). Increased expression of this protein has been observed in tumors including brain, thyroid, and colon (Jansen et al., 1998
; Landriscina et al., 1996
; Nishida et al., 1993
) in comparison to surrounding normal tissues. In agreement with these results, we show that as the tumorigenic potential of the cell series increases, p53 levels decline and MnSOD protein levels increase in the MCF-10ATG3B cells.
The MCF-10AT family of cell lines was originally generated by addition of Ha-ras into the MCF-10A cell line. We have previously shown that Ras levels continue to increase with progression of this series such that the MCF-10ATG3B cell line, which is a third generation descendant from the MCF-10AT cell line, has even higher Ras protein levels than the MCF-10AT cell line into which the Ha-ras gene was originally inserted (Starcevic et al., 2001). Irani et al. (1997)
have shown that Ha-ras (V12) transformed NIH 3T3 fibroblasts produce large amounts of reactive oxygen species. H2O2 increases intracellular ROS and triggers Ras activation (Irani et al., 1997
; Lander et al., 1996
). Ras (specifically Ha-Ras) stimulates intracellular ROS levels by activating NADPH oxidase, further amplifying the cascade initiated by H2O2. Taken together, this would predict for increased oxidative stress and potentially increased susceptibility to DNA damage with progression of the cell lines, in the absence or presence of an oxidant. However, in untreated cells, significant differences in mean olive tail moment values were not detected in the MCF-10AT series regardless of Ras levels. Additionally, the most tumorigenic cell line, the MCF-10ATG3B, was the least susceptible to DNA damage by H2O2. In agreement with our results, Deichman et al. (1998)
have shown that cells descendant from a population transformed by Ha-ras acquire a high H2O2 antioxidant activity and increased tumorigenic activity. This acquired resistance in vivo may reflect the capability of the descendants to protect against oxidative damage, presumably a contributing step in the development of tumors in vivo. In support of this possibility, comet analysis of the HCC1937 tumor cell line treated with H2O2 indicated even less DNA damage than any of the MCF-10AT series of cell lines. Thus, the generation of cells that progress to tumorigenesis most rapidly (i.e., MCF-10ATG3B) have the highest levels of Ras, the lowest levels of p53, and the shortest doubling time, factors that would predict for increased susceptibility to oxidative stress and potentially increased DNA damage. Since these cells do not exhibit increased sensitivity to oxidative DNA damage and are less sensitive to apoptosis by Fas, it is clear that compensatory cellular mechanisms have developed to limit DNA damage and ensure cell survival. Cells have evolved enzymatic and nonenzymatic mechanisms to protect cells from ROS. This is particularly important for cell survival and genomic fidelity, considering that ROS can potentially damage a wide range of cellular macromolecules including DNA. Additionally, cells are continually exposed to oxidative insult from ROS that are generated during normal metabolism. Levels of ROS are normally controlled by antioxidants including MnSOD, Cu/Zn SOD, and catalase. We show that levels of all three of these antioxidants increased with progression of this model, with the MCF-10ATG3B > MCF-10AT > MCF-10A. In agreement with our results, stably transfected V-Ha-Ras rat kidney epithelial cells have been shown to produce significantly larger amounts of superoxide radical than their corresponding wild-type cells, and the levels of Cu/Zn SOD, MnSOD and catalase were increased in these cells (Yang et al., 2001
). In breast cancer tissues, antioxidants including catalase are significantly elevated compared to corresponding adjacent uninvolved normal tissue (Kumaraguruparan et al., 2002
).
PBD is associated with a 4- to 5-fold increase in risk for developing breast cancer, and it is well recognized that breast tumor cells are generally resistant to apoptosis. In mice, the MCF-10ATG3B cells develop tumors that display the morphological characteristics of human PBD within one month and progress to ductal carcinoma in situ at a frequency of 2530%, ultimately progressing to fully invasive malignant carcinoma. Our results suggest that development of PBD may be attributed, in part, to decreased p53 levels, in conjunction with increased levels of antioxidant enzymes. Together, these events may enable tumorigenic cells to evade apoptosis, thereby allowing them to progress toward the cancer-cell phenotype.
In summary, we have demonstrated that H2O2-induced DNA damage in this cell model is concentration-dependent and decreases with progression of this cell lineage, such that the MCF-10A cells are more susceptible than the MCF-10ATG3B cells. DNA repair occurs rapidly in these breast epithelial cells as evidenced by the increase in mean olive tail moment values following pretreatment with HU/Ara-C. In the MCF-10ATG3B cells, decreased H2O2-sensitivity in the presence of increased Ras, decreased p53 levels, and a short doubling time may be, in part, attributed to compensatory antioxidant defense mechanisms, including increased expression of catalase and SOD (Cu/Zn and Mn), that develop with increasing tumorigenicity of the cell line.
ACKNOWLEDGMENTS
Supported by NIH grant ES10595 and, in part, by grant U61/ATU581481 from the ATSDR/CDC, and by EHS Center grant P30 ESO6639, through the services of the Molecular Imaging and Cytometry Core and the Cell Culture Facility Core, from the National Institute of Environmental Health Sciences.
NOTES
1 To whom correspondence should be addressed at the Institute of Environmental Health Sciences, 2727 Second Avenue, Wayne State University, Detroit, MI 48201. Fax (313) 577-0082. E-mail: r.novak{at}wayne.edu.
REFERENCES
Anderson, D., Hughes, J. A., Nizankowska, E., Graca, B., Cebulska-Wasilewska, A., Wierzewska, A., and Kasper, E. (1997). Factors affecting various biomarkers in untreated lung cancer patients and healthy donors. Environ. Mol. Mutagen. 30, 205216.[CrossRef][ISI][Medline]
Basolo, F., Elliot, J., Tait, L., Chen, X. Q., Maloney, T., Russo, I. H., Pauley, R., Momiki, S., Caamano, J., Klein-Szanto, A. J. P., et al. (1991). Transformation of human breast epithelial cells by c-Ha-ras oncogene. Mol. Carcinog. 4, 2535.[ISI][Medline]
Bravard, A., Sabatier, L., Hoffschir, F., Ricoul, M., Luccioni, C., and Dutrillaux, B. (1992). SOD2: A new type of tumor-suppressor gene? Int. J. Cancer 51, 476480.[ISI][Medline]
Cleaver, J. E., Karplus, K., Kashani-Sabet, M., and Limoli, C.L. (2001). Nucleotide excision repair "a legacy of creativity." Mutat. Res. 485, 2336.[ISI][Medline]
Cuda, G., Paterno, R., Caeravolo, R., Candigliota, M., Perrotti, N., Perticone, F., Faniello, M. C., Schepis, F., Ruocco, A., Mele, E., et al. (2002). Protection of human endothelial cells from oxidative stress: Role of Ras-ERK1/2 signaling. Circulation 105, 968974.
Dawson, P. J., Wolman, S. R., Tait, L., Heppner, G. H., and Miller, F. R. (1996). MCF10AT: A model for the evolution of cancer from proliferative breast disease. Am. J. Pathol. 148, 313319.[Abstract]
Deichman, G. I., Mateeva, V. A., Kashkina, L. M., Dyakova, N. A., Uvarova, E. N., Nikiforov, M. A., and Gudkov, A. V. (1998). Cell transforming genes and tumor progression: In vivo unified secondary phenotypic cell changes. Int. J. Cancer 75, 277283.[CrossRef][ISI][Medline]
Gedik, C. M., and Collins, A. R. (1991). The mode of action of 1-ß-D-arabinofuranosylcytosine in inhibiting DNA repair: New evidence using a sensitive assay for repair DNA synthesis and ligation in permeable cells. Mutat. Res. 254, 231237.[ISI][Medline]
Hellman, B., Vaghef, H., and Boström, B. (1995). The concepts of tail moment and tail inertia in the single-cell gel electrophoresis assay. Mutat. Res. 336, 123131.[ISI][Medline]
Irani, K., Xia, Y., Zweier, J. L., Sollott, S. J., Der, C. J., Fearon, E. R., Sundaresan, M., Finkel, T., and Goldschmidt-Clermont, P. J. (1997). Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts. Science 275, 16491652.
Jansen, A. M., Bosman, C. B., Sier, C. F., Griffioen, G., Kubben, F. J., Lamers, C. B., van Krieken, J. H., van de Velde, C. J., and Verspaget, H. W. (1998). Superoxide dismutases in relation to the overall survival of colorectal cancer patients. Br. J. Cancer 78, 10511057.[ISI][Medline]
Kumaraguruparan, R., Subapriya, R., Viswanathan, P., and Nagini, S. (2002). Tissue lipid peroxidation and antioxidant status in patients with adenocarcinoma of the breast. Clin. Chim. Acta 325, 165.[CrossRef][ISI][Medline]
Lander, H. M., Milbank, A. J., Tauras, J. M., Hajjar, D. P., Hempstead, B. L., Schwartz, G. D., Kraemer, R. T., Mirza, U. A., Chait, B. T., Burk, S. C., et al. (1996). Redox regulation of cell signaling. Nature 381, 380381.[CrossRef][ISI][Medline]
Landriscina, M., Remiddi, F., Ria, F., Palazzotti, B., De Leo, M. E., Iacoangeli, M., Rosselli, R., Scerrati, M., and Galeotti, T. (1996). The level of MnSOD is directly correlated with grade of brain tumors of neuroepithelial origin. Br. J. Cancer 74, 18771885.[ISI][Medline]
Martin, F. L., Cole, K. J., Orme, M. H., Grover, P. L., Phillips, D. H., and Venitt, S. (1999a). The DNA repair inhibitors hydroxyurea and cytosine arabinoside enhance the sensitivity of the alkaline single-cell gel electrophoresis (comet) assay in metabolically competent MCL-5 cells. Mutat. Res. 445, 2143.[ISI][Medline]
Martin, F. L., Cole, K. J., Weaver, G., Williams, J. A., Millar, B. C., Grover, P. L., and Phillips D. H. (1999b). Genotoxicity of human milk extracts and induction of DNA damage in exfoliated cells recovered from breast milk. Biochem. Biophys. Res. Commun. 259, 494504.[CrossRef][ISI]
Miller, F. R., Soule, H. D., Tait, L., Pauley, R. J., Wolman, S. R., Dawson, P. J., and Heppner, G. H. (1993). Xenograft model of progressive human proliferative breast disease. J. Natl. Cancer Inst. 85, 17251732.[Abstract]
Mirzayans, R., Dietrich, K., and Paterson, M. C. (1993). Aphidicolin and 1-ß-D-arabinofuranosylcytosine strongly inhibits transcriptionally active DNA repair in normal human fibroblasts. Carcinogenesis 14, 26212626.[Abstract]
Nishida, S., Akai, F., Iwasaki, H., Hosokawa, K., Kusunoki, T., Suzuki, K., Taniguchi, N., Hashimoto, S., and Tamura, T. T. (1993). Manganese superoxide dismutase content and localization in thyroid tumors. J. Pathol. 169, 341345.[ISI][Medline]
Offer, H., Wolkowisz, R., Matas, D., Blumenstein, S., Livneh, Z., and Rotter, V. (1999). Direct involvement of p53 in the base excision repair pathway of the DNA repair machinery. FEBS Lett. 450, 197204.[CrossRef][ISI][Medline]
Olive, P. L. (1989). Cell proliferation as a requirement for development of the contact effect in Chinese hamster V79 spheroids. Radiat. Res. 117, 7992.[ISI][Medline]
Palazzotti, B., Pani, G., Colavitti, R., De Leo, M. E., Bedogi, B., Borrello, S., and Galeotti, T. (1999). Increased growth capacity of cervical-carcinoma cells overexpressing manganese superoxide dismutase. Int. J. Cancer 82, 145150.[CrossRef][ISI][Medline]
Pani, G., Bedogi, B., Anzevino, R., Colavitti, R., Palazzotti, B., Borrello, S., and Galeotti, T. (2000). Deregulated manganese superoxide dismutase expression and resistance to oxidative injury in p53-deficient cells. Cancer Res. 60, 46544660.
Pennisi, E. (1997). Superoxides relay Ras proteins oncogenic message. Science 275, 15671568.
Russo, J., Tait, L., and Russo, I. H. (1991). Morphological expression of cell transformation induced c-Ha-ras oncogene in human breast epithelial cells. J. Cell Sci. 99, 453463.[Abstract]
Scully, R., and Puget, N. (2002). BRCA1 and BRCA2 in hereditary breast cancer. Biochimie 84, 95102.[CrossRef][ISI][Medline]
Singh, N. P., McCoy, M. T., Tice, R. R., and Schneider, E. L. (1988). A simple technique for quantification of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184191.[ISI][Medline]
Soule, H. D., Maloney, T. M., Wolman, S. R., Peterson, W. D., Brenz, R., McGrath, C. M., Russo, J., Pauley, R. J., Jones, R. F., and Brooks, S. C. (1990). Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10. Cancer Res. 50, 60756086.[Abstract]
Starcevic, S. L., Elferink, C., and Novak, R. F. (2001). Progressive resistance to apoptosis in a cell lineage model of human proliferative breast disease. J. Natl. Cancer Inst. 93, 776782.
Thompson, P. A., Kadlubar, F. F., Vena, S. M., Hill, H.L., McClure, G. H. Y., McDaniel, L. P., and Ambrosone, C. B. (1998). Exfoliated ductal epithelial cells in human breast milk: A source of target tissue DNA for molecular epidemiologic studies of breast cancer. Cancer Epidemiol. Biomarkers Prev. 7, 3742.[Abstract]
Tomlinson, G. E., Chen, T. T.-L., Stastny, V. A., Virmani, A. K., Spillman, M. A., Tonk, V., Blum, J. A., Schneider, N. R., Wistuba, I. I., Shay, J. W., et al. (1998). Characterization of a breast cancer cell line derived from a germ-line BRCA1 mutation carrier. Cancer Res. 58, 32373242.[Abstract]
Yang, J. Q., Li, S., Huang, Y., Zhang, H. J., Domann, F. E., Buettner, G. R., and Oberley, L. W. (2001). V-Ha-Ras overexpression induces superoxide production and alters levels of primary antioxidant enzymes. Antioxid. Redox Signal. 3, 697709.[CrossRef][ISI][Medline]
Yarbro, J. W. (1992). Mechanism of action of hydroxyurea. Semin. Oncol. 19, 110.[ISI][Medline]
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