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Relationship between micronuclei formation and p53 induction
Mutation Research 672 (2009) 124–128
Contents lists available at ScienceDirect
Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Relationship between micronuclei formation and p53 induction Ana María Salazar ∗ , Monserrat Sordo, Patricia Ostrosky-Wegman Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México, D.F., Mexico
a r t i c l e
i n f o
Article history: Received 15 March 2008 Received in revised form 25 October 2008 Accepted 31 October 2008 Available online 8 November 2008 Keywords: p53 protein Chromosomal damage DNA damage Micronucleus test Clastogenic and aneugenic agents
a b s t r a c t Human exposure to multiple chemicals compromises the integrity of genetic material. Hence, it is essential to determine the extent of DNA damage induced by xenobiotics. In cell lines, the induction of p53 expression in response to treatments with DNA-damaging agents has been proposed as a tool for the detection of genotoxic damage, although a direct correlation between a marker of chromosomal damage and p53 expression has not previously been studied. The micronucleus assay is a widely used genotoxicity test that has been shown to detect structural and numerical chromosomal damage. The present study was designed to characterize the relationship between micronuclei and p53 induction. RKO cells were cultured and treated with non-cytotoxic concentrations of colchicine, vinblastine, bleomycin or arsenic. Mannitol and clofibrate, which are non-genotoxic chemicals, were also included. The frequency of micronuclei was evaluated using the cytokinesis-block assay, and p53 induction was measured by Western blot assay. Our data showed that a significant induction of micronuclei and of p53 protein occurred only with the genotoxic chemicals. No differences in p53 induction were associated with the clastogenic or aneuplodogenic potential of the chemical exposure. The linear regression analysis revealed a direct relationship between p53 levels and the induction of micronuclei (p = 0.0001, r2 = 0.9372), indicating that the level of p53 is associated with chromosomal damage. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Increased exposure to xenobiotics compromises genetic integrity. Epidemiologic studies of individuals exposed to environmental hazards have shown that a high frequency of chromosome damage is associated with the development of cancer [1]. Therefore, it is important to determine the levels of DNA damage induced by hundreds of xenobiotics. Among various biomarkers of DNA damage, the micronucleus (MN) assay is one of the standard genotoxicity tests. This assay is used to measure chromosome damage both in vivo and in vitro. The in vivo MN assay in human lymphocytes has been increasingly used in the monitoring of individuals exposed to potential mutagenic agents [2]. The in vitro MN assay is used to screen genotoxic chemicals or to assess the mutagenicity of pharmaceutical agents [3–5].
∗ Corresponding author at: Department of Genomic Medicine and Environmental Toxicology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, PO BOX 70228, Ciudad Universitaria, México, D.F. 04510, Mexico. Tel.: +52 55 5622 3846; fax: +52 55 5622 3365. E-mail address: [email protected] (A.M. Salazar). 1383-5718/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2008.10.015
The tumor suppressor gene p53 (TP53) plays a key role in safeguarding the integrity of the human genome. Different stress signals, including DNA and spindle damage, have been demonstrated to increase p53 levels [6]. In the absence of cellular stress, the wild type p53 protein is maintained at low levels by rapid intracellular ubiquitination, which impedes its accumulation [7–9]. p53 activation involves an increase in p53 protein levels as well as structural changes in the protein. Post-translational modifications generally result in p53 stabilization and accumulation in the nucleus, where p53 interacts with sequence-specific sites on its target genes [10,11]. Therefore, p53 activation involves a marked increase in the cellular abundance of p53 molecules, which then execute the biochemical events important in regulatory signaling networks. Previous studies have proposed that the detection of elevated levels of p53 protein in response to treatment with DNA-damaging agents in cell lines could be used as an indicator of damage induced by genotoxic carcinogens [12–14]. However, a direct correlation between a marker of chromosomal damage and p53 expression has not been established. Since the validity of MN as a biomarker of chromosomal damage has been well established, we wanted to compare MN frequencies and p53 levels induced by the same genotoxic agents. We mea-
A.M. Salazar et al. / Mutation Research 672 (2009) 124–128
sured chromosomal damage in RKO cells with wild-type p53 using the cytokinesis-block micronucleus assay, and analysis of p53 protein was performed by western blot. In order to characterize the relationship between the induction of micronuclei and p53 levels, the chemicals evaluated in this study included non-genotoxic and genotoxic agents with different mechanisms of action and genotoxic potencies. 2. Materials and methods 2.1. Chemicals Mannitol (Sigma, CAS 69-65-8) and clofibrate (Sigma, CAS 637-07-0) were chosen to be representative of non-genotoxic agents [15]. The aneugenic chemicals tested were vinblastine (Lemery, CAS 865-21-4) and colcemid (Gibco, CAS 47730-5). As a clastogen, we used bleomycin (Sigma, CAS 9041-93-4), a DNA strand breaker. Sodium arsenite (Sigma, CAS 1327-53-3), a human carcinogen with evidence of both clastogenic and aneugenic effects, was also included. The genotoxic compounds used in the present study are clearly inducers of both MN and chromosomal aberrations in lymphocytes and in different cell lines [5,16,17]. Dose-range information was available from previous experiments in our laboratory and from published studies. Sterile water was used as the solvent for all chemicals (stock solutions) except for clofibrate, which was dissolved in dimethyl sulfoxide (DMSO, Sigma, CAS 67-68-
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5). Cytochalasin B (Sigma, CAS 14930-96-2) was dissolved in DMSO (1 mg/ml) and kept at −20 ◦ C. 2.2. Cell culture The human colorectal carcinoma cell line RKO, with wild-type p53 alleles, was grown in DMEM medium (Sigma) containing 10% fetal bovine serum (HyClone), 1% non-essential amino acids (Sigma), 1% l-glutamine (Sigma) and 1% penicillin/streptomycin (Gibco). RKO cells (0.5 × 106 cells/dish, 60 mm in diameter) were seeded and maintained at 37 ◦ C and 5% CO2 for 48 h. Cultures were treated with the chemical agents during the last 24 h. Negative controls were exposed to vehicles, either water or DMSO. When DMSO was used, the final concentration of solvent in the culture medium did not exceed 1% (v/v). 2.3. Cytotoxicity Three concentrations of each chemical were tested to define a non-cytotoxic dose. The concentrations used were 0.0005, 0.005 and 0.05 M for colcemid and vinblastine; 0.05, 0.5 and 5 M for bleomycin; and 0.1, 1 and 10 M for sodium arsenite. The concentrations used for mannitol were 5, 10 and 25 mM, and for clofibrate the concentrations were 0.002, 0.02 and 0.2 mM. RKO cells were seeded in culture plates and incubated for 24 h, and cells were treated following this stabilization period. After 24 h of treatment, RKO cells were harvested by trypsinization. An aliquot of the cell suspension was transferred to 1 ml of media for cell counting and viability determination. Cells were prepared as recommended in the Guava
Fig. 1. Dose-dependent effects on p53 levels, MN frequency and cell proliferation. (a) Relationship between MN frequency and p53 induction. The left y-axis indicates p53 induction, relative to untreated control normalized against actin protein. The right y-axis shows micronucleus frequency. (b) Analysis of cell proliferation. Left y-axis indicates the percentage of binucleated cells (BN), and right y-axis shows the nuclear proliferation index (NPI). RKO cells were incubated with mannitol, clofibrate, bleomycin, sodium arsenite, colcemid and vinblastine for 24 h. The data represent the mean ± S.E.M. calculated from three independent experiments performed in duplicated (for cytotoxicity and the MN test) and at least three independent experiments (for p53 screening). There was a significant increase *p ≤ 0.05 with respect to control cells.
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Technologies Protocol and analyzed by the Guava® ViaCount method based on the differential permeability of fluorescent reagents, using the Guava PCA system with the software CytoSoft version 2.1. Three independent experiments were done in duplicated. 2.4. Cytokinesis block micronucleus assay The micronucleus assay was performed as described [18], with some modifications. Briefly, 0.5 × 106 cells were seeded onto a cover slip in 60 mm dishes. After 24 h of culture, cells were exposed to the selected dose of each chemical agent and also to cytochalasin B (6 g/ml final concentration), which inhibits the polymerization of actin, thus blocking cytokinesis and leading to an accumulation of binucleated cells. After 24 h of exposure, the cells were harvested. Cover slips were rinsed gently one time with PBS at room temperature. Cells on the cover slips were fixed with icecold methanol–acetic acid (3:1). Air-dried cover glasses were stained with Wright’s colorant and mounted on slides for microscopic evaluation. Cell proliferation kinetics were analyzed by determining the frequency of mononucleated (Mono), binucleated (Bi) and polynucleated (Poly) cells, corresponding to 0, 1, and 2 or more in vitro cell divisions, respectively. Cytostatic activity was determined by calculating the nuclear proliferation index using the following equation: nuclear proliferation index (NPI) = [#Mono +2#Bi + 3#Poly]/200. The frequency of MN was determined in 1000 binucleated cells per treatment group, according to published criteria [19]. Three independent experiments performed in duplicate were analyzed. 2.5. p53 immunodetection The experimental design was similar to the cytotoxicity assay. Protein was extracted from the cells, and p53 expression was determined as previously described [20]. Briefly, 25 g of protein was separated on a 12% SDS-polyacrylamide gel. Proteins were transferred onto a nitrocellulose membrane. After blocking, the membrane was incubated with anti-p53 DO-1 mouse antibody (1 g/ml, Santa Cruz Biotechnology) for 2 h. Afterwards the blot was incubated with a horseradishperoxidase-coupled anti-mouse antibody. Finally, the blot was developed by the enhanced chemiluminiscence method (ECLTM , Amersham). Each blot was incubated with anti-actin (0.5 g/ml, Santa Cruz Biotechnology) for 1.5 h and with a horseradish-peroxidase-coupled anti-goat antibody for another hour. Quantification of films was performed with the Gel DocTM system (BioRad) using the Quantity One Software. Comparative analysis of separate p53 blots was performed by optical density normalization against the actin protein. Data was reported as the number of increments with respect to the corresponding negative control. At least three independent experiments were analyzed for each treatment.
Fig. 2. Correlation between p53 levels and MN frequency assessed by linear regression analysis.
evaluated by the scoring of mono-, bi- and polynucleated cells. Concentration-related decreases in the percentage of binucleated cells were observed with arsenic, colcemid and vinblastine. These effects on cell proliferation significantly decreased the nuclear proliferation index at the highest concentrations. It should be noted that low doses of arsenic, vinblastine and colchicine did not significantly impair the cell proliferation kinetics or the nuclear proliferation index. Bleomycin did not alter cell proliferation, but it did induce micronuclei for all concentrations tested. Whereas mannitol did not produce changes in cell proliferation, clofibrate reduced the cell proliferation kinetic independently of dosage (Fig. 1b). The linear regression analysis showed a direct relationship between p53 levels and MN induction (p = 0.0001, r2 = 0.9372), indicating that the level of p53 is associated with chromosomal damage (Fig. 2).
2.6. Statistical analysis
4. Discussion All data are reported as the mean ± S.E.M. of independent experiments. Normality of data was evaluated by the Kolmogorov–Smirnov test. One-way analysis of variance (ANOVA) was applied to variables. Multiple-dose experiments were analyzed by Bonferroni’s post-test. p ≤ 0.05 was considered statistically significant. The correlation between p53 levels and micronucleus frequency or cell proliferation was determined with a linear regression model.
3. Results The relationship between p53 induction and chromosomal damage was explored using three different doses of vinblastine, colcemid, bleomycin and sodium arsenite. Furthermore, representative non-genotoxic compounds were included, which have been shown to be negative for MN induction. The cellular cytotoxicity for each chemical was evaluated using a method based on the differential permeability of fluorescent chemicals. The cell viability remained >80% for concentrations tested. The genotoxic chemicals induced a dose-related increase in the frequency of MN and in the p53 levels. Statistically significant differences were observed in the MN frequency when the cells were treated with bleomycin (0.5 and 5 M), vinblastine, and cocemid (0.005 and 0.05 M), whereas the differences observed with arsenic were only significant with the higher dose (p < 0.001). With both mannitol and clofibrate, no statistically significant increases in the number of MN were observed (Fig. 1a). The levels of p53 showed a significant increase at the concentrations that were found to be statistically significant for MN formation. Since p53 activates cell cycle checkpoints to prevent the proliferation of damaged cells, the effects on cell proliferation were
The purpose of this study was to determine the relationship between the MN frequency and the levels of p53 induced by genotoxic agents in RKO cells. Mannitol and clofibrate, which are known non-genotoxic agents, did not induce MN or increase p53 expression. A highly significant increase in the MN frequency was induced by the genotoxic compounds tested. Data obtained show that the increased p53 levels in response to the drug treatment were directly associated with the extent of chromosomal damage. The results obtained with all the genotoxic chemicals tested, except bleomycin, showed a significant decrease in the proliferation index in RKO cells. When p53 levels increased, the nuclear proliferation index decreased. This observation is consistent with the role of p53, which activates cell cycle checkpoints in G1 /S and G2 /M, preventing the proliferation of damaged cells. Consequently, cell proliferation must be altered when the cells have been treated with genotoxic agents. Interestingly, bleomycin induced MN in a dose–response manner without altering the cell viability or cell proliferation. Previous studies have also shown that bleomycin produces genotoxic effects with little or no reduction in the frequency of multinucleated cells [15,21,22]. It is not clear to us how cells that are processing the DNA damage induced by bleomycin can continue proliferating. Since the p53 protein is a critical mediator of programmed cell death in response to DNA damage and genotoxic carcinogens, it is important to use p53-proficient cell lines in any study of genotoxicity [23]. Nevertheless, some cell lines commonly used for genotoxicity testing are p53 deficient. Micronuclei induction
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has been reported in two cell lines with a mutated p53 gene, L5178Y (mouse lymphoma) and WIL2-NS (human lymphoblastoid) [22,24,25]. In fact, it has been hypothesized that the p53 deficient status of cells might account for their inability to tolerate the toxic conditions imparted by long and continuous exposures to genotoxic agents [23], and the use of numerical doses when testing aneugens has been suggested. We found that RKO cells with functional p53 showed both inducible expression of the p53 protein and low spontaneous levels of micronucleated cells, Moreover, MN induction with two aneugens studied MN induction was observed using a wide dose-range. Therefore, RKO cells may become a useful model for these types of studies. No differences in the patterns of p53 induction were observed between the drugs tested, which have different mechanisms. It is possible that under our culture conditions, no differences in p53 induction were observed between the clastogenic and aneugenic agents due to the time of treatment. A study of the kinetics of p53 induction would more clearly elucidate any differences between such agents. MN are well-characterized biomarkers of chromosomal damage. However, the manual scoring of MN requires rigorous validation, and when a variety of potential genotoxic compounds or new drugs need to be evaluated, it becomes time-consuming. Thus, considerable efforts have been taken to develop an automated scoring procedure for the micronucleus assay, based on a computerized image analysis [26–28]. Furthermore, the development of automated flow cytometric methods to measure MN frequencies in nucleated cells following exposure to test chemicals [25,29] represents a technological improvement over the traditional microscopy-based micronucleus method. More efficient new methods for the assessment of genotoxic agents are constantly being developed. A new genotoxicity assay in which genotoxin-induced transcription of the GADD45a gene drives the synthesis of green fluorescent protein in TK6 cells has recently been reported [30,31]. In this context, the induction of p53 as an endpoint for in vitro genotoxicity testing should be further explored. Our results suggest that the detection of p53 levels through the Western blot assay could be useful in the screening of potential genotoxic compounds. Nevertheless, if more precise data are needed, the determination of p53 levels may also be performed by ELISA. In the present study, mannitol and clofibrate, which do not cause DNA damage, did not induce p53. Furthermore, the genotoxic compounds bleomycin, arsenic, colcemid and vinblastine, did not significantly increase p53 protein levels in RKO cells at non MN-inducing doses. Therefore, these results indicate that there is a close relationship between damage to DNA and the expression of p53. Caution must be taken, since some chemicals might behave differently depending on their mechanism of action. For example, azathioprine, an epigenetic carcinogen, did not stimulate p53 expression in mouse fibroblast cells [12], but it did induce micronuclei in bone marrow and peripheral blood erythrocytes in rats [32]. Hence, studies are needed to better understand how some chemicals are capable of producing genotoxicity without increasing the protein p53. Our results show a correlation between chromosomal damage, as measured by MN frequency, and the levels of p53 induced by genotoxic agents in RKO cells. Nevertheless, further experimental studies are needed in order to understand the sensitivity of p53 expression for detecting chromosomal damage.
Conflict of interest None.
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Acknowledgements This research was partially supported by grants from CONACYT and PAPIIT-DGAPA (UNAM). The technical help of Lucía Brito is acknowledged. References [1] S. Bonassi, A. Znaor, H. Norppa, L. Hagmar, Chromosomal aberrations and risk of cancer in humans: an epidemiological perspective, Cytogen. Genome Res. 104 (2004) 376–382. [2] M. Neri, D. Ugolini, S. Bonassi, A. Fucic, N. Holland, L.E. Knudsen, R.J. Sram, M. Ceppi, V. Bocchini, D.F. Merlo, Children’s exposure to environmental pollutants and biomarkers of genetic damage, II. Results of a comprehensive literature search and meta-analysis, Mutat. Res. 612 (2006) 14–39. [3] M.L. Garriott, J.B. Phelps, W.P. Hoffman, A protocol for the in vitro micronucleus test. I. Contributions to the development of a protocol suitable for regulatory submissions from an examination of 16 chemicals with different mechanisms of action and different levels of activity, Mutat. Res. 517 (2002) 123–134. [4] B.J. Phelps, M.L. Garriott, W.P. 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Contents lists available at ScienceDirect
Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Relationship between micronuclei formation and p53 induction Ana María Salazar ∗ , Monserrat Sordo, Patricia Ostrosky-Wegman Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México, D.F., Mexico
a r t i c l e
i n f o
Article history: Received 15 March 2008 Received in revised form 25 October 2008 Accepted 31 October 2008 Available online 8 November 2008 Keywords: p53 protein Chromosomal damage DNA damage Micronucleus test Clastogenic and aneugenic agents
a b s t r a c t Human exposure to multiple chemicals compromises the integrity of genetic material. Hence, it is essential to determine the extent of DNA damage induced by xenobiotics. In cell lines, the induction of p53 expression in response to treatments with DNA-damaging agents has been proposed as a tool for the detection of genotoxic damage, although a direct correlation between a marker of chromosomal damage and p53 expression has not previously been studied. The micronucleus assay is a widely used genotoxicity test that has been shown to detect structural and numerical chromosomal damage. The present study was designed to characterize the relationship between micronuclei and p53 induction. RKO cells were cultured and treated with non-cytotoxic concentrations of colchicine, vinblastine, bleomycin or arsenic. Mannitol and clofibrate, which are non-genotoxic chemicals, were also included. The frequency of micronuclei was evaluated using the cytokinesis-block assay, and p53 induction was measured by Western blot assay. Our data showed that a significant induction of micronuclei and of p53 protein occurred only with the genotoxic chemicals. No differences in p53 induction were associated with the clastogenic or aneuplodogenic potential of the chemical exposure. The linear regression analysis revealed a direct relationship between p53 levels and the induction of micronuclei (p = 0.0001, r2 = 0.9372), indicating that the level of p53 is associated with chromosomal damage. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Increased exposure to xenobiotics compromises genetic integrity. Epidemiologic studies of individuals exposed to environmental hazards have shown that a high frequency of chromosome damage is associated with the development of cancer [1]. Therefore, it is important to determine the levels of DNA damage induced by hundreds of xenobiotics. Among various biomarkers of DNA damage, the micronucleus (MN) assay is one of the standard genotoxicity tests. This assay is used to measure chromosome damage both in vivo and in vitro. The in vivo MN assay in human lymphocytes has been increasingly used in the monitoring of individuals exposed to potential mutagenic agents [2]. The in vitro MN assay is used to screen genotoxic chemicals or to assess the mutagenicity of pharmaceutical agents [3–5].
∗ Corresponding author at: Department of Genomic Medicine and Environmental Toxicology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, PO BOX 70228, Ciudad Universitaria, México, D.F. 04510, Mexico. Tel.: +52 55 5622 3846; fax: +52 55 5622 3365. E-mail address: [email protected] (A.M. Salazar). 1383-5718/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2008.10.015
The tumor suppressor gene p53 (TP53) plays a key role in safeguarding the integrity of the human genome. Different stress signals, including DNA and spindle damage, have been demonstrated to increase p53 levels [6]. In the absence of cellular stress, the wild type p53 protein is maintained at low levels by rapid intracellular ubiquitination, which impedes its accumulation [7–9]. p53 activation involves an increase in p53 protein levels as well as structural changes in the protein. Post-translational modifications generally result in p53 stabilization and accumulation in the nucleus, where p53 interacts with sequence-specific sites on its target genes [10,11]. Therefore, p53 activation involves a marked increase in the cellular abundance of p53 molecules, which then execute the biochemical events important in regulatory signaling networks. Previous studies have proposed that the detection of elevated levels of p53 protein in response to treatment with DNA-damaging agents in cell lines could be used as an indicator of damage induced by genotoxic carcinogens [12–14]. However, a direct correlation between a marker of chromosomal damage and p53 expression has not been established. Since the validity of MN as a biomarker of chromosomal damage has been well established, we wanted to compare MN frequencies and p53 levels induced by the same genotoxic agents. We mea-
A.M. Salazar et al. / Mutation Research 672 (2009) 124–128
sured chromosomal damage in RKO cells with wild-type p53 using the cytokinesis-block micronucleus assay, and analysis of p53 protein was performed by western blot. In order to characterize the relationship between the induction of micronuclei and p53 levels, the chemicals evaluated in this study included non-genotoxic and genotoxic agents with different mechanisms of action and genotoxic potencies. 2. Materials and methods 2.1. Chemicals Mannitol (Sigma, CAS 69-65-8) and clofibrate (Sigma, CAS 637-07-0) were chosen to be representative of non-genotoxic agents [15]. The aneugenic chemicals tested were vinblastine (Lemery, CAS 865-21-4) and colcemid (Gibco, CAS 47730-5). As a clastogen, we used bleomycin (Sigma, CAS 9041-93-4), a DNA strand breaker. Sodium arsenite (Sigma, CAS 1327-53-3), a human carcinogen with evidence of both clastogenic and aneugenic effects, was also included. The genotoxic compounds used in the present study are clearly inducers of both MN and chromosomal aberrations in lymphocytes and in different cell lines [5,16,17]. Dose-range information was available from previous experiments in our laboratory and from published studies. Sterile water was used as the solvent for all chemicals (stock solutions) except for clofibrate, which was dissolved in dimethyl sulfoxide (DMSO, Sigma, CAS 67-68-
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5). Cytochalasin B (Sigma, CAS 14930-96-2) was dissolved in DMSO (1 mg/ml) and kept at −20 ◦ C. 2.2. Cell culture The human colorectal carcinoma cell line RKO, with wild-type p53 alleles, was grown in DMEM medium (Sigma) containing 10% fetal bovine serum (HyClone), 1% non-essential amino acids (Sigma), 1% l-glutamine (Sigma) and 1% penicillin/streptomycin (Gibco). RKO cells (0.5 × 106 cells/dish, 60 mm in diameter) were seeded and maintained at 37 ◦ C and 5% CO2 for 48 h. Cultures were treated with the chemical agents during the last 24 h. Negative controls were exposed to vehicles, either water or DMSO. When DMSO was used, the final concentration of solvent in the culture medium did not exceed 1% (v/v). 2.3. Cytotoxicity Three concentrations of each chemical were tested to define a non-cytotoxic dose. The concentrations used were 0.0005, 0.005 and 0.05 M for colcemid and vinblastine; 0.05, 0.5 and 5 M for bleomycin; and 0.1, 1 and 10 M for sodium arsenite. The concentrations used for mannitol were 5, 10 and 25 mM, and for clofibrate the concentrations were 0.002, 0.02 and 0.2 mM. RKO cells were seeded in culture plates and incubated for 24 h, and cells were treated following this stabilization period. After 24 h of treatment, RKO cells were harvested by trypsinization. An aliquot of the cell suspension was transferred to 1 ml of media for cell counting and viability determination. Cells were prepared as recommended in the Guava
Fig. 1. Dose-dependent effects on p53 levels, MN frequency and cell proliferation. (a) Relationship between MN frequency and p53 induction. The left y-axis indicates p53 induction, relative to untreated control normalized against actin protein. The right y-axis shows micronucleus frequency. (b) Analysis of cell proliferation. Left y-axis indicates the percentage of binucleated cells (BN), and right y-axis shows the nuclear proliferation index (NPI). RKO cells were incubated with mannitol, clofibrate, bleomycin, sodium arsenite, colcemid and vinblastine for 24 h. The data represent the mean ± S.E.M. calculated from three independent experiments performed in duplicated (for cytotoxicity and the MN test) and at least three independent experiments (for p53 screening). There was a significant increase *p ≤ 0.05 with respect to control cells.
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Technologies Protocol and analyzed by the Guava® ViaCount method based on the differential permeability of fluorescent reagents, using the Guava PCA system with the software CytoSoft version 2.1. Three independent experiments were done in duplicated. 2.4. Cytokinesis block micronucleus assay The micronucleus assay was performed as described [18], with some modifications. Briefly, 0.5 × 106 cells were seeded onto a cover slip in 60 mm dishes. After 24 h of culture, cells were exposed to the selected dose of each chemical agent and also to cytochalasin B (6 g/ml final concentration), which inhibits the polymerization of actin, thus blocking cytokinesis and leading to an accumulation of binucleated cells. After 24 h of exposure, the cells were harvested. Cover slips were rinsed gently one time with PBS at room temperature. Cells on the cover slips were fixed with icecold methanol–acetic acid (3:1). Air-dried cover glasses were stained with Wright’s colorant and mounted on slides for microscopic evaluation. Cell proliferation kinetics were analyzed by determining the frequency of mononucleated (Mono), binucleated (Bi) and polynucleated (Poly) cells, corresponding to 0, 1, and 2 or more in vitro cell divisions, respectively. Cytostatic activity was determined by calculating the nuclear proliferation index using the following equation: nuclear proliferation index (NPI) = [#Mono +2#Bi + 3#Poly]/200. The frequency of MN was determined in 1000 binucleated cells per treatment group, according to published criteria [19]. Three independent experiments performed in duplicate were analyzed. 2.5. p53 immunodetection The experimental design was similar to the cytotoxicity assay. Protein was extracted from the cells, and p53 expression was determined as previously described [20]. Briefly, 25 g of protein was separated on a 12% SDS-polyacrylamide gel. Proteins were transferred onto a nitrocellulose membrane. After blocking, the membrane was incubated with anti-p53 DO-1 mouse antibody (1 g/ml, Santa Cruz Biotechnology) for 2 h. Afterwards the blot was incubated with a horseradishperoxidase-coupled anti-mouse antibody. Finally, the blot was developed by the enhanced chemiluminiscence method (ECLTM , Amersham). Each blot was incubated with anti-actin (0.5 g/ml, Santa Cruz Biotechnology) for 1.5 h and with a horseradish-peroxidase-coupled anti-goat antibody for another hour. Quantification of films was performed with the Gel DocTM system (BioRad) using the Quantity One Software. Comparative analysis of separate p53 blots was performed by optical density normalization against the actin protein. Data was reported as the number of increments with respect to the corresponding negative control. At least three independent experiments were analyzed for each treatment.
Fig. 2. Correlation between p53 levels and MN frequency assessed by linear regression analysis.
evaluated by the scoring of mono-, bi- and polynucleated cells. Concentration-related decreases in the percentage of binucleated cells were observed with arsenic, colcemid and vinblastine. These effects on cell proliferation significantly decreased the nuclear proliferation index at the highest concentrations. It should be noted that low doses of arsenic, vinblastine and colchicine did not significantly impair the cell proliferation kinetics or the nuclear proliferation index. Bleomycin did not alter cell proliferation, but it did induce micronuclei for all concentrations tested. Whereas mannitol did not produce changes in cell proliferation, clofibrate reduced the cell proliferation kinetic independently of dosage (Fig. 1b). The linear regression analysis showed a direct relationship between p53 levels and MN induction (p = 0.0001, r2 = 0.9372), indicating that the level of p53 is associated with chromosomal damage (Fig. 2).
2.6. Statistical analysis
4. Discussion All data are reported as the mean ± S.E.M. of independent experiments. Normality of data was evaluated by the Kolmogorov–Smirnov test. One-way analysis of variance (ANOVA) was applied to variables. Multiple-dose experiments were analyzed by Bonferroni’s post-test. p ≤ 0.05 was considered statistically significant. The correlation between p53 levels and micronucleus frequency or cell proliferation was determined with a linear regression model.
3. Results The relationship between p53 induction and chromosomal damage was explored using three different doses of vinblastine, colcemid, bleomycin and sodium arsenite. Furthermore, representative non-genotoxic compounds were included, which have been shown to be negative for MN induction. The cellular cytotoxicity for each chemical was evaluated using a method based on the differential permeability of fluorescent chemicals. The cell viability remained >80% for concentrations tested. The genotoxic chemicals induced a dose-related increase in the frequency of MN and in the p53 levels. Statistically significant differences were observed in the MN frequency when the cells were treated with bleomycin (0.5 and 5 M), vinblastine, and cocemid (0.005 and 0.05 M), whereas the differences observed with arsenic were only significant with the higher dose (p < 0.001). With both mannitol and clofibrate, no statistically significant increases in the number of MN were observed (Fig. 1a). The levels of p53 showed a significant increase at the concentrations that were found to be statistically significant for MN formation. Since p53 activates cell cycle checkpoints to prevent the proliferation of damaged cells, the effects on cell proliferation were
The purpose of this study was to determine the relationship between the MN frequency and the levels of p53 induced by genotoxic agents in RKO cells. Mannitol and clofibrate, which are known non-genotoxic agents, did not induce MN or increase p53 expression. A highly significant increase in the MN frequency was induced by the genotoxic compounds tested. Data obtained show that the increased p53 levels in response to the drug treatment were directly associated with the extent of chromosomal damage. The results obtained with all the genotoxic chemicals tested, except bleomycin, showed a significant decrease in the proliferation index in RKO cells. When p53 levels increased, the nuclear proliferation index decreased. This observation is consistent with the role of p53, which activates cell cycle checkpoints in G1 /S and G2 /M, preventing the proliferation of damaged cells. Consequently, cell proliferation must be altered when the cells have been treated with genotoxic agents. Interestingly, bleomycin induced MN in a dose–response manner without altering the cell viability or cell proliferation. Previous studies have also shown that bleomycin produces genotoxic effects with little or no reduction in the frequency of multinucleated cells [15,21,22]. It is not clear to us how cells that are processing the DNA damage induced by bleomycin can continue proliferating. Since the p53 protein is a critical mediator of programmed cell death in response to DNA damage and genotoxic carcinogens, it is important to use p53-proficient cell lines in any study of genotoxicity [23]. Nevertheless, some cell lines commonly used for genotoxicity testing are p53 deficient. Micronuclei induction
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has been reported in two cell lines with a mutated p53 gene, L5178Y (mouse lymphoma) and WIL2-NS (human lymphoblastoid) [22,24,25]. In fact, it has been hypothesized that the p53 deficient status of cells might account for their inability to tolerate the toxic conditions imparted by long and continuous exposures to genotoxic agents [23], and the use of numerical doses when testing aneugens has been suggested. We found that RKO cells with functional p53 showed both inducible expression of the p53 protein and low spontaneous levels of micronucleated cells, Moreover, MN induction with two aneugens studied MN induction was observed using a wide dose-range. Therefore, RKO cells may become a useful model for these types of studies. No differences in the patterns of p53 induction were observed between the drugs tested, which have different mechanisms. It is possible that under our culture conditions, no differences in p53 induction were observed between the clastogenic and aneugenic agents due to the time of treatment. A study of the kinetics of p53 induction would more clearly elucidate any differences between such agents. MN are well-characterized biomarkers of chromosomal damage. However, the manual scoring of MN requires rigorous validation, and when a variety of potential genotoxic compounds or new drugs need to be evaluated, it becomes time-consuming. Thus, considerable efforts have been taken to develop an automated scoring procedure for the micronucleus assay, based on a computerized image analysis [26–28]. Furthermore, the development of automated flow cytometric methods to measure MN frequencies in nucleated cells following exposure to test chemicals [25,29] represents a technological improvement over the traditional microscopy-based micronucleus method. More efficient new methods for the assessment of genotoxic agents are constantly being developed. A new genotoxicity assay in which genotoxin-induced transcription of the GADD45a gene drives the synthesis of green fluorescent protein in TK6 cells has recently been reported [30,31]. In this context, the induction of p53 as an endpoint for in vitro genotoxicity testing should be further explored. Our results suggest that the detection of p53 levels through the Western blot assay could be useful in the screening of potential genotoxic compounds. Nevertheless, if more precise data are needed, the determination of p53 levels may also be performed by ELISA. In the present study, mannitol and clofibrate, which do not cause DNA damage, did not induce p53. Furthermore, the genotoxic compounds bleomycin, arsenic, colcemid and vinblastine, did not significantly increase p53 protein levels in RKO cells at non MN-inducing doses. Therefore, these results indicate that there is a close relationship between damage to DNA and the expression of p53. Caution must be taken, since some chemicals might behave differently depending on their mechanism of action. For example, azathioprine, an epigenetic carcinogen, did not stimulate p53 expression in mouse fibroblast cells [12], but it did induce micronuclei in bone marrow and peripheral blood erythrocytes in rats [32]. Hence, studies are needed to better understand how some chemicals are capable of producing genotoxicity without increasing the protein p53. Our results show a correlation between chromosomal damage, as measured by MN frequency, and the levels of p53 induced by genotoxic agents in RKO cells. Nevertheless, further experimental studies are needed in order to understand the sensitivity of p53 expression for detecting chromosomal damage.
Conflict of interest None.
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