* Department of Life Science, Division of Molecular and Life Sciences, and School of Environmental Science and Engineering, Pohang University of Science and Technology, Pohang 790-784, Kyungbuk, Republic of Korea; School of Environmental Science and Engineering, Pohang University of Science and Technology, Pohang 790-784, Kyungbuk, Republic of Korea;
Division of Biology, California Institute of Technology, Pasadena, California 91125,
Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, Suwon 440-746, Republic of Korea; and ¶ Kwangju Institute of Science and Technology, Oryong-dong, Puk-gu, Kwangju, Republic of Korea
Received November 28, 2003; accepted January 5, 2004
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ABSTRACT |
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Key Words: polychlorinated biphenyl; p53; mitotic arrest; genetic instability.
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INTRODUCTION |
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Toxic effects of PCBs range from carcinogenesis and immunotoxicity to disruption of nerve, endocrine, and reproductive systems (Choksi et al., 1997; Hany et al., 1999
; Kato et al., 1999
; Moysich et al., 1999
; Wu et al., 1999
). PCBs are considered to be associated with cancers in animals (Silberhorn et al., 1990
). There is limited evidence for carcinogenicity in humans (Petruska and Engelhard, 1991
; Ward et al., 1997
). However, the molecular mechanisms are not clear yet.
It is accepted that cancers are caused by accumulating mutations in genes that control cell growth or death. To prevent cells from being cancerous, p53, the tumor suppressor gene product, acts as a sensor of genetic instability. Intensive studies clearly document that p53 plays an essential role in a G1 checkpoint in response to DNA-damaging agents such as radiation; thus, DNA replication is prevented until the damaged DNA is completely repaired (Levine, 1997). p53 also acts at a G2 checkpoint in response to DNA damage after replication, which inhibits cells from entering mitotic phase with damaged DNA (Taylor and Stark, 2001
). In addition, there is some evidence that p53 is activated by mitotic spindle damage to prevent endoreduplication following spontaneous exit from mitosis without cytokinesis (Lanni and Jacks, 1998
).
To investigate if PCBs affect genomic stability, we examined the effect on p53 protein that can indicate abnormality in the genome, after treatment of cells with PCBs. As a result, p53 was activated by 2,2',4,6,6'-PeCB, one of the highly ortho-substituted congeners, through mitotic spindle damage. Furthermore, in p53-deficient cells, 2,2',4,6,6'-PeCB induced polyploidy that can accelerate cancer development, which is a clue to understanding the involvement of 2,2',4,6,6'-PeCBs in cancers, considering the p53 gene is frequently mutated in many tumors.
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MATERIALS AND METHODS |
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Cell culture and establishment of a stable cell line.
Mouse embryonic fibroblasts (MEFs) derived from wild type and knockout mice were generated by spontaneous immortalization after they were isolated from murine E13.5 embryos using standard procedures. MEFs and NIH 3T3 (originally from American Type Culture Collection, ATCC, Manassas, VA) were cultured in DMEM supplemented with 10% fetal calf serum at 37°C in a humidified, 5% CO2-controlled incubator.
To establish a stable cell line, NIH 3T3 cells were cotransfected with p53-Luc plasmid that contains a firefly luciferase reporter gene driven by a basic promoter element and a TATA box, which are joined to 14 tandem repeats of a p53 enhancer element (TGCCTGGACTTGCCTGG) (Stratagene, La Jolla, CA) and pcDNA 3.1 (+) using LipofectAmine (Invitrogen, Carlsbad, CA). Positive clones were selected with 700 µg/ml G418 (Invitrogen, Carlsbad, CA), and transcriptional response was tested by luciferase reporter assay.
Luciferase reporter assay.
NIH 3T3 cells transfected with p53-Luc were seeded in 6-well plates and the next day, cells were treated with PCBs in serum-free medium for the indicated times. After washing with PBS and lysis, luciferase activity in 1 µg of lysate was assayed using a luciferase assay kit (Promega, Madison, WI) with luminometer (Labsystems, Helsinki, Finland).
Preparation of cytoplasmic and nuclear extracts.
NIH 3T3 cells were treated with 10 µM 2,2',4,6,6'- or 3,3',4,4',5-PeCB for indicated times. Cytoplasmic and nuclear extracts were prepared as described previously (Pei et al., 1999) and stored at 70°C until use.
Alkaline single cell gel electrophoresis (Comet) assay.
Comet assay was performed as described previously with slight modification (Darbon et al., 2000). After treatment of 4 x 104 cells with vehicle, 10 µM 2,2',4,6,6'- or 3,3',4,4',5-PeCB, or 5 µM etoposide for 12 h, the harvested cells were embedded in 1% low-melting-point agarose layered onto two glass slides precoated with 1% normal-melting-point agarose and dried in a warm oven for 23 h. After lysis and immersion in alkaline solution (pH > 13, 300 mM NaOH, 1 mM EDTA [pH 10.0]) at room temperature (RT) for 20 min, gels were applied to electrophoresis at 4°C, 25 V, 300 mA for 20 min in alkaline solution. DNA fragments stained with 2.5 µg/ml propidium iodide were observed under confocal microscope (LSM 510, Carl Zeiss, Jena, Germany). Analysis was performed by visual scoring based on five classes of cells, from class 0 (undamaged) to class 4 (highly damaged). 100 cells were selected at random from each slide, and each cell was given a value according to the class it was put into, so that an overall score was derived for each sample, ranging from 0 to 400 arbitrary units.
Cell cycle analysis.
Asynchronous cells were treated with vehicle, 10 µM 2,2',4,6,6'- or 3,3',4,4',5-PeCB for the indicated times in serum-free medium, harvested, and washed with PBS/5 mM EDTA twice. Approximately 1 x 106 cells were resuspended with PBS, and an equal volume of ethanol was added with vortexing. After fixation for 30 min, followed by incubation with 40 µg/ml RNase for 30 min at RT, cells were stained with 50 µg/ml propidium iodide. DNA content was determined using a FACScan flow cytometer (Becton Dickinson, San Jose, CA).
Cdc2 kinase assay.
Cells treated with 10 µM 2,2',4,6,6'- or 3,3',4,4',5-PeCB for the indicated times were harvested and sonicated in lysis buffer (40 mM Tris, pH 7.5, 120 mM NaCl, 0.1% NP-40, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM Na3VO4, 10 mM NaF). Cell lysate (200 µg) was incubated with anti-cyclin B1 antibody (sc-245, Santa Cruz biotechnology, Santa Cruz, CA) for 2 h at 4°C, followed by incubation with protein G-Sepharose beads for another 2 h. After washing three times with lysis buffer and twice with reaction buffer (25 mM Tris, pH 7.5, 10 mM MgCl2), the beads were incubated with 2µg histone H1 (Roche Molecular Biochemicals, Indianapolis, IN) and 2 µCi [-32P] ATP (NEN, Boston, MA) at 37°C for 30 min. The reaction was stopped by addition of 5x SDS sample buffer, and the samples were applied to SDS-PAGE followed by autoradiography.
Western blot analysis.
Western blotting was performed as described previously (Lee et al., 2001). Antibodies used are p53 (Ab-1, Oncogene, Uniondale, NJ), PARP (Pharmingen, San Diego, CA), phospho-Cdc 2 (New England Biolab. Beverly, MA), Cdc 2 (Santa Cruz), and MPM 2 (Upstate Biotechnology, Lake Placid, NY). Proteins were detected with ECL kit (Amersham Pharmacia Biotech, Uppsala, Sweden).
Immunocytochemistry and nuclear staining.
Immunocytochemistry was performed as described previously (Lee et al., 2001) with some modifications. Cells were treated with vehicle, 10 µM 3,3',4,4',5- or 2,2',4,6,6'-PeCB for 12 h, washed with PBS, and fixed with 4% paraformaldehyde for 30 min at RT. After incubation with 100 µg/ml RNase A and subsequent blocking with PBS containing 1% horse serum and 0.2% Triton X-100 for 30 min at RT, cells were incubated with anti-ß-tubulin antibody (Sigma) for 2 h at RT. Subsequently, cells were incubated with fluorescein isothiocyanate-labeled goat anti-mouse secondary antibody (Sigma) for 1 h at RT and then with 2.5 µg/ml propidium iodide for 10 min to visualize tubulin and nuclei, respectively.
Statistical analysis.
The results are expressed as means ± SE. Statistical significance was determined using the Student's t-test.
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RESULTS |
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2,2',4,6,6'-PeCB Does Not Cause DNA Strand Breakage
DNA damage is a well-known p53-activating signal, and there are several reports that PCBs might induce DNA damage, even though there is no conclusive evidence (Oakley et al., 1996). Therefore, we addressed if 2,2',4,6,6'-PeCB can induce DNA damage using alkaline single cell gel electrophoresis assay, the so-called comet assay. In alkaline solution (pH >13), various DNA defects, including single- and double-strand breakage, excision repair sites, cross-links, and alkali labile sites, could be detected because of the difference of the mobility between genomic DNA and fragmented DNA on gel electrophoresis (Godard et al., 1999
; Kikugawa et al., 2003
). After cells were treated with DMSO, 10 µM 2,2',4,6,6'- or 3,3',4,4',5-PeCB, or 5 µM etoposide for 12 h, DNA damage was assayed. Figure 2 shows that 2,2',4,6,6'-PeCB did not induce statistically significant DNA damage in comparison with DMSO; however, etoposide, an anticancer drug that inhibits topoisomerase II activity, exhibited obvious DNA damage and had been previously reported to cause DNA single- and double-strand breakage (Chen et al., 1984
).
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2,2',4,6,6'-PeCB Induces Mitotic Arrest
One of the important roles of p53 is to prevent damaged cells from progressing the cell cycle. Therefore, we examined the effects of 2,2',4,6,6'-PeCB on cell cycle progression to find a clue for the mechanism of p53 activation. NIH 3T3 cells were treated with 10 µM 2,2',4,6,6'- or 3,3',4,4',5-PeCB, harvested at various time points, and the population of cells with a different DNA content was measured (Fig. 3A). The result showed that the population of cells with a 4N DNA content started to increase after 3 h of treatment with 2,2',4,6,6'-PeCB. To address at which of the two phases, G2 or M, the cells were, we performed following several experiments. The onset of mitosis is triggered by dephosphorylation of Tyr15 residue of Cdc2 by phosphatase Cdc 25 (Ohi and Gould, 1999), and Cdc2 bound to cyclin B1 phosphorylates many proteins necessary to mitosis. Therefore, we first observed the phosphorylation level of Tyr15 residue of Cdc2 after treatment with 10 µM 2,2',4,6,6'- or 3,3',4,4',5-PeCB for various incubation times. As shown in Figure 3B, the level of phosphorylation at Tyr15 decreased slightly after 6 h of 2,2',4,6,6'-PeCB treatment and dramatically after 12 h. Next, the enzymatic activity of Cdc2 coprecipitated with Cyclin B1 was assayed in vitro using histone H1 as a substrate, after cells were treated with 10 µM 2,2',4,6,6'- or 3,3',4,4',5-PeCB for various incubation times. Cdc2 kinase activity rose after 3 h and reached maximum after 12 h of 2,2',4,6,6'-PeCB treatment (Fig. 3C). In addition, proteins specifically phosphorylated at the entry into mitosis were detected using MPM2 monoclonal antibody that recognizes phosphoamino acid epitopes of M phase marker proteins (Davis et al., 1983
). Figure 3D shows that phosphorylation of the proteins increase after 3 h of treatment with 2,2',4,6,6'-PeCB.
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DISCUSSION |
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It is well studied that many carcinogens initiate cancers by DNA damage that is a major stimulator of p53. 2,2',4,6,6'-PeCB, one of the highly ortho-substituted PCB congeners, activated p53-dependent transcription and induced nuclear accumulation of p53 (Fig. 1); however, it did not cause DNA damage, at least DNA strand breakage (Fig. 2). Several different groups investigated the possibility of DNA damage by PCBs (Faux et al., 1992; Oakley et al., 1996
); however, DNA damage was not detected in vivo (Schilderman et al., 2000
).
On the other hand, 2,2',4,6,6'-PeCB arrested cells at mitotic phase, which was shown by dephosphorylation of Tyr15 residue of Cdc2, activation of Cdc2 kinase, and increase in phosphorylation level of proteins (Fig. 3). Moreover, abnormal mitotic spindle was observed in all mitotic cells treated with 2,2',4,6,6'-PeCB (Fig. 4). Therefore, it is considered that defects in mitotic spindle assembly and abnormal arrangement of chromosomes caused by 2,2',4,6,6'-PeCB induced mitotic arrest. Some mitotic spindle-damaging chemicals such as nocodazole, colchicine, and paclitaxel affect dynamics of mictotubules directly (Jordan et al., 1992; Ross and Fygenson, 2003
). Therefore, the effect of 2,2',4,6,6'-PeCB on tubulin polymerization was investigated by incubation of purified
- and ß-tubulin with 2,2',4,6,6'-PeCB and subsequent centrifugation as previously reported (Mistry and Atweh, 2001
) (microtubule sedimentation assay). The amount of tubulin in pellet (polymerized tubulin) was reduced about 3050% by 50 µM 2,2,4,6,6'-PeCB and 1020% by 20 µM 2,2,4,6,6'-PeCB (data not shown). 50 µM 2,2',4,6,6'-PeCB is probably beyond the solubility limits of this chemical, but, 2,2',4,6,6'-PeCB could have some effect on microtubule polymerization at less than 50 µM. However, 2,2',4,6,6'-PeCB didn't change the amount of polymerized tubulin at 10 µM, which caused maximal induction of p53-dependent transcription. Therefore, it is probable that there are other mechanisms through which 2,2',4,6,6'-PeCB can affect mitotic spindle assembly. For example, 2,2',4,6,6'-PeCB might affect centrosome, which is involved in spindle assembly and can cause organization of aberrant spindle (Pihan et al., 1998
). In the previous report, one of the PCB congeners, 2,3,3',4,4'-PeCB induced aberrant mitosis in V79 Chinese hamster cells (Jensen et al., 2000
), but in our experimental condition, we could not detect p53 activation or mitotic arrest by this congener. It could have resulted from different cellular context.
p53 prevents cells with damage, especially in the genome, from undergoing cell cycle. Therefore, we investigated how p53 activation by 2,2',4,6,6'-PeCB is related to mitotic arrest, using wild type and p53/ MEFs. The result demonstrates that population of cells with a 2N DNA content decreased to almost zero, but population of cells with a 4N DNA content increased by treatment of p53/ MEFs with 2,2',4,6,6'-PeCB. Therefore, mitotic arrest by 2,2',4,6,6'-PeCB is independent of p53. Furthermore, the population of cells with an 8N DNA content increased to be a major population in 2,2',4,6,6'-PeCB-treated p53/ MEFs, while it was negligible in wild type cells (Fig. 5). This implies that p53 is required to prevent cells from becoming tetraploid. There are persuasive data demonstrating that p53 is involved in G1-like arrest after "mitotic slippage" in the presence of microtubule-damaging agents (Lanni and Jacks, 1998; Meek, 2000
). The cells exposed to microtubule-damaging agents do not sustain mitotic arrest but bypass mitotic block with a 4N DNA content, which may trigger signals for p53 activation. Activated p53 arrests cells at G1-like status, preventing reduplication of wrong-numbered chromosomes. However, the lack of G1-like arrest resulting from the absence of functional p53 leads to aneuploidy. Therefore, it can be inferred that, after 2,2',4,6,6'-PeCB-induced mitotic arrest, p53 was activated to prevent cells with a 4N DNA content from cell cycle progression without cytokinesis. Moreover, p53 was activated slightly after treatment for 6 h and obviously after 12 h (Fig. 1), although mitotic arrest was observed after treatment for 3 h (Fig. 3).
Numeric chromosomal imbalance, referred to as aneuploidy, is frequently found in most cancers (Sen, 2000). Although it has long been debated whether aneuploidy is a cause or consequence of cancer, accumulating evidence shows that aneuploidy contributes to malignant transformation and progression process (Vessey et al., 1999
). p53 has been reported to be mutated in many tumors, and polyploidy was induced by 2,2',4,6,6'-PeCB in p53-deficient cells.
In summary, 2,2',4,6,6'-PeCB, an ortho-substituted PCB congener, activated p53 through mitotic spindle damage during mitosis and caused polyploidy in cells deficient in functional p53; therefore, it might be related to cancer development in tumor cells that lack functional p53, through genetic instability caused by mitotic spindle damage.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Department of Life Science, Division of Molecular and Life Science, Postech Biotech Center, Pohang University of Science and Technology, San 31 Hyoja-Dong, Nam-Gu, Pohang, Kyungbuk 790784, Republic of Korea. Fax: 82542792199. E-mail: pgs{at}postech.ac.kr
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