2,2',4,6,6'-Pentachlorobiphenyl Induces Mitotic Arrest and p53 Activation

Kum-Joo Shin*,{dagger}, Sun-Hee Kim*, Dohan Kim*, Yun-Hee Kim*, Han-Woong Lee{ddagger}, Yoon-Seok Chang§, Man-Bock Gu, Sung Ho Ryu* and Pann-Ghill Suh*,1

* 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; {dagger} Division of Biology, California Institute of Technology, Pasadena, California 91125, {ddagger} 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polychlorinated biphenyls (PCBs), a class of persistent organic pollutants (POPs), have been considered to be involved in cancers, but the underlying mechanisms are not known well. Various cancers are closely related to genetic alteration; therefore, we investigated the effect of PCBs on genetic stability, through p53, a guardian of genome, in NIH 3T3 fibroblasts. Among several congeners examined, 2,2',4,6,6'-pentachlorobiphenyl (PeCB) specifically activated p53-dependent transcription. It also induced p53 nuclear accumulation, but did not cause DNA strand breakage. On the other hand, cell cycle progression that is closely connected to p53 was affected by 2,2',4,6,6'-PeCB, resulting in mitotic arrest. In the arrested cells, mitotic spindle damage was detected. Moreover, in the absence of functional p53, polyploidy was caused by 2,2',4,6,6'-PeCB. These results imply that 2,2',4,6,6'-PeCB induces mitotic arrest by interfering with mitotic spindle assembly, followed by genetic instability which triggers p53-activating signals to prevent further polyploidization. Taking these findings together, we suggest that 2,2',4,6,6'-PeCB could be involved in cancer development by causing genetic instability through mitotic spindle damage, which brings about aneuploidy in p53-deficient tumor cells.

Key Words: polychlorinated biphenyl; p53; mitotic arrest; genetic instability.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polychlorinated biphenyls (PCBs) are once widely used industrial chemicals due to their physicochemical properties. Resistance to chemical and biological degradation and high lipophilicity led them to accumulation in the environment, in animals, and even in human tissues and breast milk (Koopman-Esseboom et al., 1994;Go Safe, 1993Go). PCB congeners of different structure are considered to have distinct mechanisms of action, resulting in different cellular responses. The planar congeners without chlorine substitution at the ortho position have relatively high affinity for aryl hydrocarbon receptor, the endogenous receptor for dioxins, and thus, are considered to exhibit toxic effects through the receptor. However, the other congeners with ortho-substituted chlorine(s) have negligible binding affinity for the receptor; therefore, separate mechanisms may be involved in their toxic effects. For example, the effects on second messengers are different according to their structure (Kodavanti and Tilson, 1997Go).

Toxic effects of PCBs range from carcinogenesis and immunotoxicity to disruption of nerve, endocrine, and reproductive systems (Choksi et al., 1997Go; Hany et al., 1999Go; Kato et al., 1999Go; Moysich et al., 1999Go; Wu et al., 1999Go). PCBs are considered to be associated with cancers in animals (Silberhorn et al., 1990Go). There is limited evidence for carcinogenicity in humans (Petruska and Engelhard, 1991Go; Ward et al., 1997Go). 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, 1997Go). 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, 2001Go). 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, 1998Go).

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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents.
The PCBs (>99% pure) were purchased from AccuStandard (New Haven, CT). Propidium iodide was from Molecular Probes, Inc. (Eugene, OR). DMEM (Dulbecco’s modified Eagle’s medium) was obtained from Biowhittaker (Walkersville, MD), and fetal calf serum from Hyclone (Logan, UT). Etoposide and other chemicals were purchased from Sigma (St. Louis, MO).

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., 1999Go) 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., 2000Go). 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 2–3 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 [{gamma}-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., 2001Go). 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., 2001Go) 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2,2',4,6,6'-PeCB Activates p53
We first established a stable cell line containing luciferase reporter gene under control of p53 using NIH 3T3 fibroblasts to investigate the effect of PCBs on p53-dependent transcription. The transfected cells were treated with each PCB congener at 10 µM concentration for 12 h as indicated in Figure 1A. Among the congeners tested, 2,2',4,6,6'-PeCB caused about 3-fold increase in p53-dependent transcription compared to vehicle. To confirm transcriptional activation of p53 by 2,2',4,6,6'-PeCB, cells were treated with it at various concentrations or incubation times. p53 transcriptional activity increased over 2 µM 2,2',4,6,6'-PeCB (Fig. 1B) or after 6 h of treatment at 10 µM concentration (Fig. 1C). 3,3',4,4',5-PeCB, an isomer of 2,2',4,6,6'-PeCB that has a coplanar structure, has no effect on p53-dependent transcription.



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FIG. 1. 2,2',4,6,6'-PeCB activates p53. (A) NIH 3T3 cells were stably transfected with luciferase reporter gene under control of p53. Transfected cells were treated with DMSO (0.1%); 10 µM 2,2'-, 3,3'-, or 4,4'-DiCB; 2,2',4-, 2,2',6-, 2,4,4'-, or 3,3',4-TriCB; 2,2',4,4'-, 2,2',4,6-, 2,2',6,6'-, or 3,3',4,4'-TeCB; or 2,2',4,6,6'-, 2,3,3',4,4'-, 2,3',4,4',5-, or 3,3',4,4',5-PeCB for 12 h in serum-free medium. The transfected NIH 3T3 cells were treated with 2,2',4,6,6'- or 3,3',4,4',5-PeCB at the indicated concentrations for 18 h (B), or 10 µM for the indicated times (C). Luciferase activity in 1 µg of cell lysate was assayed as described in Materials and Methods. Data represent the means ± SE of three separate experiments, each conducted in triplicate. RLU: relative light unit, DiCB: dichlorobiphenyl, TriCB: trichlorobiphenyl, TeCB: tetrachlorobiphenyl, PeCB: pentachlorobiphenyl. *p <0.01 versus vehicle treatment. (D) NIH 3T3 cells were treated with 10 µM 2,2',4,6,6- or 3,3',4,4',5-PeCB for 6, 12, or 18 h in serum-free medium. After preparation of cytoplasmic and nuclear extracts, p53 and PARP (a nuclear marker) were detected as described in Materials and Methods. Data are representative of at least three separate experiments. N: nucleus, C: cytoplasm, NT: not treated.

 
According to intensive studies, p53 protein is quickly degraded through ubiquitin-dependent proteolysis pathway in the absence of stimuli. However, it is stabilized by stimulation and then translocates to the nucleus, followed by binding to the specific sequence in the regulatory region of target genes (Colman et al., 2000Go). Therefore, as another evidence for p53 activation by 2,2',4,6,6'-PeCB, we investigated nuclear accumulation of p53 protein after treatment with 10 µM 2,2',4,6,6'- or 3,3',4,4',5-PeCB for 6, 12, and 18 h. Cytoplasmic and nuclear proteins were prepared, and p53 protein was analyzed by Western blotting. Figure 1D demonstrates that p53 was exclusively detected in nuclear fraction of cells treated with 2,2',4,6,6'-PeCB for 12 and 18 h. On the other hand, in 3,3',4,4',5-treated cells, p53 was not detected in any fraction, which means 3,3',4,4',5-PeCB had no effect on p53 protein stability.

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., 1996Go). 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., 1999Go; Kikugawa et al., 2003Go). 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., 1984Go).



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FIG. 2. 2,2',4,6,6'-PeCB does not induce DNA strand breakage. NIH 3T3 cells were treated with DMSO (0.1%), 10 µM 3,3',4,4',5- or 2,2',4,6,6'-PeCB, or 5 µM etoposide for 12 h in serum-free medium. After cells were embedded in low-melting-point agarose, applied to gel electrophoresis, and stained with 2.5 µg/ml propidium iodide; cells containing DNA strand breakage were observed under confocal microscope (A) and quantitated (B) as described in Materials and Methods. Data are representative of at least three separate experiments.

 
While p53 nuclear accumulation and transcriptional activation were obvious after treatment with 10 µM 2,2',4,6,6'-PeCB for 12 h, there was no detectable DNA strand breakage at the same condition. This suggests that p53 is activated by 2,2',4,6,6'-PeCB with little relevance to DNA damage.

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, 1999Go), 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., 1983Go). 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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FIG. 3. 2,2',4,6,6'-PeCB induces mitotic arrest. (A) Asynchronous NIH 3T3 cells were treated with 10 µM 2,2',4,6,6'- or 3,3',4,4',5-PeCB for the indicated times in serum-free medium. After cells were harvested, fixed, and stained with 50 µg/ml propidium iodide, DNA content was analyzed. After asynchronous NIH 3T3 cells were treated with 10 µM 2,2',4,6,6- or 3,3',4,4',5-PeCB for the indicated times, phospho-Tyr15 Cdc2, Cdc2 (B) and M-phase-specific phospho-proteins (D) were detected using respective antibodies, and Cdc2 kinase activity was assayed using histone H1 as a substrate in vitro (C). All experiments were performed as described in Materials and Methods. Data are representative of at least three separate experiments.

 
2,2',4,6,6'-PeCB Damages Mitotic Spindles
Mitosis is a process for equal segregation of genome to two daughter cells, and the failure of even a single chromosome to align on the mitotic spindle is sufficient to induce mitotic arrest (Rudner and Murray, 1996Go). That is why mitotic spindle has been a target of several mitotic arrest-inducing chemicals (Huang and Lee, 1998Go; Jordan et al., 1992Go). The previous data demonstrating that 2,2',4,6,6'-PeCB induces mitotic arrest urged us to investigate the effect of 2,2',4,6,6'-PeCB on mitotic spindle. Mitotic spindle and chromosomes were visualized by staining with anti-tubulin antibody and propidium iodide, respectively. After cells were treated with DMSO, 10 µM 2,2',4,6,6'-, or 3,3',4,4',5-PeCB for 12 h, chromosomes and spindle in cells at mitotic phase were observed (Fig. 4). DMSO- or 3,3',4,4',5-PeCB-treated cells showed well-formed mitotic spindle, to which chromosomes are attached in good order. However, all the 2,2',4,6,6'-PeCB-treated mitotic cells had abnormal mitotic spindle and randomly distributed chromosomes, which were similarly observed in mitotic cells treated with nocodazole, a well-known microtubule inhibiting agent (data not shown).



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FIG. 4. 2,2',4,6,6'-PeCB causes mitotic spindle damage. NIH 3T3 cells were treated with DMSO (0.1%), 10 µM 3,3',4,4',5- or 2,2',4,6,6'-PeCB for 12 h in serum-free medium. Nuclei were stained with propidium iodide and mitotic spindles were detected by immunofluorescence using an anti-ß-tubulin antibody as described in Materials and Methods. Data are representative of at least three separate experiments. PI: propidium iodide.

 
2,2',4,6,6'-PeCB Induces Polyploidy in p53-Deficient Cells
2,2',4,6,6'-PeCB activated p53 and induced mitotic arrest, producing damaged spindle. We wondered how p53 activation is related to mitotic arrest, therefore, we investigated the effect of 2,2',4,6,6'-PeCB on cell cycle progression in p53-deficient mouse embryonic fibroblasts (MEFs). Wild type (p53+/+) and p53–/– MEFs were treated with 10 µM 3,3',4,4',5- or 2,2',4,6,6'-PeCB for 12 h, fixed, and stained with propidium iodide, and populations of cells with a different DNA content were measured. As a result, 2,2',4,6,6'-PeCB increased the population of cells with a 4N DNA content in p53–/– MEFs as well as wild type cells (Fig. 5). Furthermore, cells with an 8N DNA content are the other main population in 2,2',4,6,6'-PeCB-treated p53–/– MEFs. There is some evidence that p53 can be activated by mitotic spindle damage and then cause a G1-like growth arrest of cells containing 4N DNA, and thus p53 plays a critical role in preventing cells from endoreduplication of their DNA (Lanni and Jacks, 1998Go). On the other hand, cells lacking functional p53 underwent multiple rounds of DNA synthesis at S phase without undergoing cytokinesis, forming polyploid (Meek, 2000Go). In wild MEFs treated with 2,2',4,6,6'-PeCB, a small subpopulation of cells having a 8N DNA content was observed, which would be predicted as cycling tetraploid cells. That was also observed by Lanni and Jacks (1998).



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FIG. 5. 2,2',4,6,6'-PeCB induces tetraploidy in p53–/– cells. Wild type and p53–/– MEFs were treated with DMSO (0.1%), 10 µM 3,3',4,4',5- or 2,2',4,6,6'-PeCB for 12 h. After cells were harvested, fixed, and stained with 50 µg/ml propidium iodide, DNA content was analyzed as described in Materials and Methods. Data are representative of at least three separate experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The involvement of PCBs in cancers has been reported in animals (Dean et al., 2002Go; Silberhorn, 1990Go), and several other papers have suggested that PCBs can act as tumor promoters (Anderson et al., 1994Go; Beebe et al., 1993Go). However, the underlying mechanisms are not clear yet.

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., 1992Go; Oakley et al., 1996Go); however, DNA damage was not detected in vivo (Schilderman et al., 2000Go).

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., 1992Go; Ross and Fygenson, 2003Go). Therefore, the effect of 2,2',4,6,6'-PeCB on tubulin polymerization was investigated by incubation of purified {alpha}- and ß-tubulin with 2,2',4,6,6'-PeCB and subsequent centrifugation as previously reported (Mistry and Atweh, 2001Go) (microtubule sedimentation assay). The amount of tubulin in pellet (polymerized tubulin) was reduced about 30–50% by 50 µM 2,2,4,6,6'-PeCB and 10–20% 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., 1998Go). 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., 2000Go), 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, 1998Go; Meek, 2000Go). 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, 2000Go). 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., 1999Go). 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.


    ACKNOWLEDGMENTS
 
We thank Dr. Yusuf A. Hannun and Dr. Chang-Woo Lee for critical evaluation of the manuscript. This work was supported in part by the Ministry of Health and Welfare Grant (00-PJ1-PG1-CH13-0005) of the Republic of Korea.


    NOTES
 

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 790–784, Republic of Korea. Fax: 82–54–279–2199. E-mail: pgs{at}postech.ac.kr


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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