TOXICOLOGICAL SCIENCES 78, 215–221 (2004)
DOI: 10.1093/toxsci/kfh069
Advance Access publication January 21, 2004
2,2⬘,4,6,6⬘-Pentachlorobiphenyl Induces Mitotic Arrest
and p53 Activation
Kum-Joo Shin,* ,† Sun-Hee Kim,* Dohan Kim,* Yun-Hee Kim,* Han-Woong Lee,‡ 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; †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
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, 2,2ⴕ,4,6,6ⴕ-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 2,2ⴕ,4,6,6ⴕ-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
2,2ⴕ,4,6,6ⴕ-PeCB. These results imply that 2,2ⴕ,4,6,6ⴕ-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 2,2ⴕ,4,6,6ⴕ-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.
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 (KoopmanEsseboom et al., 1994; Safe, 1993). PCB congeners of different
structure are considered to have distinct mechanisms of action,
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: [email protected].
Toxicological Sciences vol. 78 no. 2 © Society of Toxicology 2004; all rights
reserved.
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, 1997).
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 G 1
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
G 2 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 orthosubstituted congeners, through mitotic spindle damage. Furthermore, in p53-deficient cells, 2,2⬘,4,6,6⬘-PeCB induced
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SHIN ET AL.
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
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%
CO 2-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 ␮g/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 ␮g 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 ␮M 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 ⫻ 10 4 cells with vehicle, 10 ␮M 2,2⬘,4,6,6⬘- or
3,3⬘,4,4⬘,5-PeCB, or 5 ␮M 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 ␮g/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 ␮M
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 ⫻ 10 6
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
␮g/ml RNase for 30 min at RT, cells were stained with 50 ␮g/ml propidium
iodide. DNA content was determined using a FACScan flow cytometer (Becton Dickinson, San Jose, CA).
Cdc2 kinase assay. Cells treated with 10 ␮M 2,2⬘,4,6,6⬘- or 3,3⬘,4,4⬘,5PeCB 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 ␮g/ml
aprotinin, 10 ␮g/ml leupeptin, 1 mM Na 3VO 4, 10 mM NaF). Cell lysate (200
␮g) 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 MgCl 2), the beads
were incubated with 2␮g histone H1 (Roche Molecular Biochemicals, Indianapolis, IN) and 2 ␮Ci [␥- 32P] ATP (NEN, Boston, MA) at 37°C for 30 min.
The reaction was stopped by addition of 5⫻ 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 ␮M 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 ␮g/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-␤-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 ␮g/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
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 ␮M 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 ␮M 2,2⬘,4,6,6⬘-PeCB (Fig. 1B) or after 6 h of
treatment at 10 ␮M 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.
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., 2000). 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 ␮M 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
MITOTIC ARREST AND p53 ACTIVATION BY 2,2⬘,4,6,6⬘-PENTACHLOROBIPHENYL
217
3,3⬘,4,4⬘,5-PeCB, or 5 ␮M 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 singleand double-strand breakage (Chen et al., 1984).
While p53 nuclear accumulation and transcriptional activation were obvious after treatment with 10 ␮M 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
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 ␮M 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 ␮M for the indicated times (C). Luciferase activity in 1 ␮g 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 ␮M 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.
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 ␮M 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
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.,
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 doublestrand 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 ␮M 2,2⬘,4,6,6⬘- or
FIG. 2. 2,2⬘,4,6,6⬘-PeCB does not induce DNA strand breakage. NIH 3T3
cells were treated with DMSO (0.1%), 10 ␮M 3,3⬘,4,4⬘,5- or 2,2⬘,4,6,6⬘-PeCB,
or 5 ␮M 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 ␮g/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.
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SHIN ET AL.
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, 1996). That is why mitotic spindle has
been a target of several mitotic arrest-inducing chemicals
(Huang and Lee, 1998; Jordan et al., 1992). 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
␮M 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).
FIG. 3. 2,2⬘,4,6,6⬘-PeCB induces mitotic arrest. (A) Asynchronous NIH
3T3 cells were treated with 10 ␮M 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 ␮g/ml propidium iodide, DNA content was analyzed. After
asynchronous NIH 3T3 cells were treated with 10 ␮M 2,2⬘,4,6,6- or
3,3⬘,4,4⬘,5-PeCB for the indicated times, phospho-Tyr 15 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.
two phases, G 2 or M, the cells were, we performed following several experiments. The onset of mitosis is triggered by
dephosphorylation of Tyr 15 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 Tyr 15
residue of Cdc2 after treatment with 10 ␮M 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 Tyr 15 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
␮M 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.
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
FIG. 4. 2,2⬘,4,6,6⬘-PeCB causes mitotic spindle damage. NIH 3T3 cells
were treated with DMSO (0.1%), 10 ␮M 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-␤-tubulin
antibody as described in Materials and Methods. Data are representative of at
least three separate experiments. PI: propidium iodide.
MITOTIC ARREST AND p53 ACTIVATION BY 2,2⬘,4,6,6⬘-PENTACHLOROBIPHENYL
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 ␮M 3,3⬘,4,4⬘,5- or
2,2⬘,4,6,6⬘-PeCB for 12 h. After cells were harvested, fixed, and stained with
50 ␮g/ml propidium iodide, DNA content was analyzed as described in
Materials and Methods. Data are representative of at least three separate
experiments.
p53 –/– MEFs were treated with 10 ␮M 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
G 1-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, 1998). On the other
hand, cells lacking functional p53 underwent multiple rounds
of DNA synthesis at S phase without undergoing cytokinesis,
forming polyploid (Meek, 2000). 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).
DISCUSSION
The involvement of PCBs in cancers has been reported in
animals (Dean et al., 2002; Silberhorn, 1990), and several other
papers have suggested that PCBs can act as tumor promoters
(Anderson et al., 1994; Beebe et al., 1993). However, the
underlying mechanisms are not clear yet.
219
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 Tyr 15 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 ␤-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 30 –50% by 50 ␮M
2,2,4,6,6⬘-PeCB and 10 –20% by 20 ␮M 2,2,4,6,6⬘-PeCB (data
not shown). 50 ␮M 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
␮M. However, 2,2⬘,4,6,6⬘-PeCB didn’t change the amount of
polymerized tubulin at 10 ␮M, 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 becom-
220
SHIN ET AL.
ing tetraploid. There are persuasive data demonstrating that
p53 is involved in G 1-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 G 1-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.
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.
REFERENCES
Anderson, L. M., Logsdon, D., Ruskie, S., Fox, S. D., Issaq, H. J., Kovatch,
R. M., and Riggs, C. M. (1994). Promotion by polychlorinated biphenyls of
lung and liver tumors in mice. Carcinogenesis 15, 2245–2248.
Beebe, L. E., Kim, Y. E., Amin, S., Riggs, C. W., Kovatch, R. M., and
Anderson, L. M. (1993). Comparison of transplacental and neonatal initiation of mouse lung and liver tumors by N-nitrosodimethylamine (NDMA)
and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and promotability by a polychlorinated biphenyls mixture (Aroclor 1254). Carcinogenesis 14, 1545–1548.
Chen, G. L., Yang, L., Rowe, T. C., Halligan, B. D., Tewey, K. M., and Liu,
L. F. (1984). Nonintercalative antitumor drugs interfere with the breakagereunion reaction of mammalian DNA topoisomerase II. J. Biol. Chem. 259,
13560 –13566.
Choksi, N. Y., Kodavanti, P. R., Tilson, H. A., and Booth, R. G. (1997).
Effects of polychlorinated biphenyls (PCBs) on brain tyrosine hydrox-
ylase activity and dopamine synthesis in rats. Fundam. Appl. Toxicol. 39,
76 – 80.
Colman, M. S., Afshari, C. A., Barrett, J. C. (2000). Regulation of p53 stability
and activity in response to genotoxic stress. Mutat. Res. 462, 179 – 88.
Darbon, J. M., Penary, M., Escalas, N., Casagrande, F., Goubin-Gramatica, F.,
Baudouin, C., and Ducommun, B. (2000). Distinct Chk2 activation pathways are triggered by genistein and DNA-damaging agents in human
melanoma Cells. J. Biol. Chem. 275, 15363–15369.
Davis, F. M., Tsao, T. Y., Fowler, S. K., and Rao, P. N. (1983). Monoclonal antibodies to mitotic cells. Proc. Natl. Acad. Sci. U.S.A. 80,
2926 –2930.
Dean, C. E. Jr., Benjamin S. A., Chubb, L. S., Tessari, J. D., and Keefe, T. J.
(2002). Nonadditive hepatic tumor promoting effects by a mixture of two
structurally different polychlorinated biphenyls in female rat livers. Toxicol.
Sci. 66, 54 – 61.
Faux, S. P., Francis, J. E., Smith, A. G., and Chipman, J. K. (1992). Induction
of 8-hydroxdeoxyguanosine in Ah-responsive mouse liver by iron and
Aroclor 1254. Carcinogenesis 13, 247–250.
Godard, T., Fessard, V., Huet, S., Mourot, A., Deslandes, E., Pottier,
D., Hyrien, O., Sichel, F., Gauduchon, P., and Poul, J.-M. (1999).
Comparative in vitro and in vivo assessment of genotoxic effects of
etoposide and chlorothalonil by the comet assay. Mutat. Res. 444,
103–116.
Hany, J., Lilienthal, H., Sarasin, A., Roth-Harer, A., Fastabend, A., Dunemann,
L., Lichtensteiger, W., and Winneke, G. (1999). Developmental exposure of
rats to a reconstituted PCB mixture or aroclor 1254: Effects on organ
weights, aromatase activity, sex hormone levels, and sweet preference
behavior. Toxicol. Appl. Pharmacol. 158, 231–243.
Huang, S.-C., and Lee, T.-C. (1998). Arsenite inhibits mitotic division and
perturbs spindle dynamics in HeLa S3 cells. Carcinogenesis 19, 889 –
896.
Jensen, K. G., Wiberg, K., Klasson-Wehler, E., and Onfelt, A. (2000). Induction of aberrant mitosis with PCBs: Particular efficiency of 2,3,3⬘,4,4⬘pentachlorobiphenyl and synergism with triphenyltin. Mutagenesis 15,
9 –15.
Jordan, M. A., Thrower, D., and Wilson, L. (1992). Effects of vinblastine,
podophyllotoxin and nocodazole on mitotic spindles. Implications for the
role of microtubule dynamics in mitosis. J. Cell Sci. 102, 401– 416.
Kato, Y., Haraguchi, K., Shibahara, T., Yumoto, S., Masuda, Y., and Kimura,
R. (1999). Reduction of thyroid hormone levels by methylsulfonyl metabolites of tetra- and pentachlorinated biphenyls in male Sprague-Dawley rats.
Toxicol. Sci. 48, 51–54.
Kikugawa, K., Yasuhara, Y., Ando, K., Koyama, K., Hiramoto, K., and
Suzuki, M. (2003). Protective effect of supplementation of fish oil with high
n-3 polyunsaturated fatty acids against oxidative stress-induced DNA damage of rat liver in vivo. J. Agric. Food Chem. 51, 6073– 6079.
Kodavanti, P. R., and Tilson, H. A. (1997). Structure-activity relationships of
potentially neurotoxic PCB congeners in the rat. Neurotoxicology 18, 425–
442.
Koopman-Esseboom, C., Huisman, M., Weisglas-Kuperus, N., Boersma,
E. R., de Ridder, M. A., Van der Paauw, C. G., Tuinstra, L. G.,
and Sauer, P. J. (1994). Dioxin and PCB levels in blood and human milk
in relation to living areas in The Netherlands. Chemosphere 29, 2327–
2338.
Lanni, J. S., and Jacks, T. (1998). Charaterization of the p53-dependent
postmitotic checkpoint following spindle disruption. Mol. Cell. Biol. 18,
1055–1064.
Lee, S., Park, J. B., Kim, J. H., Kim, Y., Kim, J. H., Shin, K.-J., Lee, J. S., Ha,
S. H., Suh, P. G., and Ryu, S. H. (2001). Actin directly interacts with
phospholipase D, inhibiting its activity. J. Biol. Chem. 276, 28252–28260.
Levine, A. J. (1997). p53, the cellular gatekeeper for growth and division. Cell
88, 323–331.
MITOTIC ARREST AND p53 ACTIVATION BY 2,2⬘,4,6,6⬘-PENTACHLOROBIPHENYL
Meek, D. W. (2000). The role of p53 in the response to mitotic spindle damage.
Pathol. Biol. 48, 246 –254.
Mistry, S. J., and Atweh, G. F. (2001). Stathmin inhibition enhances okadaic
acid-induced mitotic arrest. A potential role for stathmin in mitotic exit.
J. Biol. Chem. 276, 31209 –31215.
Moysich, K. B., Mendola, P., Schisterman, E. F., Frendenheim, J. L.,
Ambrosone, C. B., Vena, J. E., Shields, P. G., Kostyniak, P., Greizerstein,
H., Graham, S., Marshall, J. R. (1999). An evaluation of proposed frameworks for grouping polychlorinated biphenyl (PCB) congener data into
meaningful analytic units. Am. J. Ind. Med. 35, 223–31.
Oakley, G. G., Devanaboyina, U., Robertson, L. W., and Gupta, R. C. (1996).
Oxidative DNA damage induced by activation of polychlorinated biphenyls
(PCBs): Implications of PCB-induced oxidative stress in breast cancer.
Chem. Res. Toxicol. 9, 1285–1292.
Ohi, R., and Gould, K. L. (1999). Regulation of the onset of mitosis. Curr.
Opin. Cell Biol. 11, 267–273.
Pei, X. H., Nakanishi, Y., Takayama, K., Bai, F., and Hara, N. (1999).
Benzo[␣]pyrene activates the human p53 gene through induction of nuclear
factor ␬B activity. J. Biol. Chem. 274, 35240 –35246.
Petruska, D. A., and Engelhard, H. H. (1991). Glioblastoma multiforme
occurring in a patient following exposure to polychlorinated biphenyls. J.
Ky. Med. Assoc. 89, 496 – 499.
Pihan, G. A., Purohit, A., Wallace, J., Knecht, H., Woda, B., Quesenberry, P.,
and Doxsey, S. J. (1998). Centrosome defects and genetic instability in
malignant tumors. Cancer Res. 58, 3974 –3985.
221
Ross, J. L., and Fygenson, D. K. (2003). Mobility of taxol in microtubule
bundles. Biophys. J. 84, 3959 –3967.
Rudner, A. M., and Murray, A. W. (1996). The spindle assembly checkpoint.
Curr. Opin. Cell Biol. 8, 773–780.
Safe, S. (1993). Toxicology, structure-function relationship, and human and
environmental health impacts of polychlorinated biphenyls: Progress and
problems. Environ. Health Perspect. 100, 259 –268.
Schilderman, P. A., Maas, L. M., Pachen, D. M., de Kok, T. M., Kleinjans,
J. C., and van Schooten, F. J. (2000). Induction of DNA adducts by several
polychlorinated biphenyls. Environ. Mol. Mutagen. 36, 79 – 86.
Sen, S. (2000). Aneuploidy and cancer. Curr. Opin. Oncol. 12, 82– 88.
Silberhorn, E. M., Glauert, H. P., and Robertson, L. W. (1990). Carcinogenicity of polyhalogenated biphenyls: PCBs and PBBs. Crit. Rev. Toxicol. 20,
440 – 496.
Taylor, W. R., and Stark, G. R. (2001). Regulation of the G 2/M transition by
p53. Oncogene 20, 1803–1815.
Vessey, C. J., Norbury, C. J., and Hickson, I. D. (1999). Genetic disorders
associated with cancer predisposition and genomic instability. Prog. Nucleic
Acid Res. Mol. Biol. 63, 189 –221.
Ward, E. M., Burnett, C. A., Ruder, A., and Davis-King, K. (1997). Industries
and cancer. Cancer Causes Control 8, 356 –370.
Wu, P. J., Greeley, E. H., Hansen, L. G., and Segre, M. (1999). Immunological,
hematological, and biochemical responses in immature white-footed mice
following maternal Aroclor 1254 exposure: A possible bioindicator. Arch.
Environ. Contam. Toxicol. 36, 469 – 476.