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Environmental
Health Perspectives Supplements Volume 110, Number 5, October 2002
The Molecular Mechanisms of Arsenic-Induced Cell Transformation
and Apoptosis
Zigang Dong
The Hormel Institute, University of Minnesota, Austin, Minnesota,
USA
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Full Article in PDF
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Abstract
Arsenic is a well-documented human carcinogen associated with cancers
of the skin, lung, liver, and bladder. Interestingly, arsenic has also
been used as an effective chemotherapeutic agent in the treatment of certain
human cancers. However, the mechanisms by which arsenic induces proliferation
of cancer cells or cancer cell death are not well understood. We found
that exposure of JB6 P+ cells to low concentrations of arsenic induces
cell transformation, whereas higher concentrations of arsenic induce cell
apoptosis. Arsenite induces phosphorylation of extracellular signal-regulated
protein kinases (Erks) and c-Jun NH2-terminal kinases (JNKs).
Arsenite-induced Erk activation was markedly inhibited by introduction
of dominant-negative Erk2 into cells, whereas expression of dominant-negative
Erk2 did not inhibit JNKs or mitogen-activated protein kinase Erk kinase
1/2. Furthermore, arsenite-induced cell transformation was blocked in
cells expressing dominant-negative Erk2. In contrast, overexpression of
dominant-negative JNK1 increased cell transformation even though it inhibited
arsenite-induced JNK activation. Arsenic also induced AP-1 and nuclear
factor kappa B (NF- B)
activation. Blocking NF-B
activation by dominant-negative inhibitory kappa B
inhibited arsenic-induced apoptosis and enhanced arsenic-induced cell
transformation. Arsenic induced activation of JNKs at a similar dose range
that was effective for induction of apoptosis in JB6 cells. In addition,
we found that arsenic did not induce p53-dependent transactivation. Similarly,
apoptosis induction was not different between p53 wild-type (p53+/+)
or p53-deficient (p53-/-) cells. In contrast,
arsenic-induced apoptosis was almost totally blocked by expression of
a dominant-negative mutant of JNK. Taken together with previous findings
that p53 mutations are involved in approxmiately 50% of all human cancers
and nearly all chemotherapeutic agents kill cancer cells mainly by apoptotic
induction, we suggest that arsenic may be a useful agent for the treatment
of cancers with p53 mutations. These results suggest that the activation
of Erks is required for arsenic-induced cell transformation, whereas the
activation of JNKs and NF-B
is involved in arsenic-induced apoptosis of JB6 cells. Key words: AP-1,
apoptosis, arsenic, cell transformation, MAP kinase, NF-B,
signal transduction. Environ Health Perspect 110(suppl 5):757-759
(2002).
http://ehpnet1.niehs.nih.gov/docs/2002/suppl-5/757-759dong/abstract.html
This article is part of the monograph Molecular Mechanisms
of Metal Toxicity and Carcinogenicity.
Address correspondence to Z. Dong, The Hormel Institute,
University of Minnesota, 801 16th Ave. NE, Austin, MN 55912 USA. Telephone:
(507) 437-9600. Fax: (507) 437-9606. E-mail: zgdong@hi.umn.edu
This work was supported by The Hormel Foundation, National
Institutes of Health grants CA77646, CA81064, and CA88961, and the Eagle's
Cancer Telethon Foundation.
Received 22 April 2002; accepted 4 June 2002.
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Arsenic is a well-documented human carcinogen and is associated with an increased
risk of human cancers of the skin, lung, and bladder (Chen et al. 1985; Forkner
and McNair-Scott 1931; Kandel and Leroy 1937; Konig et al. 1997; Nriagu 1994;
Smith et al. 1992; Sommers and McManus 1953). Interestingly, arsenic-containing
compounds have been used for treatment of cancer for hundreds of years in both
traditional Chinese and Western medicine (Chen et al. 1996, 1997; Forkner and
McNair-Scott 1931; Kandel and Leroy 1937; Konig et al. 1997). Several recent
studies confirm that arsenic trioxide appears to be a valuable therapeutic tool
for patients with acute promyelocytic leukemia (Chen et al. 1996, 1997).
Although arsenic and its modes of action have been the subject of reviews
and symposia, little data exist regarding specific mechanism(s) differentiating
its action as a carcinogen to cause cancer and as a chemotherapeutic agent used
in the treatment of cancer. Recently, our laboratory findings provided mechanisms
of arsenic-induced neoplastic cell transformation and arsenic-induced apoptosis
in tumor cells (Bode and Dong 2000, 2002; Chen et al. 2000a, 2000b; Huang et
al. 1999a, 1999b, 2001) (Figure 1).
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| Figure 1. Arsenic-induced
signal transduction pathways and their role in cell transformation and apoptosis.
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Arsenic Induces Cell Transformation
Much of the lack of progress in determining mechanisms explaining the role
of arsenic as a carcinogen or as a chemotherapeutic agent has been attributed
to the availability of valid and reproducible animal models. To study whether
arsenite induces cell transformation, we exposed JB6 Cl 41 cells to arsenite
in soft agar. Anchorage-independent colonies were observed in the eighth week
after arsenite exposure. Cell transformation can only be observed in cells exposed
to 0.5-25 µM arsenite, whereas no transformed colonies were observed
at higher concentrations of arsenite (50-100 µM) because of the toxicity
of arsenic (Huang et al. 1999a, 1999b).
Induction of Apoptosis by Higher Concentrations of Arsenic
A higher concentration of arsenic appears to prevent cell transformation by
inducing apoptosis. We showed that treatment of cells with a relatively higher
concentration (200 µM) of arsenite or arsenate resulted in apoptosis by
44.5 and 61.5%, respectively (Chen et al. 2000b).
Differential Activation of Erks and JNKs by Arsenite
We found that arsenite could induce activation of both c-Jun NH2-terminal
kinases (JNKs) and extracellular signal-regulated protein kinases (Erks) (Huang
et al. 1999a, 1999b). However, the activation of JNKs and Erks by arsenite differs
in time course and dose response. During the time course and dose-response
studies, marked Erks activation could be observed 15 min after exposure and
at all dosages studied, but no significant induction of Erks by arsenite occurred
after a 30-min exposure. In contrast, arsenic activation of JNKs was observed
only at a relatively high dosage (>50 µM) and after 60 min of exposure.
Arsenic Induces AP-1 Activation in Cells and AP-1-Luciferase Transgenic
Mice
Using JB6 cells, we found that both arsenite and arsenate could induce transactivation
of AP-1 (Huang et al. 2001). This induction of AP-1 activity by arsenic appears
to occur through activation of mitogen-activated protein (MAP) kinases and protein
kinase C (PKC) because increased AP-1 activity by arsenite could be blocked
by either treating cells with PD98059, an MAP kinase Erk kinase (MEK)1 inhibitor,
or overexpression of dominant-negative PKC
(Chen et al. 2000a; Huang 2001). Furthermore, both arsenite and arsenate could
induce transactivation of AP-1 in AP-1-luciferase reporter transgenic mice (Huang
2001).
Inhibition of Erks Activation Blocks Arsenic-Induced Cell Transformation
The results described above revealed that Erks activation by arsenite may
be involved in its cell transformation activity. To test this possibility, we
used dominant-negative Erk2-K52R stable transfectants (Chen et al. 2000b; Huang
1999a, 1999b). Our results demonstrate that Erk activation but not JNK activation
is required for arsenite-induced cell transformation.
Inhibition of JNKs Blocks Arsenic-Induced Apoptosis
To investigate the role of activation of JNKs in arsenic-induced apoptosis,
we used JB6 cells and a dominant-negative mutant of JNK1 to test the effects
on arsenic-induced apoptosis (Huang et al. 1999a). Expression of the dominant-negative
mutant JNK1 blocked induction of apoptosis by arsenite (4%) or arsenate (7%)
compared with vector-transfected control cells (31.5 and 40.5% for arsenite
and arsenate, respectively) (Sommers and McManus 1953).
p53 Is Not Involved in Induction of Apoptosis by Arsenic
Arsenic had no effect on p53-dependent transcription activation in Cl 41 p53
cells treated with a wide dose range of arsenic (Huang
et al. 1999a). This suggested that p53 may not be involved in arsenic-induced
apoptosis. This hypothesis was further tested by studying the effects of arsenic
on two fibroblast cell lines derived from mouse embryos either containing wild-type
p53 (p53+/+) or deficient in p53 (p53-/-).
Results showed that treatment with arsenite or arsenate results in apoptosis
in both cell lines (Huang et al. 1999a). Therefore, arsenic may be effective
in counteracting drug resistance in p53-deficient cancers because arsenic appears
to be able to induce apoptosis in tumor cells independently of p53 activation
and thus could be specifically directed against p53-defective cancer cells.
Involvement of PKC in Arsenic-Induced Signal Transduction
Our recent data show that PKC, upstream from the MAP kinases, may be involved
in mediating arsenite-induced signal transduction (Chen et al. 2000a). Translocation
of PKC from the cytosol to the membrane is a critical step for activation of
this enzyme, and treatment of JB6 cells with arsenite resulted in an increased
translocation of PKC within 15 min. Inhibition of activation of PKC blocked
both arsenite-induced AP-1 activity and arsenite-induced phosphorylation of
Erks, JNKs, and p38 kinase, suggesting that PKC is required for arsenite-induced
activation of MAP kinases.
Tea Polyphenols Block Arsenic-Induced Signal Transduction and Cytotoxicity
Arsenite-induced apoptosis appears to be important in accounting for its toxicity.
Green tea has been used as a traditional Chinese remedy for detoxification of
arsenite-caused toxicity. In recent work, we found that tea polyphenols, (-)-epigallocatechin-3-gallate
(EGCG) and theaflavins, effectively blocked arsenite-induced apoptosis in JB6
cells and inhibited arsenite-induced AP-1 transcriptional activation and AP-1
DNA-binding activity (Chen et al. 2000b). EGCG and theaflavins potently inhibited
arsenite-induced Erk activity but not p38 kinase activity. PD98059, an inhibitor
of Erks, and dominant-negative mutant (DNM)-JNK1 blocked arsenite-induced apoptosis,
whereas DNM-p38 kinase or SB2020190, an inhibitor of p38 kinase, did not. We
conclude that Erks and JNKs may be involved in arsenite-induced apoptosis, and
the inhibition of arsenite-induced apoptosis by EGCG and theaflavins may be
mediated by a decreased phosphorylation of Erks and JNKs. Furthermore, these
results provide a possible mechanism explaining the detoxification effect of
tea on arsenite-induced toxicity (Chen et al. 2000b).
Blocking NF-B
Activation Inhibits Arsenic-Induced Apoptosis
Nuclear factor kappa B (NF-B)
is a rapidly induced stress-responsive transcription factor that may play an
important role in arsenic-induced signal transduction, cell transformation,
and apoptosis (Bode and Dong 2000, 2002). Barchowsky et al. (1996,1999) reported
that arsenic-induced oxidant stress, H2O2, and superoxide
are the predominant reactive species in endothelia cells and may be the mediators
for the activation of the NF-B
pathway. We showed that arsenic induced activation of NF-B
in different cell culture models. Expression of a dominant-negative inhibitory
kappa B
blocked arsenic-induced activation of NF-B
and apoptosis.
Conclusions
Arsenic is clearly a human carcinogen, but also acts as a beneficial chemotherapeutic
agent. The lack of information regarding its mechanisms of action has been attributed
to the availability of appropriate animal models. In the past few years we have
established cell culture models to study arsenic-induced neoplastic cell transformation
and apoptosis. Others have also reported that exposure of arsenic could induce
cell transformation in a rat liver epithelial cell line (TRL 1215) (Meng and
Meng 2000) or in blast transformation and DNA synthesis in human lymphocytes
(Zhao et al. 1997). We found that arsenic significantly affects specific signal
transduction pathways (Figure 1). Our data provided a model for the role of
signaling molecules including MAP kinases, p53, AP-1, and NF-B
in arsenic-induced cell transformation and apoptosis. More detailed studies
are needed to determine precise mechanisms of the effects of arsenic on animals
and humans.
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Last Updated: October 10, 2002
