1: Chem Biol Interact. 2005 May 30;153-154:165-70. Epub 2005 Apr 22. 

p53-dependent gene profiling for reactive oxygen species after benzene
inhalation: special reference to genes associated with cell cycle regulation.

Hirabayashi Y.

Cellular and Molecular Toxicology Division, National Institute of Health
Sciences, 1-18-1 Kamiyohga, Setagayaku, Tokyo 158-8501, Japan.
yokohira@nihs.go.jp

Benzene toxicity has long been thought to be due to its metabolites including
reactive oxygen species (ROS). However, the major toxicological effect of
benzene in wild-type mice carrying normal alleles of the p53 gene appears to be
the significant perturbation of cell cycle regulation, possibly via an indirect
signaling pathway. Other prominent genotoxic cellular damage can occur in the
absence of cell cycle arrest in p53 gene deficiency. The suppression of cell
cycle is clearly detected using a tool for stem-cell-specific cell cycle
observation by the BU-UV method. Cells (including hemopoietic progenitor cells)
in S-phase are labeled in vivo with bromodeoxyuridine (BrdU) and then exposed to
near-ultraviolet (UV) light to kill cells that incorporated BrdU. The target
fraction, the S-phase, is then evaluated on the basis of decreased numbers of
hemopoietic colonies formed in assays such as for granulomacrophage
colony-forming units (CFU-GM). Benzene toxicity was found to be more prominent
in the primitive stem-cell compartment, as first suggested more than 20 years
ago. Interestingly, when one examines the stem-cell-specific steady-state gene
expression profiling, several key genes associated with benzene exposure are
specifically identified, including CYP2E1. Benzene toxicity was found to be
mediated by aryl hydrocarbon receptor (AhR) at an expression level; thus, the
effect of benzene can be detected in nature at lower levels in the stem-cell
compartment than expected. Alterations in gene expression profiles compared with
those in steady-state gene expression profiles in the stem-cell compartment may
elucidate the mechanism underlying benzene toxicity. Functional gene expressions
after benzene exposure are not always detected, because their phenotypic
expressions are often masked by the balance of expression of genes participating
in various pathways of homeostasis, for example, p53. Thus, the actual
expressions of the above-mentioned cell cycle-related genes may not be clearly
detected. However, when one examines the genes after benzene exposure without
p53 gene participation (i.e., p53 was knocked out), various cell cycle-related
genes expressed during and after benzene exposure are identified, such as cyclin
B1, cyclin D3 and growth hormone in the bone marrow. Since age-related
impairments of p53 gene function in somatic cells are known, the possible
alteration of those genes would be based not only on a theoretical model, but
possible risks posed on the elderly should also be taken into consideration.

Publication Types:
    Review
    Review, Tutorial

PMID: 15935813 [PubMed - indexed for MEDLINE]


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2: J Pharmacol Exp Ther. 2005 Aug;314(2):603-10. Epub 2005 Apr 20. 

The p53 inhibitor pifithrin-alpha is a potent agonist of the aryl hydrocarbon
receptor.

Hoagland MS, Hoagland EM, Swanson HI.

University of Kentucky Medical Center S-305, 800 Rose Street, Lexington, KY
40536-0084, USA.

The tumor suppressor protein p53 is currently a target of emerging drug
therapies directed toward neurodegenerative diseases, such as Alzheimer's and
Parkinson's, and side effects associated with cancer treatments. Of this group
of drugs, the best characterized is pifithrin-alpha, a small molecule that
inhibits p53-dependent apoptosis through an undetermined mechanism. In this
study, we have used a number of molecular approaches to test the hypothesis that
pifithrin-alpha acts as an aryl hydrocarbon receptor (AhR) agonist and, in this
manner, inhibits the actions of p53. Toward this end, we have found that
pifithrin-alpha is a potent AhR agonist as determined by its ability to bind the
AhR, induce formation of its DNA binding complex, activate reporter activity,
and up-regulate the classic AhR target gene CYP1A1. However, examination of its
ability to inhibit p53-mediated gene activation and apoptosis revealed that
these actions occurred via an AhR-independent manner. The significance of this
study is based on the fact that activation of the AhR is typically associated
with an increase in phase I and phase II metabolizing enzymes and adverse
biological events such as tumor promotion that may contribute to untoward
effects of pifithrin-alpha. Hence, this work will aid in the future design of
more specific members of this important class of p53 inhibitors for use in a
clinical setting.

PMID: 15843497 [PubMed - indexed for MEDLINE]


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3: Comp Biochem Physiol C Toxicol Pharmacol. 2004 Jul;138(3):375-81. 

Aryl hydrocarbon receptor (AhR)-independent effects of
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on softshell clam (Mya arenaria)
reproductive tissue.

Butler RA, Kelley ML, Olberding KE, Gardner GR, Van Beneden RJ.

School of Marine Sciences, University of Maine, Orono, ME 04469-5751, USA.

A high prevalence of germinomas has been observed in certain populations of Mya
arenaria from eastern Maine. The etiology of these tumors is unknown. We are
investigating the hypothesis that exposure to environmental contaminants, such
as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) contributes to gonadal
carcinogenesis. Clams were exposed to TCDD with or without the initiating
compound diethylnitrosamine (DEN) in an attempt to induce germinomas. A
TCDD-dependent alteration in gametogenesis was observed in which 32.5+/-6.5% of
individuals exhibited undifferentiated gonads. Analyses of AhR and p53
expression were carried out to identify similarities between naturally occurring
neoplastic and TCDD (+/-DEN)-altered reproductive tissues. Neoplastic tissues
had significantly less p53 protein than matched controls, whereas TCDD-induced
undifferentiated samples exhibited no difference in p53 protein levels compared
to controls. No gender-specific differences were observed in AhR mRNA, but there
were significant differences in protein levels. AhR was undetectable in male
gonadal tissue whereas females exhibited a significant positive relationship
between AhR protein levels and stage of ovogenesis. Despite exhibiting some
morphological similarity, we conclude the TCDD-induced pathology is not a
germinoma. We further suggest the change in reproductive tissue is due to
inhibition of cell differentiation and/or development by an AhR-independent
mechanism.

PMID: 15533795 [PubMed - indexed for MEDLINE]


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4: Carcinogenesis. 2005 Jan;26(1):201-8. Epub 2004 Sep 30. 

TCDD activates Mdm2 and attenuates the p53 response to DNA damaging agents.

Paajarvi G, Viluksela M, Pohjanvirta R, Stenius U, Hogberg J.

Institute of Environmental Medicine, Karolinska Institutet, Box 210, 17177
Stockholm, Sweden.

In this study we investigated the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) on the p53 response to DNA damaging agents. Pre-treatment of rats with
TCDD attenuated the p53 liver response to diethylnitrosamine (DEN) and reduced
levels of p53 and Ser15 phosphorylated p53. In addition, there were more slowly
migrating p53 species, forming a ladder, which suggests an increased
ubiquination of p53 in TCDD-pre-treated rats. Terminal deoxynucleotidyl
transferase-mediated X-dUTP nick-end labelling analysis indicated decreased
apoptosis rates in the livers of these rats. Studies on aryl hydrocarbon
receptor (AhR) knockout mice and their wild-type littermates confirmed this
effect in AhR +/+ but not in AhR -/- mice, indicating that this effect may be
AhR-mediated. Quantitative RT-PCR analysis revealed no increased mRNA levels in
TCDD-treated rats, but immunohistological studies indicated that TCDD modulated
Mdm2 protein levels, and in particular, increased nuclear levels in rat
hepatocytes in situ. In vitro studies employing HepG2 cells confirmed the in
vivo data. Thus, TCDD increased basal levels of Mdm2 protein, but not mRNA, and
attenuated the p53 response to a variety of genotoxic and cytotoxic agents. The
increase in Mdm2 protein levels was accompanied by rapid and highly sensitive
phosphorylation of Mdm2 at Ser166, which has been associated to active Mdm2. In
summary, TCDD is a potent inhibitor of p53 that may influence the liver's
ability to handle genotoxic agents in a safe way, and may play a role in
TCDD-induced carcinogenesis.

PMID: 15459018 [PubMed - indexed for MEDLINE]


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5: Mutat Res. 2004 Aug 18;552(1-2):61-71. 

A HECT E3 ubiquitin-protein ligase with sequence similarity to E6AP does not
target p53 for degradation in the softshell clam (Mya arenaria).

Olberding KE, Kelley ML, Butler RA, Van Beneden RJ.

Department of Biochemistry, Microbiology and Molecular Biology, University of
Maine, 5751 Murray Hall, Orono, ME 04469, USA.

Numerous reports have raised the level of national concern that chemicals found
in the environment may have adverse effects on the health of humans and
wildlife. Environmental exposure to pollutants, such as dioxin, has been
implicated in gonadal tumor formation in Maine softshell clams (Mya arenaria).
Prevalence of these tumors is as high as 40% in some populations. Although their
etiology is still unknown, investigations into the mechanisms of tumor formation
have revolved around a hypothesis of dioxin-induced toxicity. The aryl
hydrocarbon receptor (AHR) was initially investigated, but was later determined
to not bind the prototypical ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),
suggesting that dioxin toxicity is mediated through an AHR-independent pathway.
An alternative mechanism of tumor formation has been investigated, involving a
protein with significant sequence similarity to mammalian E6AP, a HECT
(homologous to E6AP carboxy terminus) E3 ubiquitin-protein ligase. E6AP, in
association with the high-risk human papillomavirus (HPV) E6 protein, is
involved in the abnormal degradation of the p53 tumor suppressor protein in
human cervical cancer. Tumorigenic clam reproductive tissue revealed higher M.
arenaria E3 (MaE3) protein levels concomitant with lower M. arenaria p53 (Map53)
levels. While the function of MaE3 as a HECT E3 was verified, results from three
methods agree that MaE3 does not associate with Map53. However, alteration in
Map53 levels may still play a role in clam gonadal tumorigenesis. Due to
upregulation of MaE3 in neoplastic reproductive tissue, further investigations
will focus on determining the proteolytic targets of MaE3. In conjunction with
our previous findings that dioxin toxicity in the softshell clam is not mediated
by AHR, the results from our current investigation suggest a complex etiology
for the clam germinomas.

PMID: 15288542 [PubMed - indexed for MEDLINE]


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6: J Biol Chem. 2004 Jun 25;279(26):27187-93. Epub 2004 Apr 24. 

Dioxin-induced immortalization of normal human keratinocytes and silencing of
p53 and p16INK4a.

Ray SS, Swanson HI.

Department of Molecular and Biomedical Pharmacology, University of Kentucky
Medical Center, Lexington, Kentucky 40536, USA.

Dioxin, a potent tumor promoter, activates the aryl hydrocarbon receptor (AHR),
a ligand-activated transcription factor belonging to the basic
helix-loop-helix-PAS family, to enhance tumorigenesis via unknown mechanisms. We
undertook this study to determine the mechanisms underlying the impact of dioxin
on cell fate, in particular senescence that occurs in normal human cells and is
considered to play important tumor suppressive function. We have previously
shown that in primary human keratinocytes, dioxin attenuates senescence while
retaining the proliferative capacity and represses expression of the tumor
suppressors, p16(INK4a) and p53. Here, we show that repression of p16(INK4a) and
p53 transcriptional activity by dioxin absolutely requires the AHR and is
accompanied by promoter methylation. Furthermore, dioxin alone is sufficient to
immortalize normal human keratinocytes. Our data introduce a previously
unrecognized regulatory pathway, that of the AHR, that impacts senescence. More
importantly, this is the first report of a tumor promoter capable of inhibiting
senescence in a receptor mediated manner and introduces a novel mechanism by
which this carcinogen may contribute to human malignancies.

PMID: 15111621 [PubMed - indexed for MEDLINE]


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7: Toxicol Lett. 2004 Apr 1;149(1-3):43-50. 

Tumor promoters as inhibitors of apoptosis in rat hepatocytes.

Schrenk D, Schmitz HJ, Bohnenberger S, Wagner B, Worner W.

Food Chemistry and Environmental Toxicology, University of Kaiserslautern,
Erwin-Schroedinger-Strasse 52, D-67663 Kaiserslautern, Germany.
schrenk@rhrk.uni-kl.de

Multistage carcinogenesis in rat liver is widely used as an experimental model
for the study of the critical events in tumor promotion. After an initial
treatment with a genotoxic liver carcinogen ('initiation'), subsequent
application of certain non-genotoxic agents can lead to the clonal expansion of
putative preneoplastic cells ('promotion'). Obviously, the expansion of these
clones is correlated with an increased occurrence of benign and malignant liver
tumors at later time points. Since both proliferation and apoptosis were
reported to be enhanced in putative preneoplastic liver foci, inhibition of
apoptosis was suggested to play a critical role in tumor promotion. In rat
hepatocytes in primary culture, the liver tumor promoter
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibited apoptosis initiated by
treatment of the cultures with UV irradiation but did not affect apoptosis in
non-irradiated cultures. The suppression of apoptosis with TCDD coincided with
an attenuated increase of the tumor suppressor protein p53 observed upon UV
irradiation. Furthermore, TCDD treatment resulted in a marked
hyperphosphorylation of p53. The fact that almost identical
concentration-response curves were obtained for the phosphorylation of p53 and
the induction of cytochrome P450(CYP)1A-catalyzed 7-ethoxyresorufin O-deethylase
(EROD) activity indicates that p53 phosphorylation after TCDD treatment is
mediated by the aryl hydrocarbon receptor (AhR) signaling cascade. With
tumor-promoting 'non-dioxin-like' polychlorinated biphenyls inhibition of
UV-induced apoptosis was also observed. A comparative study investigating the
effects of various concentrations did not reveal, however, a clear correlation
between the suppression of apoptosis and the induction of CYP2B-catalyzed
7-pentoxyresorufin O-dealkylase (PROD) activity. In summary, inhibition of
UV-induced apoptosis with liver tumor promoters is observed in rat hepatocytes
in culture. Hyperphosphorylation of key proteins of apoptosis including p53
seems to play a role in this effect.

PMID: 15093247 [PubMed - indexed for MEDLINE]


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8: Toxicol Appl Pharmacol. 2004 Apr 1;196(1):136-48. 

Polycyclic aromatic hydrocarbons modulate cell proliferation in rat hepatic
epithelial stem-like WB-F344 cells.

Chramostova K, Vondracek J, Sindlerova L, Vojtesek B, Kozubik A, Machala M.

Laboratory of Cytokinetics, Institute of Biophysics, 612 65 Brno, Czech
Republic.

Although many polycyclic aromatic hydrocarbons (PAHs) are recognized as potent
mutagens and carcinogens, relatively little is known about their role in the
tumor promotion. It is known that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can
induce release of rat hepatic oval epithelial cells from contact inhibition by a
mechanism possibly involving the aryl hydrocarbon receptor (AhR) activation.
Many PAHs are AhR ligands and are known to act as transient inducers of
AhR-mediated activity. In this study, effects of 19 selected PAHs on
proliferation of confluent rat liver epithelial WB-F344 cells were investigated.
Non-mutagens that are weak activators or nonactivators of AhR-mediated activity
had no effect on cell proliferation. Relatively strong or moderate AhR ligands
with low mutagenic potencies, such as benzofluoranthenes, benz[a]anthracene, and
chrysene, were found to increase cell numbers, which corresponded to an
increased percentage of cells entering S-phase. Strong mutagens, including
benzo[a]pyrene and dibenzo[a,l]pyrene, increased a percentage of cells in
S-phase without inducing a concomitant increase in cell numbers. The treatment
with mutagenic PAHs was associated with an increased DNA synthesis and induction
of cell death, which corresponded with the activation of p53 tumor suppressor.
Apoptosis was blocked by pifithrin-alpha, the chemical inhibitor of p53. Both
weakly and strongly mutagenic PAHs known as AhR ligands were found to induce
significant increase of cytochrome P4501A activity, suggesting a presence of
functional AhR. The results of the present study seem to suggest that a release
from contact inhibition could be a part of tumor promoting effects of
AhR-activating PAHs; however, the genotoxic effects of some PAHs associated with
p53 activation might interfere with this process.

PMID: 15050415 [PubMed - indexed for MEDLINE]


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9: Toxicol Appl Pharmacol. 2003 Oct 15;192(2):131-45. 

Alteration of keratinocyte differentiation and senescence by the tumor promoter
dioxin.

Ray SS, Swanson HI.

Department of Molecular and Biomedical Pharmacology, University of Kentucky
Medical Center, 800 Rose Street, MS 305, Lexington, KY 40536, USA.

Exposure to the environmental contaminant dioxin, elicits a variety of
responses, which includes tumor promotion, embryotoxicity/teratogenesis, and
carcinogenesis in both animals and humans. Many of the effects of dioxin are
mediated by the aryl hydrocarbon receptor (AHR), a ligand-activated bHLH (basic
helix-loop-helix)/PAS transcription factor. We initiated this study to determine
whether dioxin's tumor-promoting activities may lie in its ability to alter
proliferation, differentiation, and/or senescence using normal human epidermal
keratinocytes (HEKs). Here, we report that dioxin appears to accelerate
differentiation as measured by flow cytometry and by increased expression of the
differentiation markers involucrin and filaggrin. In addition, dioxin appears to
increase proliferation as indicated by an increase in NADH/NADPH production and
changes in cell cycle. Finally, dioxin decreases SA (senescence associated)
beta-galactosidase staining, an indicator of senescence, in the differentiating
keratinocytes. These changes were accompanied by decreases in the expression
levels of key cell cycle regulatory proteins p53, p16INK4a, and p14ARF. Our
findings support the idea that dioxin may exert its tumor-promoting actions, in
part, by downregulating the expression levels of key tumor suppressor proteins,
which may impair the cell's ability to maintain its appropriate cellular status.

PMID: 14550747 [PubMed - indexed for MEDLINE]


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10: Environ Mol Mutagen. 2002;39(2-3):235-44. 

Epigenetics of breast cancer: polycyclic aromatic hydrocarbons as risk factors.

Jeffy BD, Chirnomas RB, Romagnolo DF.

Cancer Biology Interdisciplinary Program, The University of Arizona, Tucson,
Arizona 85721-0038, USA.

In the absence of a causal relationship between the incidence of sporadic breast
cancer and occurrence of mutations in breast cancer susceptibility genes,
efforts directed to investigating the contribution of environmental xenobiotics
in the etiology of sporadic mammary neoplasia are warranted. Polycyclic aromatic
hydrocarbons (PAHs) are ubiquitous pollutants, which have been shown to induce
DNA damage and disrupt cell cycle progression. In this report we discuss
published data pointing to PAHs as a risk factor in carcinogenesis, and present
findings generated in our laboratory suggesting that the mammary tumorigenicity
of PAHs may be attributable, at least in part, to disruption of BRCA-1
expression by reactive PAH-metabolites. We report that benzo[a]pyrene (B[a]P),
selected as a prototype PAH, disrupts BRCA-1 transcription in estrogen receptor
(ER)-positive but not ER-negative breast cancer cells. The reduced potential for
BRCA-1 expression in B[a]P-treated cells coincides with disruption of cell cycle
kinetics and accumulation of p53. These effects are counteracted by the
AhR-antagonist alpha-naphthoflavone (ANF), and in breast cancer cells expressing
mutant p53 or the E6 human papilloma virus protein. We suggest that exposure to
PAHs may be a predisposing factor in the etiology of sporadic breast cancer by
disrupting the expression of BRCA-1. Copyright 2002 Wiley-Liss, Inc.

PMID: 11921194 [PubMed - indexed for MEDLINE]


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11: Neoplasia. 2000 Sep-Oct;2(5):460-70. 

Disruption of cell cycle kinetics by benzo[a]pyrene: inverse expression patterns
of BRCA-1 and p53 in MCF-7 cells arrested in S and G2.

Jeffy BD, Chen EJ, Gudas JM, Romagnolo DF.

Cancer Biology Interdisciplinary Program, Arizona Cancer Center, The University
of Arizona, Tucson, USA.

The effects of a ligand of the aromatic hydrocarbon receptor (AhR), benzo[a]
pyrene (B[ a]P), and its metabolite, BPDE
(7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene), on BRCA-1
levels and cell cycle kinetics were determined in MCF-7 breast cancer cells.
Exposure of asynchronous MCF-7 cells for 72 hours to a non-cytotoxic dose of 0.5
microM B[a]P triggered a three-fold reduction in BRCA-1 protein. In MCF-7 cells
resistant (20% to 30%) to genotoxic concentrations of B[a]P (1 to 5 microM), the
loss of BRCA-1 protein was coupled with pausing in S-phase and G2/M, and
accumulation of p53, mdm2 and p21. Treatment of MCF-7 cells synchronized in
S-phase (72%) with B[a]P prolonged the arrest in S-phase, although this
checkpoint was transient since cells resumed to G2/M after 12 hours with reduced
levels of BRCA-1. In these cells, levels of p53 were increased, whereas the
cellular content of p21 remained unaltered. In contrast, the co-treatment with
the AhR antagonist, alpha-naphthoflavone (ANF), abrogated the deleterious
effects of B[a]P on BRCA-1 expression, while preventing the accumulation of p53
and disruption of cell cycle profile. These findings suggest that the AhR
mediated the inverse expression patterns of BRCA-1 and p53 upon exposure to
B[a]P. The treatment with BPDE induced S-phase arrest and reduced BRCA-1 mRNA
levels. The negative effects of BPDE on BRCA-1 expression were not transient
since removal of BPDE did not allow complete reversal of the repression. These
cumulative data suggest that the B[a]P metabolite, BPDE, may play a key role in
disruption of BRCA-1 expression and cell cycle kinetics in breast epithelial
cells.

PMID: 11191113 [PubMed - indexed for MEDLINE]


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12: J Biol Chem. 2001 Feb 16;276(7):4819-27. Epub 2000 Nov 28. 

Aromatic hydrocarbon receptor (AhR).AhR nuclear translocator- and p53-mediated
induction of the murine multidrug resistance mdr1 gene by 3-methylcholanthrene
and benzo(a)pyrene in hepatoma cells.

Mathieu MC, Lapierre I, Brault K, Raymond M.

Institut de Recherches Cliniques de Montreal, Montreal, Quebec H2W 1R7, Canada.

The mouse multidrug resistance gene family consists of three genes (mdr1, mdr2,
and mdr3) encoding P-glycoprotein. We show that the expression of mdr1 is
increased at the transcriptional level upon treatment of the hepatoma cell line
Hepa-1c1c7 with the polycyclic aromatic hydrocarbon 3-methylcholanthrene (3-MC).
This increase is not observed in the aromatic hydrocarbon receptor
(AhR)-defective TAOc1BP(r)c1 and the AhR nuclear translocator (Arnt)-defective
BP(r)c1 variants, demonstrating that the induction of mdr1 by 3-MC requires
AhR.Arnt. We show that the mdr1 promoter (-1165 to +84) is able to activate the
expression of a reporter gene in response to 3-MC in Hepa-1c1c7 but not in
BP(r)c1 cells. Deletion analysis indicated that the region from -245 to -141
contains cis-acting sequences mediating the induction, including a potential p53
binding sequence. 3-MC treatment of the cells increased the levels of p53 and
induced p53 binding to the mdr1 promoter in an AhR.Arnt-dependent manner.
Mutations in the p53 binding site abrogated induction of mdr1 by 3-MC,
indicating that p53 binding to the mdr1 promoter is essential for the induction.
Benzo(a)pyrene, a polycyclic aromatic hydrocarbon and AhR ligand, which, like
3-MC, is oxidized by metabolizing enzymes regulated by AhR.Arnt, also activated
p53 and induced mdr1 transcription. 2,3,7,8-Tetrachlorodibenzo-p-dioxin, an AhR
ligand resistant to metabolic breakdown, had no effect. These results indicate
that the transcriptional induction of mdr1 by 3-MC and benzo(a)pyrene is
directly mediated by p53 but that the metabolic activation of these compounds
into reactive species is necessary to trigger p53 activation. The ability of the
anticancer drug and potent genotoxic agent daunorubicin to induce mdr1
independently of AhR.Arnt further supports the proposition that mdr1 is
transcriptionally up-regulated by p53 in response to DNA damage.

PMID: 11096091 [PubMed - indexed for MEDLINE]


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13: Pharmacogenetics. 1999 Apr;9(2):183-8. 

Low levels of p53 are associated with resistance to tetrachlorodibenzo-p-dioxin
toxicity in DBA/2 mice.

Yang AL, Smith AG, Akhtar R, Clothier B, Robinson S, MacFarlane M, Festing MF.

MRC Toxicology Unit, University of Leicester, UK.

We show here that DBA/2 strain mice have a complex mutation/polymorphism in the
promoter region of the Trp53 locus (the mouse p53 locus). This region has
previously been shown to be essential for p53 expression. We further show that
the DBA/2 mutation is associated with approximately fourfold lower p53 levels in
thymocytes treated with the DNA-damaging agent etoposide in-vitro, and with
relative resistance of these thymocytes to apoptosis induced by the DNA-damaging
agent etoposide compared with C57BL/6 mice. When part of the promoter containing
this mutation was inserted into a plasmid containing a luciferase reporter gene
but lacking eukaryote promoter sequences and transfected into MCF-7 human breast
cell line cells, the mean luciferase activity was slightly less with the DBA/2
than with the C57BL/6 promoter-reporter construct (p < 0.01). We found that
DBA/2xC57BL/6 F2 hybrid mice with the DBA/2 genotype at the Trp53 locus were
relatively resistant to tetrachlorodibenzo-p-dioxin toxicity, and this
resistance was additive with resistance associated with the Ahr locus. DBA/2
mice are long-lived and do not have particularly high levels of cancer,
suggesting either that they carry other compensatory tumour resistance alleles
(such as Ahr(d)), or that, while there may be a p53 protein dosage effect for
acute toxicity, lower than normal levels of p53 may still be sufficient to
protect against cancer. In evolutionary terms, it may be better to maintain low
levels of p53 in order to avoid death from acute toxicity, even at the expense
of a higher incidence of cancer in later life.

PMID: 10376765 [PubMed - indexed for MEDLINE]


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14: Toxicol Appl Pharmacol. 1998 Aug;151(2):283-93. 

Alterations in the growth factor signal transduction pathways and modulators of
the cell cycle in endocervical cells from macaques exposed to TCDD.

Enan E, El-Sabeawy F, Scott M, Overstreet J, Lasley B.

Department of Environmental Toxicology, University of California, Davis 95616,
USA. eenan@ucdavis.edu

After more than a year had elapsed since a single oral exposure to 2 and 4
microgram 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg, there was an apparent
dose-related increased incidence of significant endocervical squamous metaplasia
in a group of cynomolgus macaques (Scott et al., 1998). In the present
experiments we investigated the mechanisms by which chemicals like TCDD could
induce epithelial cell transdifferentiation in the primate endocervix. One focus
of investigation was epidermal growth factor receptor (EGFR) and the key
cytosolic signaling kinases, c-Src and protein tyrosine kinase (PTK), whose
responses to TCDD are well characterized. A second focus was the distal kinase
Erk2 that transduces the cytosolic signal into a nuclear signal, and which in
combination with nuclear casein kinase II (CKII), can lead to activation of p53.
Finally, we studied three key target proteins of activated p53 (wafl/p21, Cdc2
p34, and Cdk4), whose modulation could produce cell cycle effects. The studies
were carried out using primary cell cultures prepared from endocervical
epithelium recovered at necropsy from TCDD-treated (2 and 4 microgram TCDD/kg)
and untreated macaques. There was a significant decrease in EGFR binding
activity in cells from TCDD-treated animals as compared to controls. A marked
increase in the protein amount of H-Ras and a significant increase in the
activity of c-Src kinase, PTK, and Erk2 were found in cells from TCDD-treated
animals. A significant decrease in the activity of CKII and in the protein
amount of p53, wafl/p21, and Cdc2 p34 was found. On the other hand, a
substantial increase in the protein amount of Cdk4 and DNA binding activity of
AP-1 was found in cells from TCDD-treated animals. In vitro experiments using
primary cultures of endocervical cells from untreated macaques revealed that
these cells have AhR, and that c-Src protein is functionally attached to the AhR
and is specifically activated upon ligand binding as judged by the following
criteria. (1) A structure-activity relationship study with TCDD and three dioxin
congeners revealed a rank order for their potency in activation of
AhR-associated c-Src kinase from cervical cells which was identical to that of
previously determined toxicity indices. (2) TCDD-induced, AhR-associated c-Src
kinase activity was abolished when an AhR immunoprecipitate from cervical cells
was preincubated with alpha-naphthoflavone (AhR blocker) or geldanamycin (Src
kinase inhibitor) prior to the addition of TCDD. (3) The analysis of the AhR
complex showed three proteins of molecular weights of 100 (AhR), 90, and 60 kDa.
(4) The same protein with molecular weight 60 kDa was found when the
immunoprecipitate with anti AhR-antibody was analyzed by SDS-PAGE, then
transferred into nitrocellulose membrane followed by immunobloting the membrane
with anti c-Src-antibody. Our data suggest that TCDD induced pathology in
endocervical cells through changes in growth factor receptor signaling, other
cytosolic signaling proteins, tumor suppressor proteins, and cell cycle
proteins. Copyright 1998 Academic Press.

PMID: 9707505 [PubMed - indexed for MEDLINE]


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15: J Biol Chem. 1997 Jan 31;272(5):2762-9. 

A benzo[a]pyrene-induced cell cycle checkpoint resulting in p53-independent G1
arrest in 3T3 fibroblasts.

Vaziri C, Faller DV.

Cancer Research Center, Boston University School of Medicine, Boston,
Massachusetts 02118, USA.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor
of the basic helix-loop-helix family. Although physiological ligands for the AhR
have not been identified, carcinogenic polycyclic aromatic hydrocarbons such as
Benzo[a]pyrene (B[a]P) are high affinity AhR ligands that induce nuclear
translocation and sequence-specific DNA binding of the AhR. AhR-regulated genes
include members of the cytochrome P-450 family that are known to oxidize B[a]P
to form genotoxic (DNA-damaging) metabolites. Murine Swiss 3T3 cells express
high levels of AhR. Treatment of Swiss 3T3 cells with B[a]P during the G1 phase
of the cell cycle resulted in growth arrest, as shown by inhibition of growth
factor-stimulated DNA synthesis. By contrast, other murine 3T3 fibroblasts not
expressing detectable levels of AhR did not undergo growth arrest in response to
B[a]P. The AhR antagonist alpha-naphthoflavone prevented B[a]P-induced growth
arrest, further demonstrating that cessation of cell growth was mediated by the
activated AhR. A nongenotoxic AhR ligand (2,3,7,8-tetrachlorodibenzo-p-dioxin)
did not elicit growth arrest, showing that ligand activation of the AhR alone
was insufficient to block cell cycle progression. However, genomic DNA from
B[a]P-treated Swiss 3T3 cells contained covalent adducts, whereas that from
2,3,7,8-tetrachlorodibenzo-p-dioxin-treated cells did not, showing that G1
arrest correlated with DNA damage resulting from genotoxic B[a]P metabolites.
B[a]P-induced DNA damage and growth arrest was coincident with elevated levels
of nuclear p53 protein and induction of the p53-regulated mdm-2 proto-oncogene.
However, Swiss 3T3 fibroblasts expressing "dominant negative" mutant p53, as
well as primary fibroblasts from p53-/- "knockout" mice, also underwent growth
arrest in response to B[a]P. Therefore, B[a]P-induced growth arrest occurs via
p53-independent mechanisms.

PMID: 9006915 [PubMed - indexed for MEDLINE]


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16: Geburtshilfe Frauenheilkd. 1996 Apr;56(4):177-83. 

[Value of immunohistochemical determination of receptors, tissue proteases,
tumor suppressor proteins and proliferation markers as prognostic indicators in
primary breast carcinoma]

[Article in German]

Gohring UJ, Scharl A, Ahr A.

Klinik und Poliklinik fur Frauenheilkunde und Geburtshilfe, Universitt Koln.

OBJECTIVE: We tested whether immunohistochemical detection of oestrogen and
progesterone receptor (ER, PR), the oestrogen-dependent protein pS2, the growth
hormone receptors p 185neu and EGF-R, the tumour suppressor protein p53, the
tissue proteases Cathepsin D and Urokinase, and the proliferation marker PCNA
are of prognostic relevance in breast cancer patients. METHODS: Expression of
the proteins listed above was evaluated in formalin-fixed and paraffin-embedded
sections of 311 primary breast cancer specimens using modified
Avidin-Biotin-Complex methods. Results were correlated to clinical and
morphological parameters (age, menopausal status, nodal status, tumour size,
tumour grade), and clinical course of disease (complete follow-up in 301 women,
median observation time 62 months) utilising univariate and multivariate
statistical analyses. RESULTS: If univariate analyses and multivariate
regression analyses according to the Cox-model were applied, only Cathepsin D
correlated to an elevated risk for recurrence in nodally negative patients (n =
135). In nodally positive women (n = 161), increasing tumour size, tumour grade,
lack of ER and PR, expression of p185neu, p53, and PCNA indicated a
significantly increased relative risk. CONCLUSIONS: Immunohistochemistry allows
the detection of parameters which may indicate prognosis in subgroup of breast
cancer patients.

PMID: 8682282 [PubMed - indexed for MEDLINE]


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17: Arch Gynecol Obstet. 1995;256(3):139-46. 

P53 protein in 204 patients with primary breast carcinoma--immunohistochemical
detection and clinical value as a prognostic factor.

Gohring UJ, Scharl A, Heckel C, Ahr A, Crombach G.

Department of Obstetrics and Gynecology, University of Cologne, FRG.

In a retrospective study, 204 formalin-fixed and paraffin-embedded biopsies of
primary breast carcinomas were tested immunohistochemically for the expression
of p53 protein (PAb 1801). 38% of the carcinomas were positive with respect to
p53. The expression of p53 correlated significantly with the loss of tumor
differentiation (P = 0.013), but not with menopausal status, patients' age,
tumor size, axillary lymph node involvement or hormone receptor status. The
influence of p53 expression on prognosis was evaluated in 197 patients (T1-4
N0-2 M0, median observation time 72 months). Detection of p53 protein was
associated with a significantly longer disease-free survival in node-positive
women (P = 0.03). However, p53 protein did not prove to be a prognostic factor
in node-negative patients. The results demonstrate the prognostic value of p53
expression in breast cancer which appears to be limited to patients with
node-positive tumors.

Publication Types:
    Case Reports

PMID: 7574906 [PubMed - indexed for MEDLINE]


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