Loss of heterozygosity frequency at the Trp53 locus in p53-deficient (+/) mouse tumors is carcinogen-and tissue-dependent
John E. French1,4,
Gregory D. Lacks1,2,
Carol Trempus1,
June K. Dunnick1,
Julie Foley3,
Joel Mahler3,
Raymond R. Tice2 and
Raymond W. Tennant1
1 Laboratory of Environmental Mutagenesis and Carcinogenesis, and
2 Integrated Laboratory Systems, Research Triangle Park, NC 27709, USA and
3 Laboratory of Experimental Pathology, NIEHS
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Abstract
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Mutagenic carcinogens rapidly induced tumors in the p53 haploinsufficient mouse. Heterozygous p53-deficient (+/) mice were exposed to different mutagenic carcinogens to determine whether p53 loss of heterozygosity (LOH) was carcinogen-and tissue-dependent. For 26 weeks, C57BL/6 (N5) p53-deficient (+/) male or female mice were exposed to p-cresidine, benzene or phenolphthalein. Tumors were examined first for loss of the wild-type p53 allele. p-cresidine induced p53 LOH in three of 13 bladder tumors, whereas hepatocellular tumors showed p53 LOH in carcinomas (2/2), but not in adenomas (0/3). Benzene induced p53 LOH in 13 of 16 tumors examined. Finally, phenolphthalein induced p53 LOH in all tumors analyzed (21/21). Analysis of the p-cresidine-induced bladder tumors by cold single-strand conformation polymorphism (SSCP) analysis of exon 49 amplicons failed to demonstrate polymorphisms associated with mutations in tumors that retained the p53 wild-type allele. p-cresidine induced a dose-related increase in lacI mutations in bladder DNA. In summary, these data demonstrate that loss of the wild-type allele occurred frequently in thymic lymphomas and sarcomas, but less frequently in carcinomas of the urinary bladder. In the bladder carcinomas other mechanisms may be operational. These might include (i) other mechanisms of p53 inactivation, (ii) inactivating mutations occurring outside exons 49 or (iii) p53 haploinsufficiency creating a condition that favors other critical genetic events which drive bladder carcinogenesis, as evidenced by the significant decrease in tumor latency. Understanding the mechanisms of p53 LOH and chemical carcinogenesis in this genetically altered model could lead to better models for prospective identification and understanding of potential human carcinogens and the role of the p53 tumor suppressor gene in different pathways of chemical carcinogenesis.
Abbreviations: HCA, hepatocellular adenoma; HCC, hepatocellular carcinoma; LOH, loss of heterozygosity; SCC, squamous cell carcinoma; SSCP, single-strand conformation polymorphism; TCC, transitional cell carcinoma.
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Introduction
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A defined genetic alteration in mice that results in overexpression or inactivation of a gene intrinsic to carcinogenesis, which is insufficient alone for induction of neoplasia, may provide possible models for chemical carcinogen identification and evaluation. Such models may help to shorten tumor development times where chemically induced carcinogenesis occurs via pathways requiring mutation or alteration of additional critical genes (13). Mice with only a single wild-type p53 allele present a single target for mutagens and are analogous to humans at risk of heritable forms of cancer, e.g. the LiFraumeni syndrome (4). The reduction in p53 gene alleles by this `germline first hit' greatly increases the probability for either loss of p53 tumor suppressor function or gain of transforming activity by requiring (at a minimum) only a single mutation (57).
The p53 protein is critical to cell-cycle control, DNA repair and apoptosis (810) and is often mutated or lost in human and rodent tumors (5,6,10). The p53 protein is critical for the maintenance of normal somatic mitotic recombination [e.g. V(D)J in T-cell receptors and immunoglobulin genes] (1114). Cumulative evidence also indicates the importance of p53 in maintaining normal rates of mitotic homologous recombination and fidelity in homologous recombination and in preventing non-homologous recombination (1521). Ultimately, p53 is necessary for maintaining genomic stability and preventing accumulation of multiple genetic changes required for the development of neoplasia.
The C57BL/6 (N5)Trp53 (+/)-deficient and the
liz
:Trp53 (+/)-deficient F1 mice are viable and have a low background tumor incidence for up to 1012 months of age, while Trp53 (/) homozygous null allele mice have a higher rate of sporadic tumors at sites apparently determined by the genetic background of the strain (2224). The use of the Trp53 (+/)-deficient mice in rapid (26 week exposure) cancer bioassays should reduce the incidence of concurrent background tumors that confound interpretation of 2-year carcinogenesis studies. The inherited strain susceptibility, e.g. lymphohematopoietic tumors in C57BL/6 mice, may thus influence carcinogen-induced tumor outcome unless the carcinogen mechanism of action is highly tissue-specific (organotropic). Replication of the 2-year cancer bioassay in rapid studies using these mice is possible due to the decrease in tumor latency for chemically induced tumors during a period relatively free from strain-specific sporadic tumors (3). In addition, incorporation of the recoverable
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shuttle vector transgene allows identification of tissue-specific in vivo mutagens and mechanistic studies on genomic instability and p53 function.
This investigation was undertaken to test the hypotheses that loss of the wild-type p53 allele in Trp53 (+/)-deficient mice is carcinogen-and tissue-specific. Additionally, experiments were conducted to determine whether the in vivo mutation frequency rates of the
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(lacIq) neutral reporter gene can be used to predict chemical-and target tissue-specific tumorigenicity. Three mutagenic carcinogens, p-cresidine (CAS# 120-71-8), benzene (CAS# 71-43-2) and phenolphthalein (CAS# 77-09-8) were selected because, in long and short term bioassays, they were carcinogenic in both rats and mice (3) and were positive in the Salmonella or in vivo micronuclei mutagenesis assay.
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Materials and methods
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Mouse model
Trp53 (+/)-deficient male or female mice (C57BL/6:129-Trp53tm1Dol, N5; TSG-p53TM) or Big BlueTMlacIq: Trp53 (+/)-deficient F1 C57BL/6 male mice on a C57BL/6 background were obtained from Taconic (Germantown, NY) and were included in the study at 710 weeks of age. The mice were divided into groups of 2040 mice per treatment or control group as described previously (3,25,26) and exposed for 26 weeks employing the same route of exposure as used in the 2-year cancer bioassays. Food (NIH-07 diet, Ziegler Bros., Gardners, PA) and water were provided ad libitum. Sentinel animals were maintained under the same conditions as the core animals.
Husbandry
The animals were individually housed in polycarbonate cages with absorbent hardwood bedding (Beta-chips, Northeastern Products Corp., Warrensbury, NY). This were maintained at 71 ± 7°F and 50 ± 20% relative humidity with a 12 h lightdark cycle. Body weight and food consumption were recorded twice a week, and clinical signs were recorded weekly.
Pathology
A complete necropsy was performed on all animals killed by carbon dioxide narcosis. All tissues were preserved in 10% neutral buffered formalin for 1624 h, and then transferred to 70% ethanol. Tissues were processed for histopathology within 72 h of necropsy and residual tissues were returned to formalin for storage. Major organs/tissues, target tissues identified in previous 2-year cancer bioassays and gross lesions were trimmed, embedded in paraffin, sectioned and stained with hematoxylin and eosin as described previously (3). All masses >4 mm3 were trimmed, dissected into 5 mm3 blocks when possible and flash frozen or prepared for histological examination as described. Histological examination was performed on all target tissues and gross lesions in all animals from control and exposed groups. Fisher's exact test was used for the analysis of tumor incidence data.
p53 Immunohistochemistry
Four micron, unstained, paraffin-embedded serial sections of tumor or respective control tissue (SuperFrost Plus slides, Norcross, GA) were depleted of paraffin and rehydrated (graded series of ethanol:water). Endogenous peroxidases were blocked in 3% aqueous hydrogen peroxide. Sections were immersed briefly in 0.01 M citrate buffer, pH 6, for two 5 min cycles at ~700 W for antigen retrieval. Specimens were cooled to room temperature and blocked with 1% milk, 1% normal goat serum (Vector Laboratories, Burlingame, CA), 1% bovine serum albumin (Sigma Chemical Co., St Louis, MO) and 1x automation buffer (Biomeda, Foster City, CA). Tissues were incubated overnight in primary antibody (NCL-p53-CM5p, Novacastra Laboratories, Newcastle-upon-Tyne, UK) or rabbit IgG serum (Vector Laboratories) at a 1:500 dilution. Signal detection was accomplished with a StrAviGen Super Sensitive detection kit (BioGenex Laboratories, San Ramon, CA) using diaminobenzidine (Sigma Chemical Co.) as the substrate. Tissues were counterstained with Harris Hematoxylin (Sigma Chemical Co.). Specimens from a p53 transgenic mouse (mutation in codon 135 of Trp53) served as a positive control tissue.
Cyktokeratin/vimentin immunohistochemistry
Serial sections of tumor or respective control tissues were treated as described above. Cytokeratin expression was determined with a wide spectrum-screening antibody (rabbit anti-bovine; Dako Corp, Carpinteria, CA) at 1:1000. Vimentin was screened using rabbit anti-vimentin (a gift from Dr V.Lee, Pittsburgh, PA) at 1:800. The negative control for both antibodies was normal rabbit serum (Vector Laboratories) diluted to the same concentration as the primary antibodies. Signal detection was determined as described above; issues were counterstained with Harris Hematoxylin (Sigma Chemical Co.).
DNA isolation and loss of the wild-type p53 allele by Southern analysis
High molecular weight DNA was isolated from normal non-target tissue (internal control) or tumor tissue that was isolated immediately at necropsy, trimmed free of contiguous non-tumor tissue and flash frozen for no less than 2 min in liquid nitrogen and stored at 80°C. When the frozen tissue was required to ascertain removal of normal tissue, it was cryosectioned, dehydrated and stained. Only frozen blocks free of normal tissue (except for blood vessels) were used for DNA isolation. Loss of the wild-type p53 allele was determined as described previously (1). Briefly, a 600 bp DNA probe spanning p53 exons 26 was prepared (LR10 vector, courtesy of L.Donehower), randomly labeled with [
-32P]dCTP and hybridized to BamHI-restricted normal or tumor genomic DNA after electrophoresis and transfer to a nylon membrane. A radiographic image was prepared by exposing the hybridized filter to Kodak film for 48 h at 80°C. Hybridization of the probe to the p53 pseudogene (10 kb), null allele (6.5 kb) and wild-type allele (5.5 kb) was observed. Signal intensity between the wild-type allele (1N) and the null allele (1N) formed the basis for interpretation of allele loss which was reaffirmed by image analysis of the 32P-labeled cDNA exon 26 p53 probe (600 bp) using a PhosphoImager and ImageQuant (Molecular Devices). Selection pressure for loss of the p53 null allele was considered to be less than for the wild-type p53 allele and, thus, loss of p53 signal was calculated based on the presumed approximate 1N DNA content of the null allele for the sample in question. Percent signal loss was calculated by subtracting the signal intensity of the wild-type allele from the null allele, dividing by the signal intensity of the null allele and multiplying by 100. Loss of signal intensity of
10% was considered biologically significant.
Cold single-strand conformational polymorphism (SSCP)
The non-radioactive procedure for optimized exon-specific SSCP analysis as described by Hongyo et al. (27) was used. Exon-specific PCR products (250320 bp) were prepared from normal, microdissected tumor tissue, and positive control tumor [missense mutations for each p53 exon (28)] genomic DNA using flanking intronexon sequences for exons 48 of the murine p53 tumor suppressor gene as described previously (29). Briefly, exon-specific PCR products (20100 ng DNA) were denatured using 1 M methylmercury hydroxide mixed with 15% (w/v) Ficoll 400 and loading buffer containing bromophenol blue and xylene, heating to 85°C and then rapid chilling in wet ice. This mixture was loaded onto a 20% TBE gel (39:1 acrylamide:bis-acrylamide cross-linking) and electrophoresed under exon-specific optimized conditions at 300 V in a recirculating electrophoresis chamber (Novex, San Diego, CA) until the bromophenol blue marker reached the bottom of the gel (12 h). Gels were stained with 0.5 µg/ml ethidium bromide (1x TBE) for at least 20 min and destained in distilled water for 5 min. Ethidium bromide-stained bands were visualized using a 340 nm UVR viewing box and photographed.
Neutral reporter gene mutagenesis
In vivo lacIq mutant frequency (mean±SEM; n = 6 mice/dose group) was determined using high molecular weight DNA from untreated and treated mice as described previously (30). Between 500 000 and 750 000 plaque forming units (p.f.u.) were assayed by
shuttle vector rescue.
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Results
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Microscopic pathology
Phenolphthalein
Malignant lymphomas of the thymus (Table I
and ref. 26) were observed as masses that, under a microscope, were seen to be composed of broad sheets of uniform pleomorphic to anaplastic lymphocytic cells which obliterated the normal thymic architecture. In most cases, neoplastic cells had extended through the thymic capsule and infiltrated the adjacent and intrathoracic tissues (mediastinal fat, mediastinal lymph node, heart and lungs). Although there was some variation in the cell type, most lymphomas seemed to be composed of larger and more lymphoblastic cells with little or sparse cytoplasm. Nuclei were round to oval, somewhat vesicular and contained one or more nucleoli. Mitotic figures were common in these neoplastic cells. No detectable p53 protein was observed in thymic lymphomas (0/15 examined) from treated Trp53 (+/)-deficient mice or normal thymuses (age matched) from untreated or treated controls (five of each were examined) (see Figure 1A and B
).
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Table I. Phenolphthalein (ppm in diet)-induced thymic lymphomas and the associated LOH at the p53 locus in all tumors examined from heterozygous Trp53 (+/)-deficient mice on a C57BL/6 background
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Fig. 1. Immunohistochemical analysis of p53 expression in tumors taken from heterozygous p53 (+/)-deficient mice exposed to phenolphthalein, benzene or p-cresidine. Tumors were removed from the host, trimmed free of fat and fixed overnight in 10% neutral buffered formalin (pH 7). Serial sections were analyzed for p53 expression (see Materials and methods). (A and B) Representative thymic lymphoma (phenolphthalein, 12 000 ppm) at 100 and 250x, respectively. (C and D) Representative benzene-induced sarcoma (NOS) at 100 and 250x, respectively. (E and F) Representative urinary bladder tumor at 100 and 250x, respectively. Note that p53 positive nuclei were observed only in the urinary bladder tumors (see arrows, E and F) and not in phenolphthalein-induced thymic lymphomas or benzene-induced sarcomas, which demonstrate loss of the wild-type p53 allele in the heterozygous p53-deficient mouse.
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Benzene
Tumors induced by benzene were observed as masses of subcutis around the head and neck region or thoracic cavity. When viewed under a microscope, these masses were determined to be sarcomas composed of markedly anaplastic cells ranging in overall appearance from fibrosarcomas to histiocytic sarcomas (16/39; Table II
) expressing vimentin (Figure 2
). A few thymic lymphomas were also observed (3/39; Table II
). No detectable p53 protein was observed in sarcomas or thymic lymphomas from treated Trp53 (+/)-deficient mice or from control tissue (0/15 tumors examined; see Figure 1C and D
).
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Table II. Benzene (200 mg/kg body weight/day)-induced sarcomas of the subcutis and normal lung (target tissue) and the associated LOH at the Trp53 locus in heterozygous p53 (+/)-deficient C57BL/6 mice
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Fig. 2. Origin of benzene-induced sarcomas, NOS of the subcutis. Tumors were removed from the host, trimmed free of fat and fixed overnight in 10% neutral buffered formalin (pH 7). Serial sections were analyzed for cytokeratin and vimentin expression (see Materials and methods). (A) Representative sarcoma, NOS demonstrating expression of vimentin. (B) Representative sarcoma, NOS demonstrating lack of expression of cytokeratins.
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p-Cresidine
Tumors induced by p-cresidine were observed as thickened bladder walls or large masses, which were frequently associated with hydronephrosis due to bladder occlusion. When viewed under a microscope, the carcinomas (24/24; Table III
) were invasive masses composed of markedly anaplastic cells and/or cells exhibiting squamous differentiation. A low incidence of hepatocellular tumors (5/23; Table III
) were observed grossly and microscopically under the conditions of the short-term p53-deficient mouse studies. Reproducibility of bladder tumor induction between three separate studies was very good using between 15 and 30 mice per dose group (25). Of 25 bladder carcinomas examined, heterogeneous patterns of p53-positive nuclei were observed in only three small transitional cell carcinomas (TCC) from p-cresidine-treated mice (Figure 1E and F
). The pattern (always heterogeneous) ranged from slighti.e. a few focal areas of p53 positive nucleito many, covering a wide portion of the tumor section. These tumor may represent developing TCC.
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Table III. p-Cresidine (2-methoxy-5-methylaniline; 0.5% diet)-induced hepatocellular and urinary bladder cancers and the associated LOH at the Trp53 locus in heterozygous p53 (+/)-deficient C57BL/6 mice
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Loss of the wild-type p53 allele
Loss of the wild-type allele was infrequent in p-cresidine-induced urinary bladder tumors (3/13; Figure 3A
and Table III
), whereas loss was complete (as measured by Southern analysis) in 2/2 hepatocellular carcinomas (HCCs), but not the hepatocellular adenomas (HCAs) (0/3) inTrp53 (+/)-deficient mice. In 10/10 bladder tumors the wild-type allele exhibited 3 ± 3.3% less signal intensity than the null allele. In three bladder tumors showing loss of heterozygosity (LOH), the loss of signal intensity ranged from 1089% (44% average signal loss) that of the null allele. In contrast, loss of the wild-type allele was significantly more frequent in benzene-induced sarcomas (12/15 examined; Figure 3B
and Table II
). Average p53 signal loss was 28 ± 7% in 12/15 sarcomas that of the null allele. The remaining three sarcomas showed less loss of signal (2 ± 2.8%). Signal loss for the p53 locus was complete (not detectable) in phenolphthalein-induced thymic lymphomas (21/21 tumors examined; Figure 3C
and Table I
). In untreated Trp53 (+/)-deficient mice, the null allele averaged 5% less than the wild-type allele signal intensity, suggesting a low frequency loss of the p53 null allele.

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Fig. 3. Representative loss of the wild-type allele p53 allele in carcinogen-exposed Trp53-deficient (+/) mice in rapid (26 week) cancer bioassays. (A) TCCs and SCCs of the urinary bladder and HCA and HCC observed after exposure to 0.5% p-cresidine (2-methoxy-5-methyl aniline) in the diet. Lane 1, #500, SCC; lane 2, #505, SCC; lane 3, #508, TCC; lane 4, #516, SCC; lane 5, #517, SCC; lane 6, #519, SCC; lane 7, #522, SCC; lane 8, #502, HCA; lane 9, #502, HCC; lane 10, #501, HCC. (B) Sarcomas, NOS (SaNOS) and pancreatic acinar carcinoma (PAC) observed after oral exposure (gavage, corn oil vehicle) to 100 mg benzene/kg BW. Lane 1. #564, SaNOS; lane 2, #591, SaNOS; lane 3, #572, SaNOS; lane 4, #548, SaNOS; lane 5, #565, SaNOS, lane 6, #584, SaNOS; lane 7, #587, SaNOS, lane 8, #557, PAC; lane 9, #590, SaNOS; lane 10, #592, SaNOS. (C) Thymic lymphomas observed after exposure to 75012 000 ppm phenolphthalein in the diet. Lane 1, #908, 750 ppm; lane 2, #915, 3000 ppm; lane 3, #919, 3000 ppm; lane 4, #920, 3000 ppm; lane 5, #921, 3000 ppm; lane 6, #924, 3000 ppm; lane 7, #926, 3000 ppm; lane 8, #928, 3000 ppm; lane 9, #929, 3000 ppm; lane 10, #931, 3000 ppm; lane 11, #932, 3000 ppm; lane 12, #, 3000 ppm; lane 13, #935, 3000 ppm; lane 14, #937, 3000 ppm; lane 15, #938, 3000 ppm; lane 16, #944, 12 000 ppm; lane 17, #948, 12 000 ppm; lane 18, #949, 12 000 ppm; lane 19, #953, 12 000 ppm; lane 20, #955, 12 000 ppm; lane 21, #957, 12 000 ppm. (D) Representative untreated heterozygous p53-deficient mice, homozygous null, and homozygous wild-type controls. Lane 1, het p53 def; lane 2, het p53 def; lane 3, het p53 def; lane 4, het p53 def; lane 5, het p53 def; lane 6, homozygous null; lane 7, homozygous wild-type.
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SSCP analysis
p-Cresidine-induced bladder tumors (
0.5 cm) present at the 26 week termination of the experiment were analyzed by cold SSCP (Figure 4
). No polymorphisms were observed in 24 TCCs or squamous cell carcinomas (SCCs) of the bladder in exons 48 of the p53 tumor suppressor gene. Figure 4
illustrates three different representative TCCs of the urothelium. The optimization of exon 5 was repeated under different conditions because only exon 5 may be amplified from the wild-type allele by using intronexon junctions to exclude those from the null allele and contaminating mRNA (1,22). Mutations arising in the Trp53 null allele would not be expressed and, thus, will have no effect on function.

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Fig. 4. Cold SSCP analysis of representative TCC of the urinary bladder induced after p-cresidine exposure for exon-specific p53 mutations in induced TCC in genetically altered lacI:p53(+/) F1 mice. (A) Exon 5: lane 1, positive control; lane 2, 500, TCC; lane 3, 505, TCC; lane 4, 508, TCC. (B) Exon 6: lane 1, positive control; lane 2, 500, TCC; lane 3, 505, TCC; lane 4, 508, TCC; lane 5, positive control; lane 6 positive control. (C) Exon 7: lane 1, positive control; lane 2, 500, TCC; lane 3, 505, TCC; lane 4, 508, TCC. Note the arrows that mark additional polymorphisms in the positive control (exons 5 and 6) or altered mobility (exon 7).
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In vivo lacIq mutant frequency
p-Cresidine and benzene, but not the mutagenic non-carcinogen p-anisidine (data not shown), induced a dose-related increase in lacI mutation frequency (30 days) that became exponential (180 days) in neoplastic tissue (Figure 5A and B
). The increase was 31-and 54-fold after 180 days for the low and high dose p-cresidine groups, respectively (Figure 5A
). Phenolphthalein-induced mutant frequencies in thymic tissue were not examined due to the complete loss of wild-type p53 allele and the lack of a dose-response threshold for induction of micronuclei.

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Fig. 5. In vivo lacIq mutant frequency (mean±SEM; n = 6 mice/dose group) is illustrated as an estimate of induced mutations in (A) urinary bladder (horizontal hatch), liver (solid) or skin (vertical hatch) of p-cresidine, or (B) bone marrow (vertical hatch) or spleen (solid) of lacIq:p53-deficient F1 C57BL/6 female mice (Stratagene and GenPharm International) after a 30 day exposure regimen to p-cresidine or benzene, respectively. Between 500 000 and 750 000 p.f.u. were assayed by shuttle vector arescue from high molecular weight normal or tumor tissue DNA.
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Discussion
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The p53 wild-type protein suppresses cancer in humans and mice. Protection against DNA damage via induction of growth arrest or apoptosis (810,31) is well described. Nullizygous p53 (/) are susceptible to rapid onset of cancer, whereas Trp53 (+/)-deficient mice remain free from cancer for up to 1012 months of age (23,24,32), unless exposed to mutagenic carcinogens (3,22,25,33,34). Recent evidence indicates that Atm (protein kinase), Chk2 (a protein kinase) and p53 (transcription factor) genes are critical for maintaining genomic stability (35,36). In response to ionizing radiation, Atm kinase (homologous to yeast Rad3/mec1 checkpoint kinase) phosphorylates p53 serine 15 and Chk2 (homolog of yeast Rad53/Cds1) phosphorylates p53 serine 20, resulting in p53 stabilization critical to transactivation, DNA binding and transcription. In recognition and response to DNA damage, p53 is critical to prolonged cell cycle arrest (DNA repair) or transient arrest during mitosis for maintaining integrity of the mitotic spindle. There are at least 1213 well-described phosphorylation sites on p53 required for oligerimization and tetramerization and p53 function (37). Evidence suggests that p53 half-life and different p53 functions are determined by the state of phosphorylation of multiple sites and, thus, may induce either gain or loss of p53 function through mutation.
We have reported here the outcome when Trp53 (+/)-deficient mice are exposed to different mutagenic carcinogens. Both benzene and phenolphthalein rapidly induced tumors that showed frequent or complete loss of the wild-type p53 allele in 26 weeks or less. Urinary bladder transitional cell or SCCs induced by exposure to the urinary bladder and liver carcinogen, p-cresidine, also rapidly appeared between 18 and 24 weeks, but only one out of four of the bladder tumors showed loss of the wild-type p53 allele. Too few liver tumors were observed (5/24) to determine the frequency of p53 LOH. Both HCCs observed showed p53 loss. Hepatocellular tumors also appeared around 24 weeks in C57BL/6 (N5) Trp53 (+/)-deficient mice. This represents a significant reduction in latency because bladder and liver tumors were observed at 65 weeks or later in a 2-year cancer bioassay with B6C3F1 mice (38). In spite of the infrequent loss of the wild-type p53 allele, no SSCPs indicative of point mutations were observed in the urinary bladder tumors without p53 LOH. However, LacIq mutant frequency was significantly increased in the bladder but not the liver, which demonstrated that exposure to p-cresidine was associated with bladder mutations and tissue-specific tumor induction. These observations also show the benefit of tissue-specific in vivo mutagenesis assays. Carcinogens, like the aromatic amines, may show highly specific tissue metabolism that reflects mutation specificity, and a mechanistic basis for tumor induction. Because the inactivating null allele mutation spans intron 4 and exon 5, only the intact exon 5 sequence from the wild-type allele (1,22) can be distinguished with certainty from the null allele using PCR methods. Small focal areas of the heterogeneous bladder tumors were nucleus-positive for p53 expression (Figure 1E and F
). DNA from these foci may have been lost in sampling or significantly diluted relative to the wild-type sequence present, which may have prevented observation of p53 mutations by cold SSCP analysis.
Benzene-induced sarcomas in C57BL/6 heterozygous Trp53 (+/)-deficient mice (14/39) developed within 26 weeks. These tumors were not observed until after 78 weeks in the 2-year cancer bioassay (39). Benzene-induced sarcomas demonstrated frequent loss of the wild-type allele consistent with its known aneugenic and clastogenic activity. Phenolphthalein induced thymic lymphomas in these mice (26), consistent with the 2-year cancer bioassay (40), and demonstrated a complete loss of the wild-type allele signal in all tumors examined. The potential to identify mutagenic carcinogens at maximum tolerated doses in a short period of time (
26 weeks exposure) with a potential mechanistic basis consistent with human cancer (p53 LOH) has been demonstrated.
Genomic stability appears to decrease with the loss and/or inactivation of both wild-type p53 alleles (4144), possibly because of the loss of the ability to arrest the cell cycle in order to allow sufficient time for DNA repair and/or induction of apoptosis (4145). Recent evidence suggests that LOH involving the Trp53 locus in carcinogen-induced neoplasia in p53 haploinsufficient mice may be due to illegitimate recombination (46,47). Genetic recombination occurs normally in germinal tissue (chiasmata of sister chromatids of meiosis) and bone marrow (immunoglobulin gene and T-cell receptor gene rearrangements at homologous sites on homologous chromosomes) (48). Aberrant recombination may occur at non-homologous sites within or between chromosomes due to endogenous or exogenous stressor-induced single-or double-stranded breaks and repair (49). Consequently, non-homologous chromosome translocations may result in fusion products between high level constitutive promoters (e.g. immunoglobulin genes) and protooncogenes (c-Abl, c-myc, Bcl-2, etc.) or LOH within a chromosome as a result of the formation of palindromic sequences due to single-stranded breaks or misrepaired double-stranded breaks. Ultimately, genomic instability results allowing clonal selection of preneoplastic cells to acquire multiple genetic changes, resulting in transformation and progression to malignancy.
Aging Trp53 (+/)-deficient mice demonstrate the same background tumor spectrum that arises later (>10 months) in Trp53 (/) nullizygous mice (36 months) (2224,32). Many of the aging heterozygous p53-deficient mice do not lose their wild-type allele or its function (43,44). Loss of function of the remaining wild-type allele in Trp53 (+/) mice may allow a decreased latency through an increased rate of progression to malignancy without altering tumor multiplicity or the tissue specificity of a chemical carcinogen (3). Carcinogens that induce tumors at common sites or at multiple sites in both rats and mice (or other species) in 2-year bioassays (trans-species carcinogens) may involve chemical interaction with highly conserved alleles involved in regulation of cell proliferation or differentiation (50). Mutagenic carcinogen interaction with highly conserved alleles, such as p53, in a genome may explain, at least partly, why Trp53 (+/) mice exhibit a shortened latency while retaining tissue specificity. Most known human carcinogens are trans-species carcinogens, mutagenic in Salmonella assays and possess structural alerts (51). Such chemicals represent the highest proximate risk to human health and the capability for identification of carcinogens other than through 2-year bioassays is an important goal.
In the studies reported here, mice with a single functional p53 allele exposed to three different mutagenic carcinogens demonstrated a dramatic reduction in latency and enhanced response, while retaining the carcinogen tissue specificity of wild-type mice. Both carcinogens examined (p-cresidine and benzene) also exhibited both linear and exponential increases in the in vivo lacIq mutant frequency in the target tissues. The infrequent loss of the wild-type allele and SSCP PCR products of the highly conserved exons of Trp53 suggests that mechanisms other than p53 were the basis for the rapid tumor induction. This is further illustrated by the fact that the dose-related increase in lacIq mutant frequency in the bladder demonstrated that p-cresidine was bioavailable in the bladder and its mutagenic activity was intact. Further studies are required to determine whether some other mechanism of p53 inactivation or inactivating mutations outside of the highly conserved regions are responsible for reduced tumor latency. However, both benzene and phenolphthalein are clastogens and free radical generators and, thus, may damage both DNA and the chromosome matrix. This mechanism is consistent with the frequent loss of the p53 wild-type allele in these mice and the carcinogens used in this study. Somatic tissue mitotic recombination is rare except in tumors (52,53) and it is in the thymus where pre-T lymphocytes undergo mitotic recombination of the germline T cell receptor gene sequences under RAG control. It is worth noting that rearranged T cell receptor clones that undergo apoptosis to prevent amplification of clones which recognize `self'. V(D)J recombination may be dependent upon p53 arrest for ligation of the rearranged double-stranded breaks that are induced (11,14,54). Complete loss of p53 function would severely limit repair to DNA damage and V(D)J recombination (11,5456) and could result in the rapid onset of neoplasia in the thymus, which undergoes normal mitotic V(D)J recombination in T cell development. However, normal V(D)J mitotic recombination was not required for the development of sporadically arising thymic lymphomas in mice nullizygous for Trp53 and Rag1 or Rag2 genes (57), although aberrant mitotic recombination not involving T-cell receptors in the thymus may have played a role.
These data demonstrate that loss of both p53 alleles occurred frequently in mesenchymal types of tumor of known etiology (thymic lymphomas and sarcomas), but less frequently in carcinomas of the urinary bladder. In the bladder carcinomas induced in Trp53 (+/) mice, either (i) other mechanisms of inactivation of p53 are operational, (ii) inactivating mutations are occurring outside exons 49 of the functional allele or (iii) p53 haploinsufficiency creates a condition that allows for growth selection advantage or a rapid accumulation of other critical genetic events that drive carcinogenesis as evidenced by the significant decrease in tumor latency. Further studies are underway to identify other critical genetic lesions that may drive urinary bladder carcinogenesis. Investigation of mechanisms of chemical carcinogenesis in this genetically altered model could lead to better models for prospective identification and understanding of human carcinogens and the role of the p53 tumor suppressor gene in chemical carcinogenesis. The universal nature of the p53 tumor suppressor gene and its cellular function and, thus, reduction of the p53 wild-type protein and the increased susceptibility to genotoxic stress, may allow detection of mutagenic carcinogens acting through mechanisms that do not require the loss of the remaining wild-type p53 allele. The potential to predict mutagenic risk for critical endogenous genes and determination and correlation of the mutational spectrum would provide a powerful tool for mechanism-based risk assessment and estimation.
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Notes
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4 To whom correspondence should be addressed Email: french{at}niehs.nih.gov 
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Acknowledgments
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We are grateful to Larry Donehower (Baylor College of Medicine, Houston, TX) for his valuable counsel and generous contribution of the LR10 p53 plasmid. We are also grateful to Roger Wiseman, Teddy Devereux and Jan Hulla (National Institute of Environmental Health Sciences, Research Triangle Park, NC) for their counsel and generous contribution of research materials.
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Received September 22, 2000;
revised November 10, 2000;
accepted November 13, 2000.