University of North Dakota School of Medicine, Grand Forks, ND and
1 National Institute of Environmental Health Sciences, Research Park, NC, USA
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Abstract |
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Abbreviations: HCC, hepatocellular carcinoma; LOH, loss of heterozygosity; NTP, National Toxicology Program; SSLPs, simple sequence length polymorphisms.
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Introduction |
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Epidemiological methods and conventional animal bioassays have significant limitations. Epidemiology is retrospective in nature and confounding factors reduce its sensitivity. Animal bioassays retain inherent uncertainties associated with extrapolating from inbred rodent strains to genetically diverse human populations. Therefore, the NTP continues to evaluate transgenic animal models for their usefulness in identifying human carcinogens. The p53-deficient mouse model (10) is currently under evaluation. Mice heterozygous for a null p53 allele [p53(+/)] were predicted to be selectively responsive to genotoxins in a shortened time frame since carcinogenesis is mediated by dysfunction of one wild-type Trp53. Benzene, p-cresidine and phenolphthalein were among the carcinogens selected for the first phase of the p53(+/) model evaluation because each agent was well characterized in conventional bioassays as a genotoxic, multi-site, trans-species carcinogen (5,7,8,11).
Many of the carcinogenic effects observed in conventional 2 year cancer bioassays were reproducible in the p53(+/) mice within 6 months of exposure. In the conventional 2 year bioassay for carcinogenesis increases in the incidences of bladder carcinoma were observed in both sexes of Fischer 344 rats and B6C3F1 mice after p-cresidine was administered in the feed for 104 weeks (7). In p53(+/) mice p-cresidine induced the same type of bladder tumors after only 24 weeks of feeding (12). In the 2 year phenolphthalein study treatment-related effects observed in B6C3F1 mice included malignant lymphoma of thymic origin (8,13). Thymic lymphomas were induced in p53(+/) mice after only 4 weeks of feeding phenolphthalein.
The carcinogenic effects of benzene produced in C57BL/6-p53(+/) mice did not parallel the effects observed in B6C3F1 mice. Strain-specific carcinogenic effects were observed. In a 2 year bioassay where benzene was administered by oral gavage, 344 rats and B6C3F1 mice developed dose-related Zymbal gland carcinoma. Lymphocytopenia was also observed in both rats and mice (6). The benzene-induced subcutaneous sarcomas detected in p53(+/) mice were not seen in a conventional 2 year study of benzene carcinogenesis. This strain-dependent effect was attributed to tissue-specific mechanisms involving p53 (12). In this context we have been investigating the mechanisms by which phenolphthalein, p-cresidine and benzene induce cancer in p53(+/) mice. A manuscript reporting the complete loss of one copy of chromosome 11 from phenolphthalein-induced thymic lymphomas is in preparation (J.E.Hulla, J.E.French and J.K.Dunnick, in preparation).
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Materials and methods |
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Primers
To selectively amplify the wild-type p53 allele, upstream priming was targeted to sequence located in the region of exon 5 deleted from the null allele. The upstream and downstream primer sequences were TGCCCTGTGCAGTTGTGGGTCA and ATTTCCTTCCACCCGGATAAGATG, respectively. Primers used to amplify a 386 bp fragment of the neo sequence were ATTGAACAAGATGGATTGCAC and GGTAGCCGGATCAAGCGTATG. The sets of primers used to amplify simple sequence length polymorphisms (SSLPs) were purchased from Research Genetics (Huntsville, AL).
Conditions
The final volume of each SSLP PCR reaction that used sarcoma- or bladder tumor-derived template DNA was 5 µl. The 5 µl reactions contained 6 ng template DNA, 0.15 U Taq polymerase, 0.132 µM each primer, 10 mM TrisHCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 and 0.001% gelatin. SSLP amplicons were radiolabeled with [-32P]dATP (6000 Ci/mmol, 0.130.25 µCi/5 µl reaction volume) in 5 µM non-radioactive dATP. The concentration of each of the other deoxynucleoside triphosphates was 200 µM (15). PCR amplification conditions for the analyses of DNA from the phenolphthalein-treated mice were essentially the same but scaled for a final volume of 20 µl (J.E.Hulla, J.E.French and J.K.Dunnick, in preparation). Reactions were thermal cycled: one cycle at 96°C for 3 min, 38 cycles at 92°C for 30 s, 55°C for 30 s, 72°C for 30 s, and one cycle at 72°C for 3 min. All amplifications included a negative control where sterile water was added in place of template DNA. Radiolabeled SSLP amplicons were separated under denaturing conditions on 12x16 inch, 6% polyacrylamide gels and recorded on film by autoradiography. All PCR reactions for the determination of SSLP allelotypes were run in duplicate.
The p53 amplicons were generated under slightly different conditions. An aliquot of 24 ng genomic DNA was used for each 20 µl reaction. Reactions were thermal cycled: one cycle at 96°C for 3 min, 42 cycles at 96°C for 30 s, 55°C for 30 s, 72°C for 45 s, and one cycle at 72°C for 3 min. The amplicons were electrophoretically separated on 2.0% agarose containing ethidium bromide. PCR reactions for the determination of allelotype at the p53 locus were run in duplicate for the lymphomas and sarcomas and in triplicate for the bladder tumors.
Determination of percent Trp53 loss, Trp53/pseudogene ratio and loss of heterozygosity (LOH)
Since background heterozygosity may be indicative of tissue-specific mechanisms, quantitative data for Trp53 loss are reported for the lymphomas, sarcomas and bladder tumors (Figure 2). Two parameters were determined. The percent loss was calculated from the Trp53 and pseudogene amplicon fluorescence measured from agarose gels containing ethidium bromide (Figure 2
). Measurements were made with NucleoVision software (NucleoTech Corp., Hayward, CA). Percent Trp53 loss was calculated for each tumor as [1 {Trp53/(Trp53 + pseudogene)}Tumor/{Trp53/(Trp53 + pseudogene)}Non-tumor]x100. The Trp53/pseudogene ratio was calculated by dividing the fluorescence associated with the top band by the fluorescence associated with the bottom band in each lane. The parameters for the lymphomas and sarcomas were single calculations from the gels shown. The averages reported for the bladder carcinomas were calculated from either duplicate or triplicate amplifications.
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Results |
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Figure 2 shows representative results from PCR amplifications that targeted Trp53 and the p53 processed pseudogene. All the DNA samples isolated from non-tumor tissues yielded a 260 bp amplicon from the single wild-type Trp53 allele and a 180 bp amplicon from the p53 pseudogene alleles (Figure 2AC
). DNA isolated from phenolphthalein-induced thymic lymphomas (Figure 2A
) yielded the pseudogene amplicon but only traces of the Trp53 amplicon. The low specific content of Trp53 template in the lymphomas suggested that wild-type Trp53 was lost during the early stages of lymphomagenesis (J.E.Hulla, J.E.French and J.K.Dunnick, in preparation).
Each of the sarcomas yielded a Trp53/pseudogene ratio that was much reduced from the ratio generated from the non-tumor tissue of the same animal (Figure 2B). Thus, the specific content of wild-type Trp53 present in the sarcomas was less than the specific content of wild-type Trp53 present in the non-tumor tissues. Trp53 was lost in all the sarcomas and lymphomas examined. The percent of Trp53 loss was greater in lymphomas than in sarcomas.
DNA isolated from p-cresidine-induced bladder carcinomas yielded both the Trp53 and pseudogene amplicons. However, loss of Trp53 was clearly distinguishable in bladder tumors from animals 500, 502 and 522 (Figure 2C). The average Trp53/pseudogene ratios generated from the lymphomas, sarcomas and bladder tumors were 0.02 ± 0.02 (n = 10), 0.09 ± 0.03 (n = 8) and 0.30 ± 0.15 (n = 8), respectively. The average ratios generated from the matched non-tumor tissues were 0.27 ± 0.10, 0.32 ± 0.06 and 0.41 ± 0.21, respectively. The average percent Trp53 losses for the lymphomas, sarcomas and bladder tumors were 85 ± 10, 64 ± 14 and 28 ± 29%, respectively. The differences in Trp53 specific content may reflect tissue-specific mechanisms or merely the relative difficulty in resecting the tumors from surrounding normal tissues.
LOH at SSLPs
A manuscript reporting the SSLP allelotypes from thymic lymphomas induced in p53(+/) mice by phenolphthalein is in preparation (J.E.Hulla, J.E.French and J.K.Dunnick, in preparation). Data generated from two of the phenolphthalein-treated mice, animals 928 and 931, are presented in Figure 3 for comparison with data from the benzene and p-cresidine studies. The non-tumor DNA isolated from phenolphthalein-treated mice harbored loci heterozygous for the C57BL/6 and 129Sv alleles (mouse 928 at D11MIT149, D11MIT60, D11MIT194 and D11MIT48; mouse 931 at D11MIT149, D11MIT313, D11MIT60 and D11MIT194), loci homozygous for the 129Sv allele (mouse 931 at D11MIT48) and loci homozygous for the C57BL/6 allele (mouse 928 at D11MIT313). Each of the 10 lymphomas examined showed loss of one allele at each of the 28 informative loci examined (see Figure 3
) (mouse 928 at D11MIT149, D11MIT60, D11MIT194 and D11MIT48; mouse 931 at D11MIT149, D11MIT313, D11MIT60 and D11MIT194). The SSLP LOH data were consistent with loss at the Trp53 locus. The data revealed that one complete copy of chromosome 11 was lost during lymphomagenesis. Duplication of chromosomes can occur during chemical-induced tumorigenesis. We did not attempt to determine the number of copies of chromosome 11 that remained in the tumors. However, duplication of the null p53 -harboring chromosome, if it occurred, is not inconsistent with the interpretation that the wild-type p53 allele-harboring chromosome was lost during tumorigenesis.
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The data from mice 581, 584 and 591 presented in Figure 3 are representative of results from the eight benzene-treated mice we examined. In each of the benzene-induced sarcomas LOH appeared on a background of heterozygosity and varying degrees of loss were apparent. As in the lymphomas, loss was not C57BL/6 or 129Sv specific in the sarcomas. For example, compared with the non-tumor tissue from mouse 591, the sarcoma specific content of the 129Sv allele at D11MIT48 was diminished. In the same sarcoma the specific content of the C57BL/6 allele at D11MIT313, D11MIT60 and D11MIT194 was diminished. Loss was apparent at each of the 23 informative SSLP loci examined in the benzene-induced sarcomas (Table II
). The SSLP data were consistent with selective loss of the wild-type allele at the Trp53 locus. The data showed that the Trp53-bearing copy of chromosome 11 was lost during sarcomagenesis.
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The allelotypes for each of the sarcomas and matched non-tumor tissues are summarized in Table II. Of the 368 alleles identified in non-tumor tissues 143 (39%) were 129Sv. This was higher than the expectation of <3% for a N5 generation. The overly high frequency was, in part, attributed to 129Sv linkage with the p53 selection marker (16). However, five of the eight benzene-treated mice harbored 129Sv homozygosity at one or more loci. Since the study animals were characterized as the offspring of inbred C57BL/6 females, the high frequency of 129Sv alleles and presence of 129Sv homozygosity were unexpected.
A Trp53 flanking region of C57BL/6 genetic character was lost from each sarcoma. The allelotypes at D11MIT260, D11MIT4, D11MIT368, D11MIT60, D11MIT219 and D11MIT194 showed that loss of a 10 cM region of C57BL/6 character was common among the sarcomas (Table II). Similarly, a region of C57BL/6 genetic character was lost from each of 10 lymphomas examined (J.E.Hulla, J.E.French and J.K.Dunnick, in preparation).
The allelotypes for the bladder carcinomas and matched non-tumor tissues are compiled in Table III. LOH was distinguishable only sporadically in the bladder tumors. Whether LOH in the bladder tumors from mice 505, 508 and 523 was a result of the loss of one complete chromosome or the loss of chromosome fragments was equivocal. LOH was distinguished at only two of 13, three of 18 and three of 11 informative loci, respectively (Figure 2
and Table III
). However, loss of a complete chromosome was likely in the bladder tumors from animals 502, 519 and 522. LOH was distinguishable in these tumors at 13 of 14, seven of 11 and nine of 10 informative loci, respectively (Figure 2
and Table III
). In mouse 500 LOH was distinguishable at Trp53 but there were only three other informative loci identified in this animal. LOH was not distinguishable at any of the 11 informative SSLP loci in mouse 516. Loss of Trp53 was 10 ± 7% in this mouse. These data suggest that loss of chromosome 11 did not occur in the bladder tumor from mouse 516.
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Although the patterns of C57BL/6 and 129Sv alleles were inconsistent with the breeding protocol, there was no evidence to suggest that the wild-type Trp53 in the study animals was not of maternal origin, as outline in Figure 1. Thus, we defined the spatial distribution of 129Sv and C57BL/6 alleles along chromosomes 11 for each sarcoma and matched non-tumor tissue. The allelotypes strongly suggest the loss of one complete copy of chromosome 11 from each of the eight sarcomas. We assumed a selection for cells lacking Trp53 occurred during sarcomagenesis. Since the wild-type p53 alleles were harbored on the maternal chromosomes, the maternal copies of chromosome 11 were lost. We deduced the allelotypes along each maternal chromosome 11 from allelotypes of the non-tumor tissues, which harbored maternal and paternal alleles, and tumors, which harbored only paternal alleles. The resultant arrangements of alleles are shown schematically in Figure 4
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Discussion |
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Recent evidence indicates that the Atm (a protein kinase), Chk2 (a protein kinase) and Trp53 (a transcription factor) genes are critical in maintaining genomic stability (19,20). In response to ionizing radiation Atm kinase (homologous to yeast Rad3/mec1 checkpoint kinase) phosphorylates p53 at Ser15 and Chk2 (a homolog of yeast Rad53/Cds1) phosphorylates p53 at Ser20, resulting in p53 stabilization, which is critical to transactivation, DNA binding and transcription. In the recognition and response to DNA damage p53 is critical to prolonged cell cycle arrest (DNA repair) or transient arrest during mitosis in maintaining the integrity of the mitotic spindle. There are at least 11 well-described phosphorylation sites on p53 required for oligerimization and tetramerization and p53 function. Evidence suggests that p53 half-life and different p53 functions are determined by the state of phosphorylation of the multiple sites.
p53 is critical in maintaining both normal somatic mitotic recombination [e.g. V(D)J in T cell receptors and immunoglobulin genes] (2124). Cumulative evidence also indicates the importance of p53 in maintaining normal rates of mitotic homologous recombination (2530). In summary, many lines of evidence indicate that p53 plays an important role in maintaining fidelity in homologous recombination and preventing non-homologous recombination.
We considered point mutations within wild-type Trp53 as an alternative mechanism active in the bladder tumors. A recent survey of bladder tumors that retained the wild-type p53 allele revealed no consistent mutations when wild-type Trp53 exons 58 were examined by SSCP [J.E.French, G.D.Lacks, C.Trempus, J.K.Dunnick, J.Foley, J.Mahler, R.R.Tice and R.W.Tennant (2000) Carcinogenesis, submitted for publication]. We also considered the possibility that inadvertent inclusion of non-tumor tissue from vascular epithelium or the margins of the resected tumors contributed non-tumor DNA template, increased background heterozygosity and reduced the signal-to-noise ratio, masking LOH at some loci. However, since LOH was detected at seven or more widely distributed SSLP loci in three of the eight bladder tumors examined, we believe there is good evidence that chromosome 11 was lost in these tumors. The absence of background heterozygosity in the lymphomas suggested that loss of Trp53 occurred prior to clonal expansion and/or efficient culling of DNA-damaged cells by the p53-dependent apoptotic pathway that is characteristic of the thymus.
We believe that loss of the wild-type Trp53 allele was the most significant molecular event in phenolphthalein-, benzene- and p-cresidine-induced tumorigenesis. However, in all of the lymphomas and sarcomas and in at least two bladder carcinomas examined loss of Trp53 was accompanied by loss of a flanking region of C57BL/6 character. Thus, C57BL/6-specific resistance and 129Sv-specific susceptibility cannot be dismissed from consideration. Likewise, since loss of one copy of a chromosome occurred in all of the lymphomas and sarcomas and at least three of the bladder tumors examined, a role for monoallelic expression cannot be specifically ruled out (J.E.Hulla, J.E.French and J.K.Dunnick, in preparation).
The heterozygous TSG-p53 mice used in the phenolphthalein, benzene and p-cresidine studies harbored a wild-type Trp53, a null Trp53 and a processed p53 pseudogene (10,31). The null allele is characterized by a deletion that encompasses part of intron 4 and exon 5 and harbors a selection construct containing neo. The original Trp53 null construct was introduced into 129Sv embryonic stem cells and the new p53-deficient mouse strain was introduced on a mixed 75% C57BL/6, 25% 129Sv genetic background (10).
Allelotype data for mice used in all three studies were inconsistent with the supplier's breeding protocol. The mice were reported to be the offspring of the fifth backcross (N5) of p53(/) males (N4)xinbred C57BL/6 females. In this regard, the maternally inherited chromosomes of the p53(+/) study animals should harbor only C57BL/6 alleles. However, 129Sv homozygosity was found in nine of 10 phenolphthalein study mice, five of eight benzene study mice and seven of eight p-cresidine study mice. Furthermore, the frequency of 129Sv polymorphism in the N5 generation should be 3% in the absence of linkage to a selection marker. Since 129Sv alleles were linked to the null p53 selection marker in the study mice, a somewhat higher frequency was expected. However, the overall frequencies of 129Sv in the non-tumor tissues were 50, 39 and 45% in the phenolphthalein, benzene and p-cresidine studies, respectively. Thus, the frequency of occurrence of 129Sv polymorphism and the 129Sv:129Sv allelotypes were inconsistent with the breeding protocol.
Did recombination occur? Homologous recombination in diploid tissue is restricted to specific lineages of lymphocytes undergoing either T cell receptor or immunoglobulin rearrangements during development (25,3234). However, if the breeding protocol was carried out as maintained, the only other possible explanation for the high frequency of 129Sv polymorphism, and the presence of 129Sv homozygosity, is homologous recombination during embryogenesis under p53 haploinsufficient conditions. Such recombination might result in the patterns of C57BL/6 and 129Sv alleles observed in the non-tumor tissues. Further investigation of this possible phenomenon is underway.
Although unexpected, the high frequency of occurrence of 129Sv:C57BL/6 heterozygosity led us to discover that loss of Trp53 and p53 function resulted from non-dysjunction and loss of one complete copy of chromosome 11, rather than intragenic or intrachromosomal deletion. The universality of chromosome 11 loss was striking. Loss was detected in each of 10 lymphomas and eight sarcomas examined. Although equivocal due to high levels of background heterozygosity, patterns of LOH suggested that loss of a copy of chromosome 11 also occurred in at least three of the eight p-cresidine-induced bladder carcinomas.
Loss of chromosome 11 was not tissue specific. Both mesothelium (lymphomas and sarcomas) and endothelium (bladder tumors) exhibited the phenomenon. Furthermore, the subcutaneous sarcomas induced in p53(+/) mice by benzene were not observed in benzene-treated B6C3F1 mice (12). The absence of sarcomas in benzene-treated B6C3F1 mice leads us to speculate that a p53 haploinsufficiency-related mechanism underlies loss of chromosome 11. In this regard, it is interesting that in spontaneous hepato cellular carcinoma (HCC) and HCC induced by chlordane, methylene chloride, tetrachloroethylene and vinyl carbamate in B6C3F1 mice loss of a whole chromosome was detected in only four of 142 HCC examined. Loss of chromosome 11 was not detected in any of the HCCs (35).
p53 haplo insufficiency-related rearrangement mechanisms might explain the inconsistency between breeding protocol and the allelotype data. Alternatively, a mix-up in the breeding or shipping may have occurred. It is important to determine when mix-ups have occurred because genetic background matters. p53(+/) mice on MV (36) and 129Sv backgrounds (37,38) were compared to p53-deficient mice on a mixed C57BL/6x129Sv background. Thymic lymphomas arose faster in the MV and 129Sv strains and tumor profiles varied.
Any strain-specific effects related to p53 haplo insufficiency need to be carefully assessed for positive or negative impacts on the C57BL/6-p53(+/) mouse as a model for human carcinogenesis. If, for example, the carcinogenicity of a test agent was found to be related to p53 haplo insufficiency-mediated segregation dysfunction, then the bioassay may have only succeeded in reaffirming p53 deficiency as a risk factor. Our data suggest that loss of a complete copy of chromosome 11 occurred in three different chemically induced tumor types. This heightens our interest in determining if p53 haplo insufficient-specific mechanisms are involved.
Evaluation of the p53(+/) mouse must determine how much of a dominant human pathway needs be replicated before a short-term mouse model is valid and useful. The integration of tumor allelotype data with stochastic modeling of tumorigenesis in humans and p53(+/) mice will greatly facilitate this determination (39).
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Notes |
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Acknowledgments |
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References |
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