* University of North Dakota School of Medicine, Grand Forks, ND; and
National Institute of Environmental Health Sciences, Research Park, North Carolina 27709
Received September 19, 2000; accepted November 20, 2000
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ABSTRACT |
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Key Words: allelotype; simple sequence length polymorphism; SSLP; carcinogenesis; thymus; lymphoma; phenolphthalein; recombination; LOH; chromosome loss..
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INTRODUCTION |
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To further explore the mechanisms of phenolphthalein-induced carcinogenesis and to assess the potential of p53-deficient mice to rapidly identify carcinogenic effects, heterozygous Trp53 mice on a C57BL/6 background were dosed with phenolphthalein (Dunnick et al., 1997). The p53-deficient mice were heterozygous for a null p53 allele (Donehower et al., 1992
). As in B6C3F1 mice, the thymus was a target organ in these mice. Twenty-one of 21 of the thymic lymphomas examined showed loss of wild-type Trp53 by Southern blot analyses, but the mechanism for loss was unclear. Among the other effects of phenolphthalein treatment, an increased incidence of micronucleated erythrocytes was observed. Micronucleated erythrocytes were observed at each experimental dose. Thus, a no-observable-adverse-effect-level was not estimated (Tice et al., 1998
). There was no evidence for ovarian tumors in the treated heterozygous Trp53 mice, suggesting the ovarian tumors observed in B6C3F1 mice arose through tumorigenic pathways different from those pathways present in the thymus. Alternatively, the mice expired before ovarian tumors became apparent. Lymphomagenic responses that took 2 years to develop in the conventional B6C3F1 mouse assay were evident after only 4 months in the p53-deficient mouse model.
Here we report the results from in-depth Trp53 genotyping and the results of a survey of simple sequence length polymorphisms (SSLP). The study compared DNA isolated from 10 phenolphthalein-induced thymic lymphomas from heterozygous Trp53 mice to DNA from nontumor tissues.
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MATERIALS AND METHODS |
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The TSG-p53TM mice used in the phenolphthalein study were the offspring of the fifth backcross (N5) of p53 (/) C57BL/6 males (N4) x inbred wild-type C57BL/6 females (Fig. 1). Thus, in the p53 (+/) study animals, the maternal chromosomes should have harbored only C57BL/6 alleles. The original p53-deficient strain was introduced as 75% C57BL/6: 25% 129Sv. Therefore, about 3% of the paternal inherited alleles in the N5 generation should be 129Sv, residual from the embryonic stem cells used to generate the strain.
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Conditions.
Final volume for PCR reactions was 20 µl. Each reaction contained 24 ng template DNA, 0.6 U of Taq 2000 polymerase (Stratagene), 200 µM each deoxynucleoside triphosphate, 0.132 µM each primer, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 0.001% gelatin. Reactions were thermal cycled: one cycle at 96°C for 3 min, 42 cycles at 96°C for 1 min, 55°C for 1 min, 72°C for 1 min, and one cycle at 72°C for 3 min. Amplicons generated from SSLP loci were radiolabeled for autoradiography by reducing the cold dATP to 5 µM and adding 0.5 to 1.0 µCi of [-32P]dATP, 6000 Ci/mmole per 20 µl reaction volume (Weber, 1989
). All amplifications included a negative control where sterile water was added in place of template DNA. P53 amplicons were electrophoretically separated on 2.0% agarose containing ethidium bromide. Radiolabeled amplicons were separated under denaturing conditions on 12 x 16 inch, 6% polyacrylamide gels.
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RESULTS |
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Loss of heterozygosity (LOH) occurred in the 10 phenolphthalein-induced lymphomas at each of 28 informative chromosome 11 loci. Patterns of LOH were apparent. At loci between 0 centiMorgans (cM) and 37.0 cM, each tumor DNA alternated between regions of 129Sv loss and C57BL/6 loss (Table 1 and Fig. 2
; D11MIT149). In contrast, consistent loss of the C57BL/6 allele was observed in the central region of chromosome 11, proximal to the p53 locus, and between 37.0 and 44.0 cM from the centromere (Table 1
and Fig. 2
; D11MIT4, D11MIT368, and D11MIT219). In tumors 218, 223, 026, 273, 278, 011, and 016, the C57BL/6 alleles were lost at informative loci distal to 47.0 cM. In contrast, tumors 255 and 001 retained C57BL/6 alleles in this region (Table 1
and Fig. 2
; D11MIT196 and D11MIT284).
Analyses of 21 loci on chromosomes other than 11 (D1MIT70, D3MIT169, D4MIT40, D4MIT77, D5MIT122, D6MIT86, D7MIT82, D8MIT45, D8MIT107, D9MIT194, D10MIT150, D12MIT136, D12MIT8, D12MIT158, D13MIT15, D14MIT36, D15MIT12, D16MIT20, D17MIT55, D18MIT101, and D19MIT31) did not reveal polymorphism or meaningful differences between tumor DNA and nontumor tissue DNA. A few simple intragenic repeat sequences, not known to be polymorphic, were screened for alterations. The simple repeats were targeted because they are located within cancer-associated genes. No difference between nontumor and tumor DNA was revealed by the analyses of repeat sequences within Brca1, Brca2, Gli2, Tnfr2, or Xrccl.
Figure 3 shows ethidium bromidestained PCR amplicons separated on 2.0% agarose. The amplicons were generated using wild-type Trp53specific primers and template DNA isolated from the phenolphthalein-induced thymic tumors, and nontumor tissue DNA. Reactions that used template DNA from nontumor tissues generated a 270 base pair amplicon from the wild-type Trp53 and a 190 base pair amplicon from the p53 processed pseudogene. Reactions using template DNA from the thymic lymphomas did not generate the wild-type amplicon. The pseudogene amplicon was an internal control in these reactions and confirmed that the lack of yield from the wild-type allele was due to a loss of that allele rather than inadequate amplification conditions. Although not shown, similar results were yielded from template DNA isolated from thymic tumors generated in the animals treated with 750 ppm phenolphthalein.
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DISCUSSION |
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Although unexpected, the C57BL/6:129Sv heterozygosity we detected facilitated the determination that one copy of chromosome 11 was lost from each of the 10 lymphomas. The common elements of this (maternal) chromosome that were lost included the wild-type p53 allele and a 3.0 cM flanking region of C57BL/6 character.
In consideration of the mechanism of lymphomagenesis, we speculate that cells that lost wild-type Trp53 escaped an apoptotic response elicited by phenolphthalein-induced DNA damage and that these cells expanded clonally. We extend this well-accepted mechanism and suggest a mechanism where the wild-type Trp53 was lost during tumorigenesis as a consequence of generalized genomic instability enhanced by p53 haploinsufficiency. Loss of heterozygosity at each of the 28 informative chromosome 11 loci examined showed that the wild-type p53 allele-harboring maternal chromosome 11 was lost during lymphomagenesis. Thus, the allelotypes found in the tumors defines the patterns of polymorphism on the paternal chromosomes. The paternal allelotypes, together with the heterozygous allelotypes of the nontumor tissues, allowed us to deduce the arrangements of the polymorphisms on maternal chromosomes (Fig. 4). If the breeding protocol was conducted as reported, then the maternal and paternal allelic patterns are best explained by mitotic recombination during embryogenesis. We attribute the unexpected rearrangements to generalized genomic instability in somatic tissues as a consequence of p53 haploinsufficiency. These finding parallel results from sarcomas and bladder tumors induced in benzene- and p-cresidine treated p53 (+/) mice (J. E. Hulla, J. E. French, and J. K. Dunnick, Carcinogenesis, 2000, in press).
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There was no evidence to suggest that the wild-type p53 alleles were not of maternal origin as outlined in Figure 1. Loss of the maternal chromosome was invariably selected as a consequence of harboring the wild-type Trp53. Because LOH occurred in the tumor, it is important to acknowledge that genomic imprinting effects might play a role in the mechanism of tumorigenesis. In this regard, it is noteworthy that chromosome 11 harbors imprinted genes. Mice with two maternal and no paternal copies of the proximal region of chromosome 11 showed differential fetal growth (Cattanach et al., 1996
). Two imprinted genes mapped to the proximal region of chromosome 11, Meg1Grb10 and U2af1rsl (Cattanach et al., 1998
; Miyoshi et al., 1998
). Nf1, for which there is evidence of monoallelic expression in humans (Stephens et al., 1992
), mapped to 46.06 cM, near Trp53. Nf1 is a positive regulator of Trp53 (Furlong et al., 1996
; Ginsberg et al., 1990
) and a negative regulator of ras (Bollag et al., 1996
). Loss of Nf1 may have a critical role in ras-induced cellular proliferation in the thymus. Again, we consider the loss of Trp53 to be most significant. Yet, a role for monoallelic expression could not specifically be ruled out.
Comparisons between the occurrence of spontaneous tumors in p53 (/) and (+/) mice on a C57BL/6x129Sv background and on a 129Sv background were previously reported (Donehower et al., 1995; Harvey et al., 1993
). Lymphomagenesis was most frequent in both strains and may not show strain specificity due to the rapid development of immune competence of the thymus. The Trp53-dependent requirement of the thymus for eliminating aberrant recombinants through programmed cell death might predispose the thymus for induction of a neoplasia (Lowe et al., 1993
). In this regard, lymphomagenesis was attributed to loss of Trp53 rather than to differences in genetic background. Therefore, the short time-to-tumor-development observed in the phenolphthalein study was most likely directly attributable to the loss of Trp53 and the clastogenic action of phenolphthalein rather than to genetic background.
The heterozygosity we observed on chromosome 11 should not be stable in the p53-deficient strain. The window of heterozygosity is expected to decrease to close proximity of the Trp53 locus with every C57BL/6 backcross as the differential chromosomal segment recedes (Silver, 1995). The study mice were reported to be N5 offspring of female inbred C57BL/6 mice and allelotyping data were inconsistent in this regard. This inconsistency was unresolved. However, similar inconsistency was found in the benzene and p-cresidine studies, therefore breeding errors seem unlikely. It is important to emphasize that recombination was detected in nontumor tissues in all three studies. One possible explanation is that heretofore undescribed embryonic recombination events, perhaps related to p53 haploinsufficiency, occurred. This possibility and the possibility of p53 haploinsufficiency-related environmental susceptibilities are currently under investigation.
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ACKNOWLEDGMENTS |
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NOTES |
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