Chromosome 11 Loss from Thymic Lymphomas Induced in Heterozygous Trp53 Mice by Phenolphthalein

Janis E. Hulla*,1, John E. French{dagger} and June K. Dunnick{dagger}

* University of North Dakota School of Medicine, Grand Forks, ND; and {dagger} National Institute of Environmental Health Sciences, Research Park, North Carolina 27709

Received September 19, 2000; accepted November 20, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
C57BL/6 p53 (+/–) N5 mice heterozygous for a null p53 allele were given phenolphthalein to learn more about mechanisms of carcinogenesis and to evaluate the p53-deficient mouse as a tool for identifying potential human carcinogens. DNA samples isolated from 10 phenolphthalein-induced thymic lymphomas were analyzed for loss of heterozygosity (LOH) at the Trp53 locus and simple sequence length polymorphic (SSLP) loci. The initial screening revealed remarkable results from only chromosome 11. Allelotyping at approximately five centiMorgan intervals, we found SSLP heterozygosity for C57BL/6 and 129Sv over much of chromosome 11. In the tumors, treatment-related LOH was apparent on chromosome 11 at each of the 28 informative loci examined. The strain-specific polymorphism lost from individual tumors allowed us to deduce the distribution of alleles along the length of the maternal and paternal chromosomes 11. The allelic patterns indicate that mitotic homologous recombination occurred during embryogenesis if breeding protocols were carried out as described. The mitotic recombination observed may be attributable to p53 haploinsufficiency for normal suppression of mitotic recombination.

Key Words: allelotype; simple sequence length polymorphism; SSLP; carcinogenesis; thymus; lymphoma; phenolphthalein; recombination; LOH; chromosome loss..


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phenolphthalein was identified as a multispecies and multisite carcinogen in a 2-year cancer bioassay (Dunnick and Hailey, 1996Go). However, there is no epidemiological evidence to suggest phenolphthalein is a human carcinogen. Phenolphthalein was negative in genotoxicity tests that used Salmonella typhimurium (Bonin, et al., 1981Go; Mortelmans, et al., 1986Go) and repair-deficient strains of Bacillus subtilis (Fujita et al., 1976Go; Kada et al., 1972Go). However, in B6C3F1 and Swiss CD-1 mice, phenolphthalein treatment was associated with increased incidence of micronucleated erythrocytes (Witt et al., 1995Go). Further, phenolphthalein was positive in in vitro assays for transforming activity (Tsutsui et al., 1997Go) and chromosomal aberrations (NTP, 1996Go). Because it was a widely used over-the-counter laxative with abuse potential and demonstrated in vivo genotoxicity in rodents, phenolphthalein was tested by the U. S. National Toxicology Program for carcinogenicity. In the initial chronic study, F344/N rats and B6C3F1 mice were fed phenolphthalein for 2 years. Treatment-related effects observed included histiocytic sarcoma and malignant lymphoma of thymic origin in male mice; histiocytic sarcoma, malignant lymphoma, malignant lymphoma of thymic origin, and benign sex-cord stromal tumors of the ovary in female mice; benign or malignant pheochromocytoma of male and female rats; and renal tubule adenoma and carcinoma in male rats (Dunnick and Hailey, 1996Go). Neither phenolphthalein nor its metabolites accumulated in the thymus or any of the 10 other tissues assayed from B6C3F1 mice or F344/N rats (Collins et al., 2000Go; Griffin et al., 1998Go). Because carcinogenic effects in rodents were reported, drug manufacturers reformulated their over-the-counter laxatives such that they do not now contain phenolphthalein (U.S. FDA, 1997).

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., 1997Go). The p53-deficient mice were heterozygous for a null p53 allele (Donehower et al., 1992Go). 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., 1998Go). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animal dose response.
TSG-p53TM mice, heterozygous for Trp53 (designated C57BL/6TacBR-[KO]p53 N5), were obtained from Taconic of Germantown, NY. Six groups of 20 female mice, 7–10 weeks of age, received 0, 200, 375, 750, 3000, or 12,000 ppm of phenolphthalein (CAS # 77-09-8), in their feed for up to 6 months. In the groups that received phenolphthalein, atypical hyperplasia and malignant lymphomas of thymic origin developed at a combined incidence of 5, 5, 25, 100, and 95%, respectively, as previously reported (Dunnick et al., 1997Go).

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. 1Go). 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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FIG. 1. The breeding protocol for production of the wild-type p53 (+/+) study mice and p53 (+/–) study mice heterozygous for the null allele is illustrated. N1, N2, N3, and N4 generations were produced by backcrossing C57BL/6 p53 (–/–) males homozygous for the null allele by inbred C57BL/6 females. As outlined, the maternal C57BL/6NTac animals were the sources of the wild-type p53 alleles in the p53 (+/–) N5 study mice.

 
Genomic DNA.
Genomic DNA from normal 129SvJ and C57BL/6J mice were generously provided by Dr. Norman R. Drinkwater of the McArdle Laboratory for Cancer Research, University of Wisconsin. All other DNA samples were from tissues from C57BL/6TacBR-[KO]p53 N5 study mice obtained at necropsy, flash-frozen in liquid nitrogen, and stored at –80°C. Genomic DNA was isolated from 10 thymic lymphomas from animals treated with 750 ppm (animal number 908 and 915), 3000 ppm (animal number 920, 924, 928, 931, and 938) and 12,000 ppm (animal number 948, 949, and 953) of phenolphthalein. DNA was also isolated from nontumor thymus and ear tissues from two sentinel mice (animal number 960 and 965) not treated with phenolphthalein but otherwise maintained the same as the dosed animals. The 10 tumor-derived DNA samples were paired with nontumor tissue DNA isolated from an ear or kidney of the same animal (Table 1Go).


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TABLE 1 Allelotypes of Nontumor Tissues from 2 Sentinel Mice and Thymic Lymphomas and Nontumor Tissues from 10 Phenolphthalein-treated Mice
 
Primers.
For PCR reactions specifically targeting wild-type Trp53, the upstream and downstream priming sequences were TGCCCTGTGCAGTTGTGGGTCA and ATTTCCTTCCACCCGGATAAGATG, respectively. The complementary sequence of the downstream primer is present in exon 6 of both the wild-type and null p53 alleles. The upstream priming sequence is located in the region of exon 5 deleted from the null allele and thus only primed amplification from the wild-type allele. Primers used to amplify the neo sequence harbored by the null allele were ATTGAACAAGATGGATTGCAC, located at base pair 1554, and GGTAGCCGGATCAAGCGTATG, located at base pair 1940. The sets of primers used to amplify SSLP were purchased from Research Genetics (Huntsville, AL).

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 [{alpha}-32P]dATP, 6000 Ci/mmole per 20 µl reaction volume (Weber, 1989Go). 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mice used in the study were heterozygous for C57BL/6 and 129Sv along much of chromosome 11. This was unexpected. Each scan shown in Figure 2Go is a compilation of autoradiographs generated from a single SSLP locus. Amplicons were separated on polyacrylamide gels. The amplicons were generated using template DNA isolated from C57BL/6J mice, 129SvJ mice, the phenolphthalein-induced thymic tumors, or paired nontumor tissues from the study mice. Each allele yielded multiple bands as a consequence of artifacts generated by Taq polymerase (Weber, 1989Go). The six loci shown were representative of assays from which the allelotypes reported in Table 1Go were determined. The nontumor tissues were heterozygous at most loci and yielded two distinct coamplicons. One coamplicon was identified as the C57BL/6 allele when it migrated with an amplicon generated from C57BL/6J DNA and when migration was consistent with the published length of the polymorphism harbored by the C57BL/6 mouse strain (Mouse Genome Informatics Resource, 1999Go). Likewise, the other coamplicon was identified as the 129Sv allele. The D11MIT211 and D11MIT120 loci were exceptional in that the amplicon presumed to be the 129 allele did not comigrate with amplicons generated from 129SvJ DNA. In this regard, we suggest that the 129Sv embryonic stem cells used to generate the p53-deficient mouse strain and the 129SvJ DNA were from different substrains (Donehower et al., 1992Go; Simpson et al., 1997Go).



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FIG. 2. Thymic lymphomas from p53 (+/–) mice treated with phenolphthalein showed loss of heterozygosity at each informative locus on chromosome 11. The autoradiographs are of [32P]dATP-labeled SSLP amplicons separated on 6% denaturing polyacrylamide gels. The amplification products were generated using genomic DNA isolated from wild-type 129SvJ (129) and C57BL/6J (B6) mice and from induced tumors (T) and matched nontumor tissues (N). As shown, the matched nontumor data are to the right of tumor data. At all informative loci in the region between 37.0 and 47.0 cM (D11MIT4, D11MIT368, and D11MIT219), the C57BL/6 allele was lost from the tumor. Either the C57BL/6 allele or the 129Sv allele was lost at flanking loci (D11MIT149, D11MIT196, and D11MIT284).

 
The frequency of 129Sv occurrence was approximately 50% of the loci examined. This contrasted with the expectation of about 3% 129Sv in the N5 generation. The higher than expected frequency was, in part, attributed to 129Sv linkage with Trp53, the selection marker (Donehower et al., 1992Go; Silver, 1995Go). However, the frequency of occurrence of 129Sv homozygosity was 19%. Because the study animals were characterized as the offspring of an inbred (> N20) C57BL/6 maternal animal, finding 129Sv homozygosity in nontumor tissues was inconsistent with the breeding protocol. Based on the regional distribution of informative loci, at least 25% of chromosome 11 from the C57BL/6TacBR-[KO]p53 N5 mice was heterozygous.

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 1Go and Fig. 2Go; 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 1Go and Fig. 2Go; 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 1Go and Fig. 2Go; 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 3Go shows ethidium bromide–stained PCR amplicons separated on 2.0% agarose. The amplicons were generated using wild-type Trp53–specific 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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FIG. 3. The region of exons 5–6 of wild-type Trp53 was lost during phenolphthalein-induced lymphomagenesis. The photos show agarose electrophoresis of amplicons generated using primers specific for the wild-type p53 allele. Template DNA was isolated from thymic lymphomas (T) and nontumor tissues (N) of mice treated with 3,000 and 12,000 ppm phenolphthalein. Also shown are results for template DNA isolated from nontumor tissues of sentinel mice. DNA from the sentinels and DNA from the nontumor tissues of the treated animals generated amplicons from both the gene and the pseudogene. The DNA from tumors did not yield the longer gene amplicon. Thus, the wild-type p53 allele was lost from each of the phenolphthalein-induced tumors.

 
Unlike the wild-type allele, the null allele was not lost during phenolphthalein-induced tumorigenesis. Each of the tumor- and nontumor tissue- derived DNA samples generated a targeted 386 base pair amplicon from the null allele neo (not shown). The results were consistent with previous Southern analyses that showed loss of wild-type Trp53 and retention of the null allele in each of 21 phenolphthalein-induced tumors (Dunnick et al., 1997Go).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Heterozygous TSG-p53TM mice harbor a wild-type Trp53, a null Trp53, and a processed p53 pseudogene (Donehower et al., 1992Go; Zakut-Houri et al., 1983Go). The null allele is characterized by a deletion that encompasses part of intron 4 and exon 5 and it 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 (Donehower et al., 1992Go). The phenolphthalein study mice were reported to be on a C57BL/6 background (Fig. 1Go).

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. 4Go). 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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FIG. 4. The spatial arrangements of alleles along the chromosomes 11 from matched nontumor (N) and tumor (T) tissues are shown. Tumor allelotypes were consistent with loss of the maternal wild-type p53-bearing chromosomes. The maps of the maternal chromosomes were deduced from the homozygous allelotypes of the paternal chromosomes present in the tumor and the heterozygous allelotypes of the matched nontumor tissues. Wt indicates the wild-type p53 locus on the maternal chromosome.

 
A second but less significant common feature of the tumors was consistent loss of C57BL/6 genetic elements. C57BL/6 elements closely linked to the wild-type Trp53 in the nontumor tissue were absent from the lymphoma from the same animal. Thus, we considered 129Sv-specific cancer susceptibility and C57BL/6-specific resistance that map to this region as possibly having roles in tumorigenesis. Except for the teratoma susceptibility that mapped to chromosome 18, 129Sv mice have a relatively low incidence of spontaneous tumors (Mouse Genome Informatics Resource, 1999Go). With regard to C57BL/6-specific resistance, it is interesting that C57BL/6 mice are less susceptible to the development of micronuclei than BALB/c mice following treatment with clastogenic base analogues and nucleosides (Sato, et al., 1993Go). The C57BL/6 strain also demonstrated resistance to colon, pulmonary, mammary, and skin tumors induced by dimethylhydrazine (Evans et al., 1974Go, 1977Go), dimethylbenzanthracene (Flaks, 1968Go), urethane (Bentvelzen et al., 1970Go) and methylcholanthrene (Andervont and Edgcomb, 1956Go) respectively. Although we feel loss of the wild-type Trp53 is most significant, a role for loss of C57BL/6specific resistance could not strictly be ruled out.

There was no evidence to suggest that the wild-type p53 alleles were not of maternal origin as outlined in Figure 1Go. 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., 1996Go). Two imprinted genes mapped to the proximal region of chromosome 11, Meg1Grb10 and U2af1rsl (Cattanach et al., 1998Go; Miyoshi et al., 1998Go). Nf1, for which there is evidence of monoallelic expression in humans (Stephens et al., 1992Go), mapped to 46.06 cM, near Trp53. Nf1 is a positive regulator of Trp53 (Furlong et al., 1996Go; Ginsberg et al., 1990Go) and a negative regulator of ras (Bollag et al., 1996Go). 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., 1995Go; Harvey et al., 1993Go). 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., 1993Go). 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, 1995Go). 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.


    ACKNOWLEDGMENTS
 
We acknowledge the excellent technical support of Mary Kovarik, Sherry Levandowski, Tanya Schill, Jamie Hanrahan, Isaac Grindeland, Sam Rodriguez, Elena Rodgers, and Robin Adams-Hays at the University of North Dakota School of Medicine and Health Sciences. Expert review and advice from Drs. Norman Drinkwater, University of Wisconsin, and Roger Wiseman, the National Institute of Environmental Health Sciences, were invaluable. This project was funded through NIH grant 5-P20RR11817-03 and the NIEHS Division of Intramural Research (ES21207-05).


    NOTES
 
1 To whom correspondence should be addressed at National Institute of Environmental Health Sciences, National Institutes of Health, 111 T. W. Alexander Drive, Box 12233, F1-05, Research Triangle Park, NC, 27709. Fax: (919) 541-1460. E-mail: hulla{at}niehs.nih.gov. Back


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