Laboratory of Environmental Carcinogenesis and Mutagenesis, F1-05 National Institutes of Health National Institute of Environmental Health Sciences P.O. Box 12233 Research Triangle Park, N.C. 27709
To the Editor:
I am writing to you with regard to the paper entitled, "Chromosome 11 Loss from Thymic Lymphomas Induced in Heterozygous Trp53 Mice by Phenolphthalein." This paper was published in Toxicological Sciences 60, 264270. The authors are J. E. Hulla, J. E. French, and J. K. Dunnick. In this paper we stated that, "The allelic patterns indicate that mitotic homologous recombination occurred during embryogenesis if breeding protocols were carried out as described." This letter is written to report that the study mice were not bred as we reported. The breeding protocol that appears as Fig. 1 of the paper is correct. However the TSG-p53TM mice were N4 and not N5 as stated in the paper.
The generation number is very significant in the context of our findings. N4 mice are generated from C57BL/6TacBR-[KO]p53 N4 intercrosses whereas each N5 mouse is the offspring of a C57BL/6TacBR-[KO]p53(-/-) N4 male and an inbred C57BL/6 p53 (+/+) female. All N5 study mice harbor a C57BL/6 chromosome 11 while both copies of chromosome 11 in N4 mice are mixtures of C57BL/6 and 129Sv genetic character. In our study, the loss of the wildtype p53-bearing maternal copy of chromosome 11 revealed the linkage of alleles along the paternal chromosome 11 that remained in each lymphoma. The linkage on the maternal chromosome was deduced from allelotypes determined from the mouse-matched normal tissues. The parental linkages were inconsistent with the breeding protocol for N5 mice. None of the ten study mice harbored the expected C57BL/6 chromosome. In each of the animals, both chromosomes harbored 129Sv and C57BL/6 alleles. As explanation we proposed that the p53 (+/-) mice were p53 haploinsufficient for suppression of mitotic recombination. While the linkages are inconsistent with N5 mice they are not inconsistent with N4 mice. Thus, there is no evidence for mitotic recombination.
Evidence for recombination related to p53 haploinsufficiency is reported in two other papers:
Misidentification of N4 mice as N5 mice impacts the Carcinogenesis paper. The impact is similar to what is discussed in the previous paragraph. The investigation reported by Boley et al. attributed patterns of LOH in lymphomas from p53 (+/-) mice to mitotic recombination related to p53 haploinsufficiency. This paper is brought to your attention for two reasons. First, the study mice were reported to be p53 (+/-) N5 mice and it now seems possible that the mice were N4. Second, there are methodological weaknesses. For example, examination of normal liver tissue from the tumor-bearing mice revealed C57BL/6 homozygosity at many loci. These loci should have been considered uninformative for LOH. However, the Boley et al. made the assumption that both a 129Sv allele and a C57BL/6 allele should reside at each locus and attributed the absence of a 129Sv allele in normal liver to common instability of repeat sequences. Thus, they reported the C57BL/6 homozygosity found in the animal's induced tumor as LOH in the face of evidence that the locus was uninformative for LOH. The loci that define the tumor LOH patterns purported to be evidence of mitotic recombination were those same loci found in the normal liver to be uninformative for LOH. The authors also assumed that a 129Sv allele and a C57BL/6 allele were present at each polymorphic locus in mice bearing spontaneous tumors. Spontaneous tumor loci at which only one allele was detected were reported as LOH without a determination from mouse-matched normal tissue that those loci were informative for LOH. The paper also reports duplication of the null p53 allele. This finding is unfounded because the determination was based on the false assumption that the p53 pseudogene is a single copy gene.
In summary, there is little or no evidence from the three investigations to support the contention that p53 (+/-) mice are haploinsufficient for suppression of mitotic recombination.