Mutagenesis in vivo in T cells of p21-deficient mice

Abstract

Mice that are deficient in p53 exhibit an early onset of multiple types of tumors, especially thymic lymphoma. However, it remains unclear to what extent each of the p53-regulated pathways exerts its tumor suppressor activity. p21^Cip1/Waf1, acting down stream of p53, is a major G1/S checkpoint protein that restricts cell cycle progression into S phase in the presence of DNA damage. While at old ages p21−/− mice have a higher incidence of many types of tumors than p21+/+ mice, they are more resistant to thymic lymphomagenesis. In this study, we characterized mutagenesis in vivo in T cells of p21-deficient mice, using loss of heterozygosity (LOH) at Aprt locus as an indicator. We found that the spontaneous Aprt mutant frequency in T cells of p21−/− mice is lower than that in p21+/+ mice. The mutational spectra, however, are similar, with mitotic recombination being the predominant pathway. In contrast to the remarkable induction of LOH events in T cells of p53−/− mice exposed to X-rays, LOH in T cells of p21−/− mice is not significantly induced by X-rays. Correspondingly, lymphoid cells of p21−/− mice are more sensitive to IR-induced apoptosis than those of p21+/+ mice, in contrast to the radioresistance of p53-deficient lymphocytes. Reduction in mutation load in T cell lineages may contribute to the suppression of thymic lymphomagenesis in p21−/− mice.

Introduction

The tumor suppressor p53 is a master regulator of the cellular responses to DNA damage. The activation of p53 up-regulates an array of genes that initiates cell cycle checkpoints, DNA repair and apoptosis [1]. While it has been firmly established that loss or mutation of p53 can lead to a broad spectrum of human malignancy, it remains controversial which of the p53-regulated pathways, or a combination of them, functions as a tumor-suppressing mechanism [2], [3]. Whereas there is an early onset of many types of tumors, particularly thymic lymphomas, in mice that lack p53, disruption in each of the p53-regulated pathways only has limited tumor-enhancing effect [3], [4], [5], [6], [7], [8]. For example, p53 mutant mice that are engineered to be deficient in induction of apoptosis, but not in other functions of p53, were found to escape the early onset of thymic lymphomas that characterize p53-null mice [3]. Moreover, the lymphomas and sarcomas that eventually developed in this particular strain of mice retained a diploid chromosome number, in sharp contrast to aneuploidy observed in tumors and cells from p53-null mice, suggesting that apoptosis is dispensable for the maintenance of chromosome euploidy in lymphoid cells. Also, knockout mice for Puma, a gene required for apoptosis, were shown to be relatively tumor free [7], [8].

Studies involving mice that lack Cdkn1a (p21), a protein that arrests cell cycle progression at G1/S checkpoint in the presence of DNA damage, showed that p21 has tumor suppressor activity in a tissue-dependent manner. While lack of p21 predisposes mice to tumor development in skin, colon, intestine, pituitary, thyroid, mammary gland, salivary gland, connective tissue and histiocystic sarcomas, p21-deficient mice are more resistant to thymic lymphomas [9], [10], [11]. For example, p21−/− mice are more protected than wild type mice from irradiation-induced lymphomas. The absence of p21 also significantly reduces the incidence of spontaneous and radiation-induced thymic lymphomas in p53−/− and p53+/− mice. Increased rate of apoptosis in thymic cells has been proposed to be responsible for the decreased lymphomagenesis [11].

Lack of p53 is associated with increased chromosome instability in vivo [12], [13]. Furthermore, chromosome instability is induced more prominently by IR in the absence of p53. For example, X-rays induce loss of heterozygosity (LOH) events more dramatically in T cells of p53−/− and p53+/− mice than in those of p53+/+ mice [14]. However, since p53 is involved in multiple cellular processes, it is unclear to what extent each of the p53-regulated pathways is responsible for the suppression of genetic instability.

In this study we addressed how a defective G1/S checkpoint contributes to mutagenesis in vivo by characterizing the somatic mutations that arise at Aprt locus in T cells of 129XC57F1 p21 null mice that are also Aprt heterozygous. In Aprt heterozygous mice, multiple pathways of LOH, including deletion, mitotic recombination and chromosome loss, can be evaluated [13], [15], [16]. The APRT-deficient mutant cells are recoverable by the virtue of their resistance to adenine analogs such as 2, 6-diaminopurine (DAP) and are designated as DAP^r.