Selection for loss of p53 function in T-cell lymphomagenesis is alleviated by Moloney murine leukemia virus infection in myc transgenic mice

Abstract

Thymic lymphomas induced by Moloney murine leukemia virus (MMLV) have provided many examples of oncogene activation, but the role of tumor suppressor pathways in these tumors is less clear. These tumors display little evidence of loss of heterozygosity, and MMLV is only weakly synergistic with the Trp53 null genotype, suggesting that viral lymphomagenesis involves mechanisms which do not require mutational loss of Trp53 function. To explore this relationship in greater depth, we infected CD2-myc transgenic mice with MMLV and examined the role of Trp53 in the genesis of these tumors. Most (19 of 27) of the tumors from MMLV-infected, CD2-myc Trp53(+/-) mice retained the wild-type Trp53 allele in vivo while tumors of uninfected CD2-myc Trp53(+/-) mice invariably showed allele loss from a significant fraction of primary tumor cells. The functional integrity of the Trp53 gene in these tumors was indicated by ongoing allele loss or selection for mutational stabilization during in vitro propagation and by the radiosensitivity of selected Trp53(+/-) tumor cell lines. An inverse correlation was noted between retention of the wild-type Trp53 allele and expression of p19(ARF), providing further evidence of negative-feedback control of the latter by p53. However, expression of p19(ARF) does not appear to be counterselected in the absence of p53, and its integrity in Trp53(+/-) tumors was indicated by its transcriptional upregulation on Trp53 wild-type allele loss in vitro in selected tumor cell lines. The role of MMLV was investigated further by analysis of proviral insertion sites in tumors of CD2-myc transgenic mice sorted for Trp53 genotype. A proportion of tumors showed insertions at Runx2, an oncogene which has been shown to collaborate independently with CD2-myc and with the Trp53 null genotype, and at a novel common integration site (ptl-1) on chromosome 8. Genotypic analysis of the panel of tumors suggested that neither of these integrations is functionally redundant with loss of p53, but it appears that the combination of the MMLV oncogenic program with the CD2-myc oncogene relegates p53 loss to a late step in tumor progression or in vitro culture. While the means by which these tumors preempt the p53 tumor suppressor response remains to be established, this study provides further evidence that irreversible inactivation of this pathway is not a prerequisite for tumor development in vivo.

FIG. 1

(A) MMLV infection accelerates the development of thymic lymphoma in Trp53^−/− CD2-myc mice. Tumor-free survival was significantly decreased in MMLV-infected Trp53^−/− CD2-myc mice compared to that in uninfected mice of the same genotype (P < 0.001). (B) Loss of p53 influences the latency of tumor development in infected CD2-myc mice. Trp53^−/− CD2-myc MMLV-infected mice develop tumors significantly faster than Trp53^+/− or Trp53^+/+ CD2-myc MMLV-infected mice (P < 0.001 for both).

FIG. 2

(A) Loss of the Trp53 wild-type allele (WT) is uncommon in MMLV-induced, Trp53^+/− CD2-myc primary tumors (T) and in kidney metastases (K) but is common in the resulting established cell lines (C). The pseudogene (ψ) and homologously recombined, knocked-out allele (KO) are indicated. Tumors judged not to have lost the wild-type allele were found to have wt Trp53 alleles with intensity of at least 90% of that of the knockout allele by Southern blot analysis. Loss of the Trp53 wild-type allele is evident in p/m63i cell line, tumor, and kidney DNA but retention of the Trp53 wild-type allele is seen in p/m48i, p/m51i, p/m61i, and p/m62i tumor and kidney DNA. (B) Apoptosis response to gamma irradiation in Trp53^+/− cell lines by flow cytometry. Induction of apoptosis, as identified by annexin V staining, was found in early passages (E) of p/m51i and p/m69i cell lines following irradiation (hatched bar). A greatly reduced apoptotic response to gamma irradiation was observed in later passages (L) of these cell lines, coinciding with loss of the Trp53 wt allele as shown by Southern blot analysis. Gamma-irradiated Trp53^−/− cells were included as a negative control, and dexamethasone-treated lymphoma cells were used as a positive control.

FIG. 3

Western analysis of p19^ARF. (A) Expression of p19^ARF is commonly down-regulated in MMLV-induced CD2-myc primary tumors from Trp53^+/− and Trp53^+/+ mice but not in those from Trp53^−/− littermates. In contrast, p19^ARF expression is strong in spontaneous primary tumors from Trp53^−/− and Trp53^+/− mice. It should be noted that the spontaneous tumors (p/m28, 30, 47, 48) from Trp53^+/− mice lost the Trp53 wt allele. Gels were routinely run in parallel and stained using Coomassie blue to ensure that there were equal amounts of protein in each lane on the Western blots. The presence or absence of the Trp53 wild-type allele is indicated in each case. (B) Repression of p19^ARF expression in the presence of p53. p19^ARF expression is repressed in early passages (E) of two cell lines which maintain functional p53, but the repression is not irreversible since on later passage (L), p53 is lost and p19^ARF expression is comparable to that of a control cell line (C) that lacks functional p53. Amounts of protein loaded are indicated by β-actin staining.

FIG. 4

Chromosomal localization of ptl-1. The locus was mapped to chromosome 8 with a logarithm of odds score of 13. ptl-1 mapped close to markers D8Mit6 and D8Mit228, 40 centimorgans from the centromere.