Conditional inactivation of p53 in mature B cells promotes generation of nongerminal center-derived B-cell lymphomas

Abstract

The p53 tumor suppressor exerts a central role in protecting cells from oncogenic transformation. Accordingly, the p53 gene is mutated in a large number of human cancers. In mice, germ-line inactivation of p53 confers strong predisposition to development of different types of malignancies, but the early onset of thymic lymphomas in the majority of the animals prevents detailed studies of tumorigenesis in other tissues. Here, we use the Cre/Lox approach to inactivate p53 in mature B cells in mice (referred to as "CP" B cells) and find that such p53 inactivation results in the routine development of IgM-positive CP peripheral B-cell lymphomas. The CP lymphomas generally appear to arise, even in mice subjected to immunization protocols to activate germinal center reaction, from naive B cells that had not undergone immunoglobulin (Ig) heavy chain gene class switching or somatic hypermutation. In contrast to thymic lymphomas that arise in p53-deficient mice, which generally lack clonal translocations, nearly all analyzed CP B-cell tumors carried clonal translocations. However, in contrast to spontaneous translocations in other mouse B-cell tumor models, CP B-cell tumor translocations were not recurrent and did not involve Ig loci. Therefore, CP tumors might provide models for human lymphomas lacking Ig translocations, such as splenic marginal zone B-cell lymphoma or Waldenstrom macroglobulinemia. Our studies indicate that deletion of p53 is sufficient to trigger transformation of mature B cells and support the notion that p53 deficiency may allow accumulation of oncogenic translocations in B cells.

Fig. 1.

CP mice develop B-cell lymphomas. (A) Southern blot analysis demonstrating deletion of p53 gene in mature B cells. A schematic map with the position of relevant restriction sites and probes used is shown at top. Position of the bands corresponding to the wild-type (wt), floxed (fl), and deleted (del) p53 alleles is indicated. Bc-day4, purified splenic B cells cultured for 4 d with anti-CD40/IL4; Bcells, purified splenic B cells; Tcells, purified splenic T cells. (B) Kaplan-Meier curve of the CP (n = 22) and control CH (n = 17) cohorts. Curves represent total survival. (C) (Left) Example of enlarged spleen frequently observed in CP mice succumbing to B-cell lymphomas; (Right) normal control spleen.

Fig. 2.

CP B-cell lymphomas are IgM+ and show features of MZL. (A) Representative histologic sections from indicated CP tumors, stained with H&E. (B) Representative FACS analysis on CP220 and CP301 tumors, using antibodies against B220 and IgM, Igκ, or Igλ, as indicated. (Left) Normal spleen sample. pLN, peripheral lymph nodes; Spl, total spleen.

Fig. 3.

CP B-cell tumors harbor clonal IgH and Igκ loci rearrangements, but are not somatically hypermutated. (A) Schematic of the IgH and Igκ loci showing restriction sites and probes used for Southern blot analyses. RI, EcoRI; HIII, HinDIII. (B) Southern blot analysis of DNA from CP tumors indicated on top demonstrating clonal rearrangements in the J_H (Upper) and Jκ (Lower) regions. (C) Southern blot analysis of DNA from indicated CP tumors with the Iμ probe, which detects rearrangements in the Sμ region. (B and C), probes and restriction enzyme used are indicated at the bottom of each panel. Position of the germ-line bands (gl) is shown. DNA from normal spleen was used as control. (D) Table summarizing the results of experiments to verify levels of SHM in DNA from CP tumors. The diagram on the top shows the region of the IgH locus used for PCR amplification and sequencing.

Fig. 4.

CP B-cell tumors harbor clonal, nonrecurrent translocations that do not involve Ig loci and c-myc. (A) SKY analysis of selected CP tumors. One representative metaphase is shown. The arrows indicate chromosomes involved in clonal translocations. Detailed view of these chromosomes is presented in the panels at the side, showing DAPI, spectral, and computer-classified staining for each chromosome. (B) FISH and chromosome paint analyses on CP tumors carrying chr 12 and chr 6 translocations. Sequential hybridization with the set of probes indicated on the left was performed. Only chromosomes involved in translocations are shown, with corresponding normal (n) counterparts. The whole metaphases are presented in Fig. S1. (C) Northern blot analysis on RNA from indicated CP tumors with probes specific for c-myc (Upper) or GAPDH (Lower) as loading control. RNA from normal spleen (norm spl) and from a pro-B-cell tumor with c-myc overexpression (+ control) is included for comparison.