Exon 1 disruption alters tissue-specific expression of mouse p53 and results in selective development of B cell lymphomas

Abstract

p53 is a tumor suppressor gene mutated in >50% of human cancers, while p53 deficiency in mice results in cancers and accelerated mortality. Thymic T cell lymphoma is the most common malignancy in p53-deficient mice, making it difficult to study the role of p53 in other malignancies. To overcome this limitation, we attempted to generate mice with a reversible p53 knockout (p53(rev/rev)) by inserting a floxed transcriptional stop into the first exon of p53, anticipating that this would allow tissue-specific Cre-mediated expression of p53. Contrary to expectations, functional p53 protein was expressed in the thymus and multiple other tissues of p53(rev/rev) mice in the absence of Cre, whereas B cells expressed p53 protein only in the presence of B cell-specific CD19-Cre. In the absence of Cre, 76% of p53(rev/rev) mice developed splenic marginal zone B cell lymphomas, indicating sensitivity of this B cell subset to transformation caused by p53 deficiency. 5'-RACE identified p53 mRNA transcribed from a novel start site utilized in thymocytes but not normal B cells or B cell lymphomas from p53(rev/rev) mice. The p53(rev/rev) mouse thus demonstrates an effect of p53 deficiency in development of splenic marginal zone lymphomas and provides a model for study of p53-deficient human B cell lymphomas.

Figure 1. Generation of p53^rev/rev mice.

A) Gene targeting strategy and restriction map of the p53 gene. Filled boxes indicate exons; labeled boxes indicate neomycin resistance or herpesvirus thymidine kinase (tk) genes; and arrows indicate loxP sites. B) Southern blot analysis of ES cell DNA. The 5 kb band represents the germ line allele and 7 kb band represents the targeted allele after Kpn I digestion and hybridization with probe A. C) PCR analysis for the p53^rev/rev mouse genotype. The 300 and 500-bp PCR products represent the wt and gene-targeted alleles, respectively.

Figure 2. Expression of p53 in lymphocytes of p53^rev/rev mice.

A) Western blot analysis was used to determine p53 protein expression in thymocytes of wt and p53^rev/rev mice as indicated. Actin protein expression was used as a loading control. The results shown are representative of four independent experiments. B) Western blot analysis was used to determine p53 protein expression in spleen cells of wt and p53^rev/rev mice as indicated. Actin protein expression was used as a loading control. The results shown are representative of four independent experiments. C) Western blot analysis was used to determine p53 protein expression in total spleen cells (SP), purified splenic B cells (B) and purified splenic T cells (T) of p53^+/+ and p53^rev/rev mice with (IR) or without (C) irradiation as indicated. Actin protein expression was used as a loading control. The results shown are representative of two independent experiments.

Figure 3. The expression of p53 mRNA and p53REV mRNA in thymocytes and splenocytes.

A) RT-PCR analysis for p53 mRNA expression in thymocytes, splenocytes, spleen B cells and spleen T cells of wt and p53^rev/rev mice as indicated. Actin was used as a loading control. The results shown are representative of three independent experiments. The primers are shown relative to their position in the p53 gene. The numbers at the bottom indicate the dilution of RNA templates. B) RT-PCR analysis for p53REV and neomycin mRNA expression in thymocytes, spleen cells, splenic T cells and splenic B cells of p53^rev/rev mice as indicated. Actin was used as a loading control. The results shown are representative of two independent experiments. The primers are shown relative to their positions in p53REV and neomycin genes. E4 is also the RACE start site. The numbers at the bottom indicate the dilution of RNA templates. Filled boxes indicate exons; labeled boxes indicate neomycin resistance or herpesvirus thymidine kinase (tk) genes; and arrows indicate loxP sites.

Figure 4. Apoptosis of thymocytes and splenocytes in response to irradiation.

A) Apoptosis of littermate p53^−/− and p53^+/+ thymocytes after irradiation and overnight culture. The results shown are representative of three independent experiments. B) Apoptosis of littermate p53^rev/rev and wt thymocytes after irradiation and overnight culture. The results shown are representative of three independent experiments. C) Apoptosis of littermate p53^−/− and p53^+/+ spleen cells after irradiation and overnight culture. The results shown are representative of two independent experiments. D) Apoptosis of littermate p53^rev/rev and wt spleen cells after irradiation and overnight culture. The results shown are representative of two independent experiments.

Figure 5. B cell lymphomas developed in p53^rev/rev mice.

A) Survival of p53^rev/rev and control (p53^+/rev and p53^+/+) mice. B) Pie chart shows percentages of cause of death for p53^rev/rev mice. C, E, G, I) Structure of splenic marginal zone for normal B6, normal p53^rev/rev, and two individual tumor-bearing p53^rev/rev mice, respectively (H&E, 10×). D, F, H, J) High magnification of red pulp of and marginal zone for the spleen of a normal B6 mouse, normal p53^rev/rev, and lymphoma-affected spleens from two different p53^rev/rev mice, respectively (H&E, 63×).

Figure 6. FACS analysis of cell surface markers of B cell lymphomas of p53^rev/rev mouse.

A) Spleen cells of p53^+/+ mouse (upper row) and B cell lymphomas of p53^rev/rev mice (bottom row) were stained with B220, IgM, IgD, CD21, CD23, CD19 and CD5 antibodies, and analyzed by FACS. B) Peripheral blood lymphocytes were isolated from p53^+/rev and p53^rev/rev mice, stained with B220, CD19 and CD5 antibodies, and analyzed by FACS.

Figure 7. B cell lymphomas of p53^rev/rev mice did not express p53REV mRNA and p53 protein.

A) RT-PCR analysis for p53REV and neomycin mRNA expression in B cell lymphomas. Actin was used as control. The results shown are representative of two independent experiments. B) Western blot analysis of p53 protein expression in six independent B cell lymphomas with (IR) or without (C) irradiation as indicated. Actin protein expression was used as loading control.

Figure 8. B cell lymphomas do not develop in p53^rev/revCD19-Cre⁺ mice.

A) The B cells of p53^rev/revCD19-Cre⁺ and p53^rev/revCD19-Cre⁻ mice were isolated from splenocytes with MACS systems. The purified B cells were irradiated and cultured for three hours at 37°C. The lysates of B cells were immunoblotted with anti-p53 antibodies. B) B cell lymphoma incidence was determined in p53^rev/revCD19-Cre⁺ and p53^rev/revCD19-Cre⁻ mice followed for 9 months.