Apoptosis-stimulating protein of p53 (ASPP2) heterozygous mice are tumor-prone and have attenuated cellular damage-response thresholds

Abstract

The expression of ASPP2 (53BP2L), a proapoptotic member of a family of p53-binding proteins, is frequently suppressed in many human cancers. Accumulating evidence suggests that ASPP2 inhibits tumor growth; however, the mechanisms by which ASPP2 suppresses tumor formation remain to be clarified. To study this, we targeted the ASPP2 allele in a mouse by replacing exons 10-17 with a neoR gene. ASPP2(-/-) mice were not viable because of an early embryonic lethal event. Although ASPP2(+/-) mice appeared developmentally normal, they displayed an increased incidence of a variety of spontaneous tumors as they aged. Moreover, gamma-irradiated 6-week-old ASPP2(+/-) mice developed an increased incidence of high-grade T cell lymphomas of thymic origin compared with ASPP2(+/+) mice. Primary thymocytes derived from ASPP2(+/-) mice exhibited an attenuated apoptotic response to gamma-irradiation compared with ASPP2(+/+) thymocytes. Additionally, ASPP2(+/-) primary mouse embryonic fibroblasts demonstrated a defective G(0)/G(1) cell cycle checkpoint after gamma-irradiation. Our results demonstrate that ASPP2 is a haploinsufficient tumor suppressor and, importantly, open new avenues for investigation into the mechanisms by which disruption of ASPP2 pathways could play a role in tumorigenesis and response to therapy.

Fig. 1.

Generation of ASPP2^+/− mice. (A) Schema of ASPP2 allele and targeting vector. Black boxes indicate coding exons; white boxes indicate untranslated regions. Neo indicates neomycin resistance gene; TK indicates thymidine kinase gene. (B) Strategies for detecting a wild-type ASPP2 allele (Upper) or integration of the targeting vector (Lower). Probe locations for Southern blot analysis are shown as gray bars. Open arrows indicate PCR set 1 primers. Neo-PCR indicates Neo^R gene amplicon. WT-PCR indicates ASPP2 exon 13 amplicon. (C) Genotyping using PCR (Upper) and Southern blot analysis (Lower). (D) Western blots on equivalent amounts of total protein from ASPP2^+/− and ASPP2^+/+ MEFs (Upper), or thymuses and livers (Lower), using anti-ASPP2 Ab1 or anti-ASPP2 Ab2. Fold-expression (relative to +/+) normalized to tubulin.

Fig. 2.

ASPP2^−/− mice are not viable. Indicated genotypes of newborn pups from ASPP2^+/− matings are shown. The expected Mendelian frequencies are indicated by black columns; observed ASPP2 genotypes are indicated by white columns.

Fig. 3.

ASPP2^+/− mice have an increased incidence of spontaneous tumors. (A) Kaplan–Meier tumor-free survival curves for ASPP2^+/+ and ASPP2^+/− mice (P = 0.011, log-rank test). (B) H&E-stained microscopic sections of representative tumors found in ASPP2^+/− mice and a graph of tumor type frequency between genotypes (P = n.s., Fisher exact test).

Fig. 4.

ASPP2^+/− mice have an increased incidence of γ-irradiation-induced lymphomas. (A) Kaplan–Meier lymphoma-free survival curves for ASPP2^+/+ and ASPP2^+/− mice after 6.0-Gy or 10.5-Gy (P = 0.024 or P = 0.045, log-rank test) total γ-irradiation delivered in divided weekly fractions starting at 6 weeks old. (B) Kaplan–Meier lymphoma-free survival curves for tet-o-MYC;EμSR-tTA mice (29) after conditional c-Myc expression in an ASPP2^+/+ or ASPP2^+/− background (P = 0.97, log-rank test).

Fig. 5.

High-grade thymic T cell lymphomas induced in ASPP2^+/− mice after γ-irradiation. (A) Thymic lymphoma (red arrow) in an ASPP2^+/− mouse. H&E-stained microscopic sections of lymphoma-infiltrated liver, kidney, and lung from this mouse. (B) Immunophenotyping of tumor cells in unfractionated bone marrow from this mouse. A total of 2% of normal myeloid progenitors coexpressed Mac-1 and Gr-1 (Upper Right). A total of 98% of the bone marrow was replaced by tumor cells expressing CD5 and CD8 (Lower), but not B220 (Upper Left), CD4, and Nk1.1 markers (Lower).

Fig. 6.

ASPP2^+/− thymocytes have an attenuated apoptotic response to γ-irradiation. Flow cytometry on ASPP2^+/+ or ASPP2^+/− primary thymocytes 4 h after 5-Gy γ-irradiation (right column) or 0-Gy γ-irradiation (left column). The percentage of cells in each quadrant (relative to total cells) is indicated. The fold-increase in apoptosis between 0-Gy and 5-Gy γ-irradiated thymocytes was determined by the increase in percentage of Annexin V-stained cells (lower right plus upper right quadrants).

Fig. 7.

ASPP2^+/− MEFs have an attenuated G₀/G₁ checkpoint. Cell cycle distribution shows an increase in G₀/G₁-arrested ASPP2^+/+ MEFs but not ASPP2^+/− MEFs 24 h after 5-Gy γ-irradiation (P = 0.0052 and P = 0.9, unpaired 2-tailed Student's t test, respectively). Mean values of triplicate experiments are shown with SDs.