p27 deficiency cooperates with Bcl-2 but not Bax to promote T-cell lymphoma

Abstract

The effect of Bcl-2 on oncogenesis is complex and expression may either delay or accelerate oncogenesis. The pro-oncogenic activity is attributed to its well characterized anti-apoptotic function while the anti-oncogenic function has been attributed to its inhibition of cellular proliferation. Recent studies demonstrate that p27 may mediate the effects of Bcl-2 on cellular proliferation. We hypothesized that p27 may suppress tumor formation by Bcl-2 family members. To test this hypothesis, cell cycle inhibition and lymphoma development were examined in Lck-Bcl-2 and Lck-Bax38/1 transgenic mice deficient in p27. Strikingly, p27 deficiency synergistically cooperates with Bcl-2 to increase T cell hyperplasia and development of spontaneous T cell lymphomas. Within 1 year, >90% of these mice had developed thymic T cell lymphomas. This high penetrance contrasts with a one year incidence of <5% of thymic lymphoma in Lck-Bcl-2 or p27 -/- mice alone. In contrast, p27 deficiency had no effect on tumor formation in Lck-Bax38/1 transgenic mice, another model of T cell lymphoma. Histologically the lymphomas in p27 -/- Lck-Bcl-2 mice are lymphoblastic and frequently involve multiple organs suggesting an aggressive phenotype. Interestingly, in mature splenic T cells, Bcl-2 largely retains its anti-proliferative function even in the absence of p27. T cells from p27 -/- Lck-Bcl-2 mice show delayed kinetics of CDK2 Thr-160 phosphorylation. This delay is associated with a delay in the up regulation of both Cyclin D2 and D3. These data demonstrate a complex relationship between the Bcl-2 family, cellular proliferation, and oncogenesis and demonstrate that p27 up-regulation is not singularly important in the proliferative delay observed in T cells expressing Bcl-2 family members. Nonetheless, the results indicate that p27 is a critical tumor suppressor in the context of Bcl-2 expression.

Figure 1. Lymphoid hyperplasia in p27 −/− Lck-Bcl-2 mice.

Spleens were dissected and T cells isolated from p27 −/− Lck-Bcl-2 transgenic mice and analyzed for viability and total splenic T cells as described in the Materials and Methods. Control groups included p27 −/−, p27 +/−, and p27 +/− Lck-Bcl-2 mice. A) Representative images of spleens from mice of the indicated genotypes. B) Total splenic T cells were determined by staining with an anti-CD3 antibody as described in the Materials and Methods. The Mean±SD of at least three mice is shown for the genotypes indicated. * P value <0.01 compared to all three other groups. C) Purified T cells from mice of the indicated genotypes were prepared and cultured as described in the Materials and Methods. Viability was determined over time by using the Guava Flow cytometer and Viacount reagent. The mean±SD for duplicate samples are shown.

Figure 2. Splenic T cell hyperplasia and T cell size in p27 −/− Lck-Bcl-2 mice.

A) Total splenic cells from mice of the indicated genotypes were isolated and stained with anti-B220-PE and anti-CD3-FITC antibodies as described in the Materials and Methods. The percentage of CD3-positive cells and the total number of splenic T cells (total # of splenic cells X %CD3) is shown. The data are representative of at least three mice from each genotype. B) The Mean Forward Scatter of CD3 positive splenic T cells was determined by staining with an anti-CD3 antibody as described in the Materials and Methods. The Mean±SD of at least 5 mice is shown for the genotypes indicated. * P<0.01 versus both the p27 +/− and p27 −/− mice using the unpaired Students T test. The p27 +/− Lck-Bcl-2 mice were not significantly different than the P27 −/− Lck-Bcl-2 mice (P = 0.89).

Figure 3. Thymocyte cellularity and cell cycle in p27 −/− Lck-Bcl-2 mice.

A) Total thymocyte cellularity from mice of the indicated genotypes (Mean +/− SEM) between 4 and 12 weeks of age is shown. B) DNA content analysis was performed on freshly isolated thymocytes on mice of the indicated genotypes between 4 and 6 weeks of age. The mean and SD of at least 3 mice per group is shown. * P<0.01 versus the control p27 +/−, p27+/− Lck-Bcl-2 and p27 −/−mice using the unpaired Students T test. C) DNA content analysis was performed on freshly isolated thymocytes on mice of the indicated genotypes between 7 and 12 weeks of age. The mean and SD of at least 3 mice per group is shown. # P<0.01 versus both the p27 +/− and p27 −/− mice using the unpaired Students T test. The p27 +/− Lck-Bcl-2 mice were not significantly different than the P27 −/− Lck-Bcl-2 mice in this age group (P = 0.94).

Figure 4. Bcl-2 retains anti-proliferative activity in p27 −/− mice.

Splenic T cells were isolated, purified, and activated with immobilized anti-CD3 antibody as described in the Materials and Methods. A) At the time points indicated, T cells were harvested and analyzed for cell cycle using PI staining. The %S/G2/M at each time point is shown. This experiment is representative of at least three independent experiments. B) Shown is the %S/G2/M (mean±SD) cell cycle analysis at 24 and 40–42 hours following activation from multiple independent experiments from mice of the indicated genotypes. The differences between the Lck-Bcl-2 samples (p27 +/− vs p27 −/−) were not significant (NS). * P<0.01 versus the control p27 +/− and p27 −/− mice using the unpaired Students T test. C) Proliferation of T cells of the indicated genotypes was determined by ³H-thymidine uptake. Values represent Mean±SD of quintuplet samples. All three groups are significantly different from one another with a P value of <0.01 using the Students T test.

Figure 5. Bcl-2 inhibition of Cdk2 phosphorylation is independent of p27.

T cells from mice of the indicated genotypes were purified and activated with immobilized anti-CD3 antibody prior to harvesting for immunoblot analysis at the time indicated. Immunoblots for total Cdk2, Phospho Thr-160, and Actin as a loading control are shown.

Figure 6. Bcl-2 delays the upregulation of Cyclins D2 and D3 independent of p27.

T cells from mice of the indicated genotypes were purified and activated with immobilized anti-CD3 antibody prior to harvesting for immunoblot analysis at the time indicated. Immunoblots for total Cyclin D1, Cyclin D2, Cyclin D3, and Actin as a loading control are shown.

Figure 7. Bcl-2 synergizes with p27 deficiency to rapidly promote lymphoma formation.

Mice of the indicated genotype were followed for thymic tumor free survival as described in the Materials and Methods. Kaplan-Meier lymphoma free survival analysis of mice of the indicated genotypes is shown. Lymphoma formation in p27 −/− Lck-Bcl-2 mice was significantly (P<0.01) accelerated compared to all three of the control groups (p27 +/−, p27 −/−, and p27 +/− Lck-Bcl-2). The other three groups were not significantly different from one another with regards to survival due to thymic tumors. As previously reported, a subset of p27 −/− mice did succumb to pituitary adenomas but these animals had no evidence of thymic lymphomas and were censored from this analysis of lymphoma formation (data not shown).

Figure 8. Tumors from p27 −/− Lck-Bcl-2 mice are aggressive T cell lymphoblastic lymphomas.

Histological analysis of the thoracic tumors from the p27 −/− Lck-Bcl-2 mice is shown as indicated. H&E staining shows a characteristic lymphoblastic lymphoma (Top left) that were frequently locally invasive (Heart) and involved distant organs such as the kidney and liver. Immunohistochemistry staining of the liver confirms the tumors are of T cell origin (Thy1 positive) and retain expression of human BCL-2.

Figure 9. p27 deficiency does not accelerate lymphoma formation in Lck-Bax38/1 mice.

A) Thymic cellularity from young mice of the indicated genotypes is shown. Data represent the Mean±SD of at least four animals from each group. In all cases, Lck-Bax38/1 mice had significantly reduced cellularity compared to the three transgene negative control groups shown on the left. *P<0.05 vs the respective p27 +/+ and p27 +/− controls. B) Kaplan-Meier analysis for lymphoma free survival comparing p27 −/− Lck-Bax38/1 (♦) and p27 +/− Lck-Bax38/1 (Δ) mice is shown.