Bcl11b mutations identified in murine lymphomas increase the proliferation rate of hematopoietic progenitor cells

Abstract

Background: The telomeric region of mouse chromosome 12 has previously shown frequent allelic loss in murine lymphoma. The Bcl11b gene has been identified and suggested as a candidate tumor suppressor gene within this region. In this study, we aimed to elucidate whether Bcl11b is mutated in lymphomas with allelic loss, and whether the mutations we detected conferred any effect on cell proliferation and apoptosis.

Methods: Mouse lymphomas induced by 1,3-butadiene or 2',3'-dideoxycytidine were analysed for mutations in the Bcl11b gene using single strand conformation analysis and direct DNA sequencing. Effects on cell proliferation by the detected mutations were studied by expressing wild-type and mutant Bcl11b in the cytokine-dependent hematopoietic progenitor cell line FDC-P1, lacking endogenous Bcl11b expression.

Results: Missense and frameshift (FS) mutations were identified in 7 of 47 tumors (15%). Interestingly, all mutations were found between amino acids 778-844 which encode the three C-terminal DNA-binding zinc fingers. In FDC-P1 cells, wild-type Bcl11b suppressed cell proliferation, whereas the mutated versions (S778N, K828T, Y844C and FS823) enhanced proliferation several-fold.

Conclusion: The genetic alterations detected in this study suggest that the three C-terminal zinc fingers of Bcl11b are important for the DNA-binding. Cell proliferation was suppressed by overexpression of wild-type Bcl11b but enhanced by mutant Bcl11b, indicating that these mutations may be an important contributing factor to lymphomagenesis in a subset of tumors.

Figure 1

Overexpression of Bcl11b inhibits proliferation of the hematopoietic progenitor cell line FDC-P1 but has no effect on apoptosis. (A) Expression of Bcl11b in FDC-P1 and cells transfected with the mutated versions K828T, S778N, Y844C and FS823 was detected by Western blot and RT-PCR. In the Western blot analysis, the human cell line Jurkat was included as a positive control. The two bands correspond to different isoforms of Bcl11b. (B) Cell viability was determined by staining with propidium iodide followed by flow cytometric analysis. Data shown are the mean ± SD (n = 6). (C) Proliferation of non-transfected FDC-P1 cells and FDC-P1 cells transfected with wild-type Bcl11b or the mutated versions (K828T, S778N or Y844C) was measured by [³H]-TdR incorporation (cpm × 10^-3) after 72 hours of SCF-stimulation. Results are presented as mean ± SD (n = 3). FDCP1: 11.8 ± 1.7; wild-type Bcl11b: 4.4 ± 1.1; K828T: 37.5 ± 3.3; S778N: 19.3 ± 1.9; Y844C: 32.4 ± 0.9. (D) Cells were stained with CFSE and cultured for 72 hours in SCF, after which CFSE fluorescence was determined by flow cytometry. Histograms for the four different mutants K828T, S778N, Y844C and FS823 (K, S, Y and FS, respectively) are shown. CFSE fluorescence declines (shift to the left on the X-axis) as cell numbers increase. In each histogram, data from non-transfected FDC-P1 cells (F) and cells transfected with wild-type Bcl11b (wt) are overlaid for a comparison. Results are from one representative experiment of three performed.

Figure 2

Allelic loss of the C3H allele or the B6 allele on chromosome 12 in BLF and DLF. Two polymorphic sites (729A>G and 1386G>A) were found in Bcl11b, confirming previously detected allelic loss in D12Mit181 and D12Mit133 [17]. Markers analyzed are shown on the left side of the chromosome and the distance of the marker from the centromere is presented on the right side of the chromosome. Black square indicates loss of the C3H allele; white circle indicates loss of the B6 allele; absence of circle or square indicates retention of heterozygosity. CEN, centromere; TEL, telomere.