Expression of constitutively nuclear cyclin D1 in murine lymphocytes induces B-cell lymphoma - PubMed (original) (raw)

Expression of constitutively nuclear cyclin D1 in murine lymphocytes induces B-cell lymphoma

A B Gladden et al. Oncogene. 2006.

Abstract

Mantle cell lymphoma (MCL) is a B-cell lymphoma characterized by overexpression of cyclin D1 due to the t(11;14) chromosomal translocation. While expression of cyclin D1 correlates with MCL development, expression of wild-type (WT) cyclin D1 transgene in murine lymphocytes is unable to drive B-cell lymphoma. As cyclin D1 mutants that are refractory to nuclear export display heighten oncogenicity in vitro compared with WT D1, we generated mice expressing FLAG-D1/T286A, a constitutively nuclear mutant, under the control of the immunoglobulin enhancer, Emu. D1/T286A transgenic mice universally develop a mature B-cell lymphoma. Expression of D1/T286A in B lymphocytes results in S phase entry in resting lymphocytes and increased apoptosis in spleens of young premalignant mice. Lymphoma onset correlates with perturbations in p53/MDM2/p19Arf expression and with BcL-2 overexpression suggesting that alterations in one or both of these pathways may contribute to lymphoma development. Our results describe a cyclin D1-driven model of B-cell lymphomagenesis and provide evidence that nuclear-retention of cyclin D1 is oncogenic in vivo.

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Figures

Figure 1

Figure 1. D1/T286A associates with CDK4 and p27 in transgenic splenocytes

a) Cyclin D1 direct Western of whole tissue protein lysates from either 6-week-old non-transgenic wild type (WT) or Eµ-D1/T286A (Eµ) mice. L.E. denotes longer exposure of the Western blot. b) Western analysis of protein lysates from NIH-3T3 cells (3T3), wild type spleens (WT), D1/T286A spleens (Eµ) or two human MCL cell lines performed with antibodies specific for β- Tubulin, human and mouse cyclin D1 (DCS6) or mouse D1 only (7213G). A non-specific background band recognized by the DCS6 antibody is noted by the (*). c) Western analysis with the indicated antibodies was performed on splenic extracts prepared from non-transgenic wild type (WT), D1/T286A (Eµ) mice, or NIH-3T3 fibroblasts. d) Protein lysates from (c) were precipitated using either a cyclin D1 antibody (D1), the M2 antibody (FLAG), or with normal rabbit serum (NRS). Proteins were separated by electrophoresis and probed with the indicated antibodies.

Figure 2

Figure 2. D1/T286A mice have a decreased lifespan due to the onset of B-cell lymphoma

a) Survival curve representing mean survival of either wild type (WT) (Green Line) or D1/T286A (Eµ) (Red Line) cohort over a 24-month period. b) Southern blot analysis of immunoglobulin heavy chain gene rearrangements in transgenic splenic tumors and available peripheral tumors from the identical mouse. Bold line represents the germline size of the IgH gene and (*) notes rearranged gene products. c) Representative histology of either wild type spleen (a, b, c) or a tumor burden D1/T286A spleen (d, e, f) stained with hematoxylin and eosin (a, d) with arrowheads denoting mitotic figures. Immunohistochemistry for either total cyclin D1 (b, e) or B220 (c, f). d) Higher magnification of D1/T286A tumor burden spleens stained with hematoxylin and eosin (A,B), for cyclin D1 (C) or B220 (D).

Figure 3

Figure 3. D1/T286A splenic B-cells display an altered surface immunoglobulin profile

a) Bone marrow cells from either non-transgenic wild type (WT) or D1/T286A (Eµ-TA) six-week old mice were analyzed by flow cytometry with antibodies specific for either B220 or surface IgM. Likewise total splenic lymphocytes from either wild type or Eµ-D1/T286A mice were stained with B220, surface IgD and surface IgM. B220 positive cells were gated on and surface IgM and IgD expression was analyzed. b) Splenic cells from six-week-old pre-malignant mice were stained with B220, surface IgM and IgD, B220 cells were gated on and surface immunoglobulin levels were analyzed. c) Cells from a tumor burden spleen (EµTA) or an age-matched mouse were stained with B220, surface IgM or IgD. B220 positive cells were gated on and surface immunoglobulin levels were analyzed by flow cytometry.

Figure 4

Figure 4. D1/T286A B-cells have a decreased dependence on mitogen stimulation

a) Purified splenic B-cells from six-week old wild type or D1/T286A mice were stained with propidium iodide and DNA content was determined by flow cytometry. b) Six-week-old wild type or D1/T286A mice were injected with BrdU 24 hours prior to sacrifice. BrdU incorporation was quantitated by ELISA from purified splenic B or thymic T-cells. c) Purified splenic B-cells from pre-malignant transgenic (Eµ) and age matched wild type (WT) mice were stimulated with the indicated concentrations of LPS for 48 hrs. Proliferation was determined by incorporation of 3H-Thymidine. d) Cells from a tumor burden spleen (Eµ) or an age-matched mouse were stained with propidium iodide and DNA content was determined by flow cytometry.

Figure 5

Figure 5. D1/T286A transgenic spleens display an increased proportion of apoptotic cells

Representative TUNEL analysis performed on a) non-transgenic spleens, b) age matched pre-malignant D1/T286A spleens, and c) tumors from malignant transgenic mice. d) Quantitation of TUNEL analysis.

Figure 6

Figure 6. The p53 pathway is targeted D1/T286A tumors

a) Direct western analysis was performed on protein lysates from either wild type spleen (WT) or D1/T286A (TA) tumors using antibodies specific for MDM2 (top panel) (*) denotes cross reacting band, p53 (middle panel) or p19ARF (bottom panel). b) Total protein lysates from wild type spleens or D1/T286A tumors were separated electrophoretically, transferred to nitrocellulose and probed with antibodies for either BcL-2 (top panel) or BcL-XL (bottom panel).

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References

    1. Adams JM, Harris AW, Pinkert CA, Corcoran LM, Alexander WS, Cory S, Palmiter RD, Brinster RL. Nature. 1985;318:533–538. - PubMed
    1. Akiyama N, Tsuruta H, Sasaki H, Sakamoto H, Hamaguchi M, Ohmura Y, Seto M, Ueda R, Hirai H, Yazaki Y, et al. Cancer Res. 1994;54:377–379. - PubMed
    1. Alt FW, Yancopoulos GD, Blackwell TK, Wood C, Thomas E, Boss M, Coffman R, Rosenberg N, Tonegawa S, Baltimore D. Embo J. 1984;3:1209–1219. - PMC - PubMed
    1. Alt JR, Cleveland JL, Hannink M, Diehl JA. Genes Dev. 2000;14:3102–3114. - PMC - PubMed
    1. Argatoff LH, Connors JM, Klasa RJ, Horsman DE, Gascoyne RD. Blood. 1997;89:2067–2078. - PubMed

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