Susceptibility to autoimmunity and B cell resistance to apoptosis in mice lacking androgen receptor in B cells - PubMed (original) (raw)

G-ARKO B cells are resistant to apoptosis. A, Bone marrow (BM) immature B cells from G-ARKO and B-ARKO mice were more resistant to apoptosis than cells from Wt littermates. Total BM cells were cultured in vitro for 14 d in MesenCult medium supplemented with IL-7 and then stained with fluorochrome-conjugated anti-B220, anti-IgM, and 7-AAD and analyzed by flow cytometry. Histograms show 7-AAD staining profiles of gated B220low IgMlow immature B cells. B, Total BM cells were cultured in vitro for 48 h in MesenCult medium supplemented with IL-7. The TUNEL assay was used to detect apoptotic cells, with these events gated on B220low. C and D, Caspase-3 activity was analyzed in B cells from Wt and G-ARKO BM cultures cultured in vitro for 14 d in MesenCult medium supplemented with IL-7, stained with fluorochrome-conjugated anti-B220 and anti-caspase-3, and then analyzed by FACS gating on B220low developing B cells. E, Caspase-8 analysis of immature B cells from Wt, G-ARKO, and B-ARKO BM cells cultured in vitro for 14 d in MesenCult medium containing IL-7. We analyzed levels of caspase-8 in BM B cell lysates by ELISA and used general caspase inhibitor Z-VAD-fmak to reduce caspase-8 activity. F, Distribution of Fas (CD95)-positive B220+lowcells in Wt and G-ARKO BM cells cultured in vitro for 14 d in MesenCult medium containing IL-7 and then stained with fluorochrome-conjugated anti-B220 and anti-CD95 (Fas). Flow cytometry analysis and Western blot revealed that BM B220low cells from G-ARKO, B-ARKO mice have less Fas-positive cells than Wt littermates. G, BrdU staining of B cells from G-ARKO and Wt littermates. G-ARKO and Wt littermates were injected ip with 1 mg of BrdU twice a day for 4 d. BM cells were stained with fluorochrome-conjugated anti-B220 and anti-BrdU and analyzed by FACS for B220low BrdU+ cells. H, The absolute number of BrdU-positive B220low cells was increased in G-ARKO (n = 9) and B-ARKO (n = 6) compared with Wt mice (n = 9). I, Proliferation analysis for bone marrow B cells from Wt and G-ARKO littermates in the presence of 20 n

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IL-7. J, Western blot analysis of NF-kB/p65 and Bcl2 from BM B cell lysates of Wt, G-ARKO, and B-ARKO mice littermates. K, BM B cell proliferation was measured by [3H]thymidine incorporation in the presence of 20 n

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IL-7 (see Materials and Methods). Thymidine incorporation is greater in immature B cells from G-ARKO (n = 5) and B-ARKO mice (n = 5) compared with Wt (n = 5). L, AR activity is knocked down by AR siRNA and restored by AR-cDNA. CV-1 cells were cotransfected with ARE4-Luc, pBabe-AR (or pBabe vector), and AR-siRNA using SuperFect. phRL-TK was cotransfected as the control for normalization. Total plasmid DNA amounts were balanced by empty vectors. After 24 h transfection, cells were treated with or without 10 n

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DHT. After 16–18 h incubation, cells were harvested for the luciferase reporter assay. Each Luc activity is presented relative to the transactivation observed in the absence of DHT and is the mean ±

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of four experiments. M, We plated BM cells infected with retroviruses harboring pBabe-AR, pSuperior-ARsiRNA, and vectors in methylcellulose-containing media supplemented with IL-7. Colonies containing more than 30 cells were counted after 14 d. AR knockdown in Wt BM cells by siRNA induces colony forming, and AR restoration in G-ARKO BM cells reduces colony forming. N, Western blot revealed AR restored by AR-cDNA in G-ARKO BM cells. Data are presented as mean ±

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*, P < 0.05; **, P < 0.01. PCNA, Proliferating cell nuclear antigen.