Limits...
Haematopoietic stem cell survival and transplantation efficacy is limited by the BH3-only proteins Bim and Bmf.

Labi V, Bertele D, Woess C, Tischner D, Bock FJ, Schwemmers S, Pahl HL, Geley S, Kunze M, Niemeyer CM, Villunger A, Erlacher M - EMBO Mol Med (2012)

Bottom Line: Thereby, we revealed a critical role for Bim and Bmf as regulators of HSPC dynamics both during early engraftment and long-term reconstitution.HSPCs derived from wild-type donors were readily displaced by Bim- or Bmf-deficient or Bcl-2-overexpressing HSPCs as early as 10 days after engraftment.Finally, we provide proof of principle that RNAi-based reduction of BIM or BMF, or overexpression of BCL-2 in human CD34(+) cord blood cells may be an attractive therapeutic option to increase stem cell survival and transplantation efficacy.

View Article: PubMed Central - PubMed

Affiliation: Division of Developmental Immunology, Biocenter, Innsbruck Medical University, Innsbruck, Austria. verena.labi@mdc-berlin.de

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Bim- and Puma-mediated LSK cell killing upon growth factor deprivationWt LSK cells were isolated from murine BM and cultured for 14 h in the presence or absence of SCF, TPO and Flt3L (100 ng/ml each). mRNA levels of the indicated BH3-only proteins, Bax and Bak as well as the anti-apoptotic Bcl-2 proteins were determined by RT-MLPA®. Blots show mRNA changes in the absence of cytokines when compared to mRNA levels in the presence of cytokines. Bars represent means of n = 5–6 from four independent experiments ± SEM. Significant p values: Bim p = 0.01; Puma p = 0.01; Bmf p = 0.01; Bak p = 0.01; Bcl-2 p = 0.04 (Mann–Whitney-test).LSK cells isolated from mice of the indicated genotypes were cultured for 48 h in the presence or absence of cytokines. Apoptosis was determined by combined staining with AnnexinV and 7-AAD, and specific apoptosis triggered by cytokine withdrawal was calculated by the following equation: (induced apoptosis − spontaneous apoptosis)/(100 – spontaneous apoptosis). Bars represent mean values of n = 3–4/genotype from three independent experiments ± SEM. Significant differences are indicated (Student t-test with Welch's correction).Eight- to ten-week-old mice of the indicated genotypes were sacrificed and BM was analysed by flow cytometry. Percentages of LSK as well as MPP and LT-HSC were determined by cell surface staining.Total numbers of LSK, MPP and LT-HSC were determined by multiplying BM cellularity (2 femurs) by the indicated percentages. Bars represent means from three to four mice/genotype from two independent experiments ± SEM. No significant differences were observed (Mann–Whitney-test).
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fig01: Bim- and Puma-mediated LSK cell killing upon growth factor deprivationWt LSK cells were isolated from murine BM and cultured for 14 h in the presence or absence of SCF, TPO and Flt3L (100 ng/ml each). mRNA levels of the indicated BH3-only proteins, Bax and Bak as well as the anti-apoptotic Bcl-2 proteins were determined by RT-MLPA®. Blots show mRNA changes in the absence of cytokines when compared to mRNA levels in the presence of cytokines. Bars represent means of n = 5–6 from four independent experiments ± SEM. Significant p values: Bim p = 0.01; Puma p = 0.01; Bmf p = 0.01; Bak p = 0.01; Bcl-2 p = 0.04 (Mann–Whitney-test).LSK cells isolated from mice of the indicated genotypes were cultured for 48 h in the presence or absence of cytokines. Apoptosis was determined by combined staining with AnnexinV and 7-AAD, and specific apoptosis triggered by cytokine withdrawal was calculated by the following equation: (induced apoptosis − spontaneous apoptosis)/(100 – spontaneous apoptosis). Bars represent mean values of n = 3–4/genotype from three independent experiments ± SEM. Significant differences are indicated (Student t-test with Welch's correction).Eight- to ten-week-old mice of the indicated genotypes were sacrificed and BM was analysed by flow cytometry. Percentages of LSK as well as MPP and LT-HSC were determined by cell surface staining.Total numbers of LSK, MPP and LT-HSC were determined by multiplying BM cellularity (2 femurs) by the indicated percentages. Bars represent means from three to four mice/genotype from two independent experiments ± SEM. No significant differences were observed (Mann–Whitney-test).

Mentions: To analyse which BH3-only proteins are induced in the absence of cytokine-mediated survival signals and may therefore mediate HSPC apoptosis, we isolated LSK cells from BM of wt mice and cultured them in the presence or absence of the cytokines TPO, Flt3L and SCF. Employing RT-MLPA® analysis allowed us to screen relative changes in the mRNA expression of all known Bcl-2 family members as well as different additional apoptosis-related genes (Eldering et al, 2003). We observed cytokine deprivation-induced changes in the mRNA levels of Bim (2.0-fold), Bmf (3.3-fold), Puma (2.3-fold) and Noxa (1.9-fold), but not Bad, Bid (Fig 1A) or Bik (not expressed). Induction of mRNA levels was independently confirmed by qRT-PCR (Supporting Information Fig 1A). No upregulation of Bax or Bak was observed whereas their pro-survival antagonists Bcl-2 and Bcl-xL were downregulated at the mRNA level (0.5-fold, both; Fig 1A and Supporting Information Fig 1A). The levels of Mcl-1 or A1 mRNA (Fig 1A) as well as of all remaining genes monitored were found to be largely unchanged (Supporting Information Table II).


Haematopoietic stem cell survival and transplantation efficacy is limited by the BH3-only proteins Bim and Bmf.

Labi V, Bertele D, Woess C, Tischner D, Bock FJ, Schwemmers S, Pahl HL, Geley S, Kunze M, Niemeyer CM, Villunger A, Erlacher M - EMBO Mol Med (2012)

Bim- and Puma-mediated LSK cell killing upon growth factor deprivationWt LSK cells were isolated from murine BM and cultured for 14 h in the presence or absence of SCF, TPO and Flt3L (100 ng/ml each). mRNA levels of the indicated BH3-only proteins, Bax and Bak as well as the anti-apoptotic Bcl-2 proteins were determined by RT-MLPA®. Blots show mRNA changes in the absence of cytokines when compared to mRNA levels in the presence of cytokines. Bars represent means of n = 5–6 from four independent experiments ± SEM. Significant p values: Bim p = 0.01; Puma p = 0.01; Bmf p = 0.01; Bak p = 0.01; Bcl-2 p = 0.04 (Mann–Whitney-test).LSK cells isolated from mice of the indicated genotypes were cultured for 48 h in the presence or absence of cytokines. Apoptosis was determined by combined staining with AnnexinV and 7-AAD, and specific apoptosis triggered by cytokine withdrawal was calculated by the following equation: (induced apoptosis − spontaneous apoptosis)/(100 – spontaneous apoptosis). Bars represent mean values of n = 3–4/genotype from three independent experiments ± SEM. Significant differences are indicated (Student t-test with Welch's correction).Eight- to ten-week-old mice of the indicated genotypes were sacrificed and BM was analysed by flow cytometry. Percentages of LSK as well as MPP and LT-HSC were determined by cell surface staining.Total numbers of LSK, MPP and LT-HSC were determined by multiplying BM cellularity (2 femurs) by the indicated percentages. Bars represent means from three to four mice/genotype from two independent experiments ± SEM. No significant differences were observed (Mann–Whitney-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig01: Bim- and Puma-mediated LSK cell killing upon growth factor deprivationWt LSK cells were isolated from murine BM and cultured for 14 h in the presence or absence of SCF, TPO and Flt3L (100 ng/ml each). mRNA levels of the indicated BH3-only proteins, Bax and Bak as well as the anti-apoptotic Bcl-2 proteins were determined by RT-MLPA®. Blots show mRNA changes in the absence of cytokines when compared to mRNA levels in the presence of cytokines. Bars represent means of n = 5–6 from four independent experiments ± SEM. Significant p values: Bim p = 0.01; Puma p = 0.01; Bmf p = 0.01; Bak p = 0.01; Bcl-2 p = 0.04 (Mann–Whitney-test).LSK cells isolated from mice of the indicated genotypes were cultured for 48 h in the presence or absence of cytokines. Apoptosis was determined by combined staining with AnnexinV and 7-AAD, and specific apoptosis triggered by cytokine withdrawal was calculated by the following equation: (induced apoptosis − spontaneous apoptosis)/(100 – spontaneous apoptosis). Bars represent mean values of n = 3–4/genotype from three independent experiments ± SEM. Significant differences are indicated (Student t-test with Welch's correction).Eight- to ten-week-old mice of the indicated genotypes were sacrificed and BM was analysed by flow cytometry. Percentages of LSK as well as MPP and LT-HSC were determined by cell surface staining.Total numbers of LSK, MPP and LT-HSC were determined by multiplying BM cellularity (2 femurs) by the indicated percentages. Bars represent means from three to four mice/genotype from two independent experiments ± SEM. No significant differences were observed (Mann–Whitney-test).
Mentions: To analyse which BH3-only proteins are induced in the absence of cytokine-mediated survival signals and may therefore mediate HSPC apoptosis, we isolated LSK cells from BM of wt mice and cultured them in the presence or absence of the cytokines TPO, Flt3L and SCF. Employing RT-MLPA® analysis allowed us to screen relative changes in the mRNA expression of all known Bcl-2 family members as well as different additional apoptosis-related genes (Eldering et al, 2003). We observed cytokine deprivation-induced changes in the mRNA levels of Bim (2.0-fold), Bmf (3.3-fold), Puma (2.3-fold) and Noxa (1.9-fold), but not Bad, Bid (Fig 1A) or Bik (not expressed). Induction of mRNA levels was independently confirmed by qRT-PCR (Supporting Information Fig 1A). No upregulation of Bax or Bak was observed whereas their pro-survival antagonists Bcl-2 and Bcl-xL were downregulated at the mRNA level (0.5-fold, both; Fig 1A and Supporting Information Fig 1A). The levels of Mcl-1 or A1 mRNA (Fig 1A) as well as of all remaining genes monitored were found to be largely unchanged (Supporting Information Table II).

Bottom Line: Thereby, we revealed a critical role for Bim and Bmf as regulators of HSPC dynamics both during early engraftment and long-term reconstitution.HSPCs derived from wild-type donors were readily displaced by Bim- or Bmf-deficient or Bcl-2-overexpressing HSPCs as early as 10 days after engraftment.Finally, we provide proof of principle that RNAi-based reduction of BIM or BMF, or overexpression of BCL-2 in human CD34(+) cord blood cells may be an attractive therapeutic option to increase stem cell survival and transplantation efficacy.

View Article: PubMed Central - PubMed

Affiliation: Division of Developmental Immunology, Biocenter, Innsbruck Medical University, Innsbruck, Austria. verena.labi@mdc-berlin.de

Show MeSH
Related in: MedlinePlus