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A transgenic mouse model to inducibly target prosurvival Bcl2 proteins with selective BH3 peptides in vivo.

Lee EF, Takiguchi M, Pettikiriarachchi A, Evangelista M, Huang DC, Dickins RA, Fairlie WD - Cell Death Dis (2015)

View Article: PubMed Central - PubMed

Affiliation: 1] Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia [2] Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia.

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Dear Editor, BH3-mimetic drugs that antagonize Bcl-2 family prosurvival proteins are effective against some cancers, particularly those with abnormally high expression of the prosurvival protein target... Despite the recent success of BH3-mimetics targeting Bcl-2, Bcl-xL and Bcl-w, mechanism-based cell killing in vivo by targeting other important prosurvival proteins such as Mcl-1 or Bfl-1 has yet to be demonstrated... BimS is an intrinsically unstructured protein that tolerates extensive mutation of its BH3 sequence, and BimSBH3 chimeras display the prosurvival protein specificity profile of the replacement BH3 domain... In this study we focussed on the BH3 domain of Bad because it targets the prosurvival proteins Bcl-2, Bcl-xL and Bcl-w... As ABT-737/263 induces thrombocytopenia in mice and humans due to antagonism of Bcl-xL, we measured platelet levels as a biomarker for functional expression of the ligand... Notably, blood cell analysis of these mice revealed a significant reduction (~65% decrease) in platelet counts (Figure 1c and d), a degree of thrombocytopenia comparable with that seen in patients administered with ABT-263... Importantly, platelet counts rebounded to normal levels following removal of Dox for 7 days (Figure 1d), illustrating the reversibility of the system... We also generated an additional transgenic mouse strain allowing inducible expression of a BimSBad construct possessing a BH3 sequence mutation (BimSBad; Figure 1a) that decreases its affinity for Bcl-xL by >40-fold (KD 8.5 nM versus <0.2 nM as measured by surface plasmon resonance)... Induction of BimSBad expression (Figure 1b) did not alter blood cell or platelet counts (Figure 1d), indicating that the thrombocytopenia observed in BimSBad mice is due to Bcl-xL inhibition... For example, tet-regulated expression of different BimS variants in mice could reveal toxicities associated with neutralization of their prosurvival protein targets (individually or in combination), and tissue-specific effects could be addressed by crossing onto mice where rtTA expression is driven by different promoters.

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Expression of BimSBad reduces platelet levels in mice. (a) Schematic of the CMV-rtTA and TRE-BimSBad transgenes. Upon Dox treatment of bitransgenic mice, the rtTA (tet-on) protein transactivates the TRE promoter to drive expression of N-terminally FLAG tagged BimSBad. Targeting vectors were generated by cloning MluI-flanked FLAG-BimSBad/BimSBadmut PCR amplicons into the MluI site of a modified version of the pgkATGfrt vector6 in which the TRE promoter has been replaced with TREtight. BH3 domain numbering refers to the amino acid residue position within the respective BimS or Bad protein sequences. The BadBH3 residue in red (F121) was mutated to alanine in the BimSBadmut mice. Targeted ES cell clones were injected into C57Bl/6 blastocysts and chimeras crossed to C57Bl/6 female mice. All mouse colonies were maintained by C57Bl/6 backcrossing. (b) Western blot of white blood cells isolated from TRE-BimSBad/BimSBadmut; CMV-rtTA bitransgenic mice or wild-type or BimSBad/BimSBadmut single transgenic control mice following 7 days of Dox food (600 mg/kg). Blood (100 μl) was cleared of red blood cells by 10-fold dilution in red cell lysis buffer for 5 min followed by centrifugation. The white blood cell-containing pellet was then resuspended in lysis buffer (20 mM Tris pH 7.4, 135 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton X100 and 10% glycerol) for 1 h on ice, followed by centrifugation. The supernatant was then analyzed by Western blot probed with an anti-FLAG antibody and reprobed with anti-β-actin as loading control. * Indicates a non-specific band (c) Blood cell parameters after Dox treatment of bitransgenic and control animals for 7 days. ****P<0.0001 (t-test) compared with littermate controls (d) Platelet levels in TRE-BimSBad bitransgenic mice treated with Dox for 7 days rebound to normal levels after 7 days without Dox treatment. No changes in platelet levels were observed in BimSBadmut mice following Dox treatment
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fig1: Expression of BimSBad reduces platelet levels in mice. (a) Schematic of the CMV-rtTA and TRE-BimSBad transgenes. Upon Dox treatment of bitransgenic mice, the rtTA (tet-on) protein transactivates the TRE promoter to drive expression of N-terminally FLAG tagged BimSBad. Targeting vectors were generated by cloning MluI-flanked FLAG-BimSBad/BimSBadmut PCR amplicons into the MluI site of a modified version of the pgkATGfrt vector6 in which the TRE promoter has been replaced with TREtight. BH3 domain numbering refers to the amino acid residue position within the respective BimS or Bad protein sequences. The BadBH3 residue in red (F121) was mutated to alanine in the BimSBadmut mice. Targeted ES cell clones were injected into C57Bl/6 blastocysts and chimeras crossed to C57Bl/6 female mice. All mouse colonies were maintained by C57Bl/6 backcrossing. (b) Western blot of white blood cells isolated from TRE-BimSBad/BimSBadmut; CMV-rtTA bitransgenic mice or wild-type or BimSBad/BimSBadmut single transgenic control mice following 7 days of Dox food (600 mg/kg). Blood (100 μl) was cleared of red blood cells by 10-fold dilution in red cell lysis buffer for 5 min followed by centrifugation. The white blood cell-containing pellet was then resuspended in lysis buffer (20 mM Tris pH 7.4, 135 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton X100 and 10% glycerol) for 1 h on ice, followed by centrifugation. The supernatant was then analyzed by Western blot probed with an anti-FLAG antibody and reprobed with anti-β-actin as loading control. * Indicates a non-specific band (c) Blood cell parameters after Dox treatment of bitransgenic and control animals for 7 days. ****P<0.0001 (t-test) compared with littermate controls (d) Platelet levels in TRE-BimSBad bitransgenic mice treated with Dox for 7 days rebound to normal levels after 7 days without Dox treatment. No changes in platelet levels were observed in BimSBadmut mice following Dox treatment

Mentions: Here we have generated transgenic mice in which peptide-based BH3 ligands can be inducibly expressed to evaluate their effects in vivo and provide proof-of-principle for similar-acting drugs. To achieve this we adapted a previously described strategy allowing FLP-recombinase-mediated insertion of an expression cassette at an frt ‘landing pad' at the type I collagen (Col1a1) locus in mouse embryonic stem cells.6 The cassette comprises sequences encoding a BH3-domain protein under control of a tetracycline (tet)-regulated element (TRE) promoter. To develop a system that is broadly applicable to BH3 domains with different specificities, we employed the BimS BH3-only protein as a scaffold in which BH3 domains with different specificities could replace the native BH3 sequence (Figure 1a). BimS is an intrinsically unstructured protein that tolerates extensive mutation of its BH3 sequence, and BimSBH3 chimeras display the prosurvival protein specificity profile of the replacement BH3 domain.7 In this study we focussed on the BH3 domain of Bad because it targets the prosurvival proteins Bcl-2, Bcl-xL and Bcl-w.7 Hence BimSBad expression should mimic the BH3-mimetics ABT-737 and ABT-263 that have been extensively studied in vivo.1


A transgenic mouse model to inducibly target prosurvival Bcl2 proteins with selective BH3 peptides in vivo.

Lee EF, Takiguchi M, Pettikiriarachchi A, Evangelista M, Huang DC, Dickins RA, Fairlie WD - Cell Death Dis (2015)

Expression of BimSBad reduces platelet levels in mice. (a) Schematic of the CMV-rtTA and TRE-BimSBad transgenes. Upon Dox treatment of bitransgenic mice, the rtTA (tet-on) protein transactivates the TRE promoter to drive expression of N-terminally FLAG tagged BimSBad. Targeting vectors were generated by cloning MluI-flanked FLAG-BimSBad/BimSBadmut PCR amplicons into the MluI site of a modified version of the pgkATGfrt vector6 in which the TRE promoter has been replaced with TREtight. BH3 domain numbering refers to the amino acid residue position within the respective BimS or Bad protein sequences. The BadBH3 residue in red (F121) was mutated to alanine in the BimSBadmut mice. Targeted ES cell clones were injected into C57Bl/6 blastocysts and chimeras crossed to C57Bl/6 female mice. All mouse colonies were maintained by C57Bl/6 backcrossing. (b) Western blot of white blood cells isolated from TRE-BimSBad/BimSBadmut; CMV-rtTA bitransgenic mice or wild-type or BimSBad/BimSBadmut single transgenic control mice following 7 days of Dox food (600 mg/kg). Blood (100 μl) was cleared of red blood cells by 10-fold dilution in red cell lysis buffer for 5 min followed by centrifugation. The white blood cell-containing pellet was then resuspended in lysis buffer (20 mM Tris pH 7.4, 135 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton X100 and 10% glycerol) for 1 h on ice, followed by centrifugation. The supernatant was then analyzed by Western blot probed with an anti-FLAG antibody and reprobed with anti-β-actin as loading control. * Indicates a non-specific band (c) Blood cell parameters after Dox treatment of bitransgenic and control animals for 7 days. ****P<0.0001 (t-test) compared with littermate controls (d) Platelet levels in TRE-BimSBad bitransgenic mice treated with Dox for 7 days rebound to normal levels after 7 days without Dox treatment. No changes in platelet levels were observed in BimSBadmut mice following Dox treatment
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fig1: Expression of BimSBad reduces platelet levels in mice. (a) Schematic of the CMV-rtTA and TRE-BimSBad transgenes. Upon Dox treatment of bitransgenic mice, the rtTA (tet-on) protein transactivates the TRE promoter to drive expression of N-terminally FLAG tagged BimSBad. Targeting vectors were generated by cloning MluI-flanked FLAG-BimSBad/BimSBadmut PCR amplicons into the MluI site of a modified version of the pgkATGfrt vector6 in which the TRE promoter has been replaced with TREtight. BH3 domain numbering refers to the amino acid residue position within the respective BimS or Bad protein sequences. The BadBH3 residue in red (F121) was mutated to alanine in the BimSBadmut mice. Targeted ES cell clones were injected into C57Bl/6 blastocysts and chimeras crossed to C57Bl/6 female mice. All mouse colonies were maintained by C57Bl/6 backcrossing. (b) Western blot of white blood cells isolated from TRE-BimSBad/BimSBadmut; CMV-rtTA bitransgenic mice or wild-type or BimSBad/BimSBadmut single transgenic control mice following 7 days of Dox food (600 mg/kg). Blood (100 μl) was cleared of red blood cells by 10-fold dilution in red cell lysis buffer for 5 min followed by centrifugation. The white blood cell-containing pellet was then resuspended in lysis buffer (20 mM Tris pH 7.4, 135 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton X100 and 10% glycerol) for 1 h on ice, followed by centrifugation. The supernatant was then analyzed by Western blot probed with an anti-FLAG antibody and reprobed with anti-β-actin as loading control. * Indicates a non-specific band (c) Blood cell parameters after Dox treatment of bitransgenic and control animals for 7 days. ****P<0.0001 (t-test) compared with littermate controls (d) Platelet levels in TRE-BimSBad bitransgenic mice treated with Dox for 7 days rebound to normal levels after 7 days without Dox treatment. No changes in platelet levels were observed in BimSBadmut mice following Dox treatment
Mentions: Here we have generated transgenic mice in which peptide-based BH3 ligands can be inducibly expressed to evaluate their effects in vivo and provide proof-of-principle for similar-acting drugs. To achieve this we adapted a previously described strategy allowing FLP-recombinase-mediated insertion of an expression cassette at an frt ‘landing pad' at the type I collagen (Col1a1) locus in mouse embryonic stem cells.6 The cassette comprises sequences encoding a BH3-domain protein under control of a tetracycline (tet)-regulated element (TRE) promoter. To develop a system that is broadly applicable to BH3 domains with different specificities, we employed the BimS BH3-only protein as a scaffold in which BH3 domains with different specificities could replace the native BH3 sequence (Figure 1a). BimS is an intrinsically unstructured protein that tolerates extensive mutation of its BH3 sequence, and BimSBH3 chimeras display the prosurvival protein specificity profile of the replacement BH3 domain.7 In this study we focussed on the BH3 domain of Bad because it targets the prosurvival proteins Bcl-2, Bcl-xL and Bcl-w.7 Hence BimSBad expression should mimic the BH3-mimetics ABT-737 and ABT-263 that have been extensively studied in vivo.1

View Article: PubMed Central - PubMed

Affiliation: 1] Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia [2] Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Dear Editor, BH3-mimetic drugs that antagonize Bcl-2 family prosurvival proteins are effective against some cancers, particularly those with abnormally high expression of the prosurvival protein target... Despite the recent success of BH3-mimetics targeting Bcl-2, Bcl-xL and Bcl-w, mechanism-based cell killing in vivo by targeting other important prosurvival proteins such as Mcl-1 or Bfl-1 has yet to be demonstrated... BimS is an intrinsically unstructured protein that tolerates extensive mutation of its BH3 sequence, and BimSBH3 chimeras display the prosurvival protein specificity profile of the replacement BH3 domain... In this study we focussed on the BH3 domain of Bad because it targets the prosurvival proteins Bcl-2, Bcl-xL and Bcl-w... As ABT-737/263 induces thrombocytopenia in mice and humans due to antagonism of Bcl-xL, we measured platelet levels as a biomarker for functional expression of the ligand... Notably, blood cell analysis of these mice revealed a significant reduction (~65% decrease) in platelet counts (Figure 1c and d), a degree of thrombocytopenia comparable with that seen in patients administered with ABT-263... Importantly, platelet counts rebounded to normal levels following removal of Dox for 7 days (Figure 1d), illustrating the reversibility of the system... We also generated an additional transgenic mouse strain allowing inducible expression of a BimSBad construct possessing a BH3 sequence mutation (BimSBad; Figure 1a) that decreases its affinity for Bcl-xL by >40-fold (KD 8.5 nM versus <0.2 nM as measured by surface plasmon resonance)... Induction of BimSBad expression (Figure 1b) did not alter blood cell or platelet counts (Figure 1d), indicating that the thrombocytopenia observed in BimSBad mice is due to Bcl-xL inhibition... For example, tet-regulated expression of different BimS variants in mice could reveal toxicities associated with neutralization of their prosurvival protein targets (individually or in combination), and tissue-specific effects could be addressed by crossing onto mice where rtTA expression is driven by different promoters.

Show MeSH
Related in: MedlinePlus