a role for proapoptotic bid in the dna-damage response
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A Role for Proapoptotic BID in the DNA-Damage Response. Zinkel S.S. et al., (2005) Cell 122,579-591. 銘傳大學 生物科技系 學生:徐鉦竤 指導教授:陳秀儀老師. Introduction. BH. Introduction. 1.The BH3-only member BID interconnect the intrinsic pathway and extrinsic Death receptor pathway. - PowerPoint PPT PresentationTRANSCRIPT
A Role for Proapoptotic BIDin the DNA-Damage Response
Zinkel S.S. et al., (2005) Cell 122,579-591
銘傳大學生物科技系學生:徐鉦竤
指導教授:陳秀儀老師
Introduction
BH
Introduction
Introduction
1.The BH3-only member BID interconnect the intrinsic pathway and extrinsic Death receptor pathway .
2.BID in maintaining myeloid homeostasis and suppressing leukemogenesis.
3.In a resting cell, BID is predominantly cytoplasmic. Following TNFαor Fas treatment,BID is cleaved by caspase 8 in an unstructured loop, facilitating its translocation to the mitochondria.
material
Cell line:Hox11-immortalized premalignant myeloid progenitor cell line(MPCs)
1.BID 有穩定染色體的功能
HOX 11 gene 會造成 t (7:10) (q35;q24) or t(10:14)(q24;qll) 的translocations. 與 T-cell leukemia 和 lymphoid neoplasias 有關。欲證明 BID 的存在時會抑制此現象發生。
Mataphase Spreads
Method
wild-type and Bid−/− MPCs
arrested in metaphase
fixed
visualized
mytomycin C, 24hr
0.1 mg/ml of colchicine,
Giemsa staining
Result
Fig.1
Result
藉由每個細胞中所增加的 breaks 數來計數 in 50 metaphase spreads
Each chromosomes break was given a score of +1Each tri- or quadriradial was given a score of +2total 50 metaphase spreads
Fig.1
Result
Increased chromosal instability is a general feature of Bid dificiency
Fig.1
Result
Each chromosomes break was given a score of +1Each tri- or quadriradial was given a score of +2total 50 metaphase spreads
Fig.1
Bid-/- MPCs display increased sensitivity to DNA-damage agents
Result
Fig.2
Fig.2
Bid−/− primary activated T cells in response to IR (data not shown), a cell type that is less dependent upon the BID-mediated mitochondrialamplification loop for death (Scaffidi et al., 1998).
Result
Result
Aphidicolin is Reversible inhibitor of eukaryotic nuclear DNA replication.
Fig.3
Result
Fig.3C
To verify that this S phase phenotype is attributable to the absence of BID.
Fig.3D
The death function of proapoptotic BCL-2 family membersis localized to the BH3 domain. To determine whether an intact BH3 domain is required for the BID deficient S phasedefect.
Conclusion
Result
Fig.4
Result
Fig.4
Result
Fig.4
A region of BID distinct from its proapoptotic BH3 domain mediates the BID’s role downstream of DNA damage induced by replicative stress.
To further elucidate the kinase responsible for Regulation of BID phosphorylation, the authors evaluated a series of MEFs deficient for the DNA repair kinases ATM, ATR,and DNA-PKcs.
MEF display significant genotypic and phenotypic variations dependingon the strain and the genetic background of the mice they were isolatedfrom as well as the immortalization strategy used.
BID has at least two ATM/ATR/DNA-PKcs consensus Phosphorylation sites at residues S61 and S78
Fig.6A
Fig.S6 Wild-type MPCs
Result
1mM 10mM
1 μM 10 μM
1 μM 10 μM
1 μM
1 μM
10 μM
10 μM 1mM 10mM
Fig.6B
ATM/ATR/DNA-PKcs 是在同一的位置對 BID 的磷酸化,表示 BID 磷酸化是受這些 DNA repair kinase 所調控。
Result
缺少 ATM , BID 磷酸化幾乎完全消失
Fig.6EMEFs
Result
甚至部份的 ATR 或 DNA-PKcs 失活, BID 還是可以被磷酸化
Fig.6H Fig.S8
Result
在 Bid-/- MPCs 中的 Bid S78A mutant 無法恢復由 Aphidicolin造成的 intra-S phase arrest ,表示 BID 的 S78A 可能在 intra-SPhase checkpoint 起作用。
The S Phase Role of BID Is Mediated by Phosphorylation at Position 78
Fig.7BResult
Conclusion
Conclusion
1. BID plays an unexpected role in the intra-S phase checkpoint downstream of DNA damage.
5.ATM/ATR 在 BID 的 S78A 磷酸化可以調控 intra-S phase checkpoint
4.this role is mediated through BID phosphorylation by the DNA-damage kinase ATM.
2.BID 有穩定染色體的功能3.A Functional BID BH3-Domain Is Not Required to Rescue S Phase Accumulation.
Thank you for your attention
A mixture of glycerin, methanol, methylene azure, and eosin used tostain chromosomes.
Giemsa staining
Subcellular Fractionation
Cells
sucrose lysis buffer
lysed
centrifuged at 1200 RPM for 5 minutes
Supernatantcentrifuged at 8000 RPM 12minutes
cytosolic fraction
pellet
low salt buffer
an equal volume of high salt buffer
The soluble nuclear fraction
centrifugation,at 12,000 RPM for 10 minutes
resuspend
pellet
resuspend
10 mM HEPES pH 8.01.5 mM MgCl210% glycerol
with Benzonase nuclease (Novagen)
The nuclear pellet fraction
centrifugation at 12000 RPM
digested
Subcellular fractionation of MPCs at 2 hr following hydroxyureatreatment (1 mM). BID is found in the insoluble chromatin fractionfollowing hydroxyurea.
Result
Fig.5C
Manganese superoxide dismutase (MnSOD)
Under normal physiological conditions, mitochondria are the major source of O2
- production. Numerous studies have indicated that MnSOD plays an important role in preventing cells from oxidative stress and inhibiting tumorigenicity.
The human RAD9 gene partially complemented the hydroxyurea sensitivity, radiosensitivity, and checkpoint defects of rad9-null mutant cells. In vivo, the human RAD9 protein was phosphorylated in response to DNA damage, suggesting that it participates in a DNA damage-inducible signaling pathway.
O2- H2O2 + O2 (mitochondria)
MethodCell cycle analysis
(propidium iodide staining)
One million cellsResuspended in 200μl Krishan’s reagent
Incubated
Analyzed on a Becton-Dicknson FACS machineusing Flojo analysis software
15min, Room Temperature, in the dark
Krishan’s reagent
• 0.1% NaCitrate
• 0.03%NP40
• 0.05mg/ml propidium iodide
• 0.02mg/ml RNase a
background
Method
BrDU analysisCells were incubated with 10 μl BrDU for 45min
Washed
Incubated with 100mM hydroxyurea or0.1 μM aphidicolin
Murine Stem Cell Virus retroviral vector (MSCV)
1.The Murine Stem Cell Virus (MSCV) vectors were derived from the Murine Embryonic Stem Cell Virus (MESV).2. The vectors achieve stable, high-level gene expression in hematopoietic and embryonic stem cells through a specifically designed 5' long terminal repeat (LTR).
293T is a human embryonic kidney cell line commonly used for transfection assays.The expression of the large T antigen in the cell, plasmids with SV40 origin of replication can be transiently transfected and give extremely high levels of expression of AP fusion proteins.
293T cells
In Vitro Kinase Assays
293T cells
treated with 10Gy IR , 30 minutes
24 hours post transfection
harvested and lysed in TNE buffer
Kinase assays
10Gy IR for 30 minutes
adding 30 μl of kinase buffer
incubating for 30 minutes at 30°C
293T is a human embryonic kidney cell line commonly used for transfection assays.The expression of the large T antigen in the cell, plasmids with SV40 origin of replication can be transiently transfected and give extremely high levels of expression of AP fusion proteins.
293T cells
Bid is a substrate for ATM and ATR in vitro
Fig.6I
An ATRflox targeting construct containing a duplicated exon 2 as well as an exon 2 disrupted in frame with the coding region of the neomycinresistance gene and polyadenylation sequence was created using ATRgenomic DNA cloned from a lambda genomic library.
Cre systemthe system has been developed as a means to efficiently edit and manipulate the genome. It has been successfully applied in studiesof yeast, plants, mammalian cell culture and mice.1 Targetedmutations, transgenics, allelic modification, deletions, andchromosomal aberrations can all be produced using variations of a common reaction.