a role for proapoptotic bid in the dna-damage response
DESCRIPTION
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
![Page 1: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/1.jpg)
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
![Page 2: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/2.jpg)
Introduction
BH
![Page 3: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/3.jpg)
Introduction
![Page 4: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/4.jpg)
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.
![Page 5: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/5.jpg)
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。
![Page 6: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/6.jpg)
Mataphase Spreads
Method
wild-type and Bid−/− MPCs
arrested in metaphase
fixed
visualized
mytomycin C, 24hr
0.1 mg/ml of colchicine,
Giemsa staining
![Page 7: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/7.jpg)
Result
Fig.1
![Page 8: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/8.jpg)
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
![Page 9: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/9.jpg)
![Page 10: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/10.jpg)
Result
Increased chromosal instability is a general feature of Bid dificiency
Fig.1
![Page 11: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/11.jpg)
Result
Each chromosomes break was given a score of +1Each tri- or quadriradial was given a score of +2total 50 metaphase spreads
Fig.1
![Page 12: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/12.jpg)
Bid-/- MPCs display increased sensitivity to DNA-damage agents
Result
Fig.2
![Page 13: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/13.jpg)
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
![Page 14: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/14.jpg)
Result
Aphidicolin is Reversible inhibitor of eukaryotic nuclear DNA replication.
Fig.3
![Page 15: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/15.jpg)
Result
Fig.3C
To verify that this S phase phenotype is attributable to the absence of BID.
Fig.3D
![Page 16: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/16.jpg)
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
![Page 17: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/17.jpg)
Result
Fig.4
![Page 18: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/18.jpg)
Result
Fig.4
![Page 19: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/19.jpg)
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.
![Page 20: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/20.jpg)
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.
![Page 21: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/21.jpg)
BID has at least two ATM/ATR/DNA-PKcs consensus Phosphorylation sites at residues S61 and S78
Fig.6A
![Page 22: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/22.jpg)
Fig.S6 Wild-type MPCs
Result
![Page 23: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/23.jpg)
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
![Page 24: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/24.jpg)
缺少 ATM , BID 磷酸化幾乎完全消失
Fig.6EMEFs
Result
![Page 25: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/25.jpg)
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
![Page 26: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/26.jpg)
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
![Page 27: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/27.jpg)
Conclusion
![Page 28: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/28.jpg)
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.
![Page 29: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/29.jpg)
Thank you for your attention
![Page 30: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/30.jpg)
A mixture of glycerin, methanol, methylene azure, and eosin used tostain chromosomes.
Giemsa staining
![Page 31: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/31.jpg)
Subcellular Fractionation
Cells
sucrose lysis buffer
lysed
centrifuged at 1200 RPM for 5 minutes
Supernatantcentrifuged at 8000 RPM 12minutes
cytosolic fraction
![Page 32: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/32.jpg)
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
![Page 33: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/33.jpg)
resuspend
10 mM HEPES pH 8.01.5 mM MgCl210% glycerol
with Benzonase nuclease (Novagen)
The nuclear pellet fraction
centrifugation at 12000 RPM
digested
![Page 34: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/34.jpg)
Subcellular fractionation of MPCs at 2 hr following hydroxyureatreatment (1 mM). BID is found in the insoluble chromatin fractionfollowing hydroxyurea.
Result
Fig.5C
![Page 35: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/35.jpg)
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)
![Page 36: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/36.jpg)
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
![Page 37: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/37.jpg)
Krishan’s reagent
• 0.1% NaCitrate
• 0.03%NP40
• 0.05mg/ml propidium iodide
• 0.02mg/ml RNase a
background
![Page 38: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/38.jpg)
Method
BrDU analysisCells were incubated with 10 μl BrDU for 45min
Washed
Incubated with 100mM hydroxyurea or0.1 μM aphidicolin
![Page 39: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/39.jpg)
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).
![Page 40: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/40.jpg)
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
![Page 41: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/41.jpg)
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
![Page 42: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/42.jpg)
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
![Page 43: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/43.jpg)
Bid is a substrate for ATM and ATR in vitro
Fig.6I
![Page 44: A Role for Proapoptotic BID in the DNA-Damage Response](https://reader036.vdocuments.net/reader036/viewer/2022081501/56815032550346895dbe2e21/html5/thumbnails/44.jpg)
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.