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.