A Role for Proapoptotic BID in the DNA-Damage Response

1. 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

2. Introduction BH

3. Introduction

4. 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 TNFα or Fas treatment,BID is cleaved by caspase 8 in an unstructured loop, facilitating its translocation to the mitochondria. Introduction

5. 1.BID有穩定染色體的功能 material Cell line: Hox11-immortalized premalignant myeloid progenitor cell line(MPCs) 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。

6. Method Mataphase Spreads wild-type and Bid−/− MPCs mytomycin C, 24hr 0.1 mg/ml of colchicine, arrested inmetaphase fixed visualized Giemsa staining

7. Result Fig.1

8. Result 藉由每個細胞中所增加的breaks數來計數in 50 metaphase spreads Fig.1 Each chromosomes break was given a score of +1 Each tri- or quadriradial was given a score of +2 total 50 metaphase spreads

10. Result Fig.1 Increased chromosal instability is a general feature of Bid dificiency

11. Result Fig.1 Each chromosomes break was given a score of +1 Each tri- or quadriradial was given a score of +2 total 50 metaphase spreads

12. Result Bid-/- MPCs display increased sensitivity to DNA-damage agents Fig.2

13. Bid−/− primary activated T cells in response to IR (data not shown), a cell type that is less dependent upon the BID-mediated mitochondrial amplification loop for death (Scaffidi et al., 1998). Result Fig.2

14. Result Fig.3 Aphidicolin is Reversible inhibitor of eukaryotic nuclear DNA replication.

15. Result To verify that this S phase phenotype is attributable to the absence of BID. Fig.3C Fig.3D

16. Conclusion The death function of proapoptotic BCL-2 family members is localized to the BH3 domain. To determine whether an intact BH3 domain is required for the BID deficient S phase defect.

17. Result Fig.4

18. Result Fig.4

19. 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.

20. 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 depending on the strain and the genetic background of the mice they were isolated from as well as the immortalization strategy used.

21. Fig.6A BID has at least two ATM/ATR/DNA-PKcs consensus Phosphorylation sites at residues S61 and S78

22. Result Wild-type MPCs Fig.S6

23. Result Fig.6B 1mM 10mM 1 μM 10 μM 1mM 10mM 1 μM 10 μM 1 μM 10 μM 1 μM 10 μM 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。

24. Result Fig.6E MEFs 缺少ATM，BID磷酸化幾乎完全消失

25. Result Fig.6H Fig.S8 甚至部份的ATR或DNA-PKcs失活，BID還是可以被磷酸化

26. The S Phase Role of BID Is Mediated by Phosphorylation at Position 78 Result Fig.7B 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-S Phase checkpoint起作用。

27. Conclusion

28. Conclusion 1. BID plays an unexpected role in the intra-S phase checkpoint downstream of DNA damage. 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. 4.this role is mediated through BID phosphorylation by the DNA-damage kinase ATM. 5.ATM/ATR在BID的S78A磷酸化可以調控intra-S phase checkpoint

29. Thank you for your attention

30. Giemsa staining A mixture of glycerin, methanol, methylene azure, and eosin used to stain chromosomes.

31. Subcellular Fractionation Cells sucrose lysis buffer lysed centrifuged at 1200 RPM for 5 minutes Supernatant centrifuged at 8000 RPM 12minutes cytosolic fraction

32. pellet low salt buffer resuspend an equal volume of high salt buffer centrifugation, at 12,000 RPM for 10 minutes pellet The soluble nuclear fraction

33. 10 mM HEPES pH 8.0 1.5 mM MgCl2 10% glycerol resuspend with Benzonase nuclease (Novagen) digested centrifugation at 12000 RPM The nuclear pellet fraction

34. Result Fig.5C Subcellular fractionation of MPCs at 2 hr following hydroxyurea treatment (1 mM). BID is found in the insoluble chromatin fraction following hydroxyurea.

35. 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. O2 O2- · H2O2 + (mitochondria) The human RAD9 gene partially complemented the hydroxyurea sensitivity, radiosensitivity, and checkpoint defects of rad9-null mutant cells. In vivo, the humanRAD9 protein was phosphorylated in response to DNA damage, suggesting that it participates in a DNA damage-inducible signaling pathway.

36. Method Cell cycle analysis (propidium iodide staining) One million cells Resuspended in 200μl Krishan’s reagent Incubated 15min, Room Temperature, in the dark Analyzed on a Becton-Dicknson FACS machine using Flojo analysis software

37. background Krishan’s reagent • 0.1% NaCitrate • 0.03%NP40 • 0.05mg/ml propidium iodide • 0.02mg/ml RNase a

38. Cells were incubated with 10 μl BrDU for 45min Method BrDU analysis Washed Incubated with 100mM hydroxyurea or 0.1 μM aphidicolin

39. Murine Stem Cell Virus retroviral vector (MSCV) 1.TheMurine 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).

40. 293T cells 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.

41. In Vitro Kinase Assays 293T cells 24 hours post transfection 10Gy IR for 30 minutes treated with 10Gy IR , 30 minutes harvested and lysed in TNE buffer adding 30 μl of kinase buffer Kinase assays incubating for 30 minutes at 30°C

42. 293T cells 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.

43. Fig.6I Bid is a substrate for ATM and ATR in vitro

44. An ATRflox targeting construct containing a duplicated exon 2 as well as an exon 2 disrupted in frame with the coding region of the neomycin resistance gene and polyadenylation sequence was created using ATR genomic DNA cloned from a lambda genomic library. Cre system the system has been developed as a means to efficiently edit and manipulate the genome. It has been successfully applied in studies of yeast, plants, mammalian cell culture and mice.1 Targeted mutations, transgenics, allelic modification, deletions, and chromosomal aberrations can all be produced using variations of a common reaction.