1

Thesis for the degree

Doctor of Philosophy

By

Iris Kamer

Advisor

Prof Atan Gross

August, 2008

Submitted to the Scientific Council of the

Weizmann Institute of Science

Rehovot, Israel

DNA בתגובה התאית לנזקי BIDתפקידו של החלבון

חבור לשם קבלת התואר

דוקטור לפילוסופיה

מאת

איריס קמר

ח"תשס, אב

ת שלמוגש למועצה המדעי

מכון ויצמן למדע

The role of full-length BID in the

DNA damage response

מנחה

איתן גרוס' ופפר

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Table of contents

Table of contents ......................................................................................................................2 List of figures............................................................................................................................5 Abbreviations ...........................................................................................................................6 Abstract.....................................................................................................................................7 Scientific background ..............................................................................................................9

The BCL-2 family................................................................................................................10 BID.......................................................................................................................................12

The objective of the research ................................................................................................15 Materials and Methods..........................................................................................................16

Tissue culture.......................................................................................................................16 Mouse embryonic fibroblasts ...........................................................................................16 hTERT transformation of primary MEFs ........................................................................16 Generation of hTERT BID-/- stable clones expressing wtBID or the BID-S61A/S78A mutant ..............................................................................................................................17 Preparation of BID recombinant adenoviruses and infection of MEFs ..........................17 Human cell lines and transient transfection ....................................................................18 HeLa BID KD cells ..........................................................................................................18 ATM KD cells...................................................................................................................18 A-T Lymphoblasts ............................................................................................................18 A-T Fibroblasts ................................................................................................................18 Treatments........................................................................................................................18 Clonogenic survival assays..............................................................................................19 Immunocytochemistry (immunofluoresence) ...................................................................19

FACS analysis......................................................................................................................19 BrdU labeling and analysis .............................................................................................19 Cell viability assays .........................................................................................................20 Cell cycle assays ..............................................................................................................20 Apoptosis assays ..............................................................................................................20 Synchronization of cells in G1-S phase ...........................................................................20 Determination of mitochondrial membrane potential .....................................................21 Determination of cellular ROS levels ..............................................................................21

Proteins analysis ..................................................................................................................21 Generation of phospho-specific antibodies .....................................................................21 HA-affinity chromatography............................................................................................21 Cross-linking with BS3 .....................................................................................................21 Cross-linking with Formaldehyde....................................................................................22 Formaldehyde treatment and subcellular fractionation ..................................................22 Western blot .....................................................................................................................23 Alkaline or potato-acid phosphatase treatment ...............................................................23 Purification of the HA-BID cross-linked complex ...........................................................23 Mass spectrometry analysis .............................................................................................24

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Chapter I - Pro-apoptotic BID is an ATM effector in the DNA damage response..........25 Introduction..........................................................................................................................25

ATM and the response of cells to DNA DSBs .................................................................25 BCL-2 family members and the response to DSBs ..........................................................26

Results..................................................................................................................................28 BID is important for DNA damage-induced apoptosis....................................................28 DSBs induce the phosphorylation of BID, and this phosphorylation is mediated by the ATM kinase ......................................................................................................................30 Mouse and human BID are phosphorylated on PIKK consensus sites............................32 Characterization of endogenous BID phosphorylation by using the phospho-specific BID antibodies .........................................................................................................................35 Phosphorylation of S61 and S78 is transient, rapid and occurs many hours before the onset of Etop- induced apoptosis .....................................................................................37 Phosphorylation of S78 does not depend on phosphorylation of S61 .............................38 The phosphorylation of BID occurs in response to extremely low, non-apoptotic levels of IR and it is dose-dependent..............................................................................................39 BID-/- MEFs expressing a non-phosphorylatable BID mutant (S61A/S78A) are more susceptible to Etop- induced apoptosis than those expressing wtBID.............................40 BID-/- MEFs fail to accumulate in the S and G2 phases of the cell cycle following Etop treatment ..........................................................................................................................41 BID-/- MEFs expressing BID-S61A/S78A do not accumulate in the S phase following Etop treatment..................................................................................................................44 Phosphorylation of BID is not cell cycle dependent ........................................................46 BID-/- MEFs show a delayed time course of cell death following low levels of DNA damage.............................................................................................................................48 Knocking down the expression of BID in HeLa cells partially impairs Etop-induced S phase arrest......................................................................................................................49 Cellular BID partially localizes to the nucleus................................................................50 BID might be involved in the immediate cellular response to DNA damage ..................52 CDC25A degradation seems to be less efficient in BID-/- and BID-S61A/S78A MEFs ...54 The phosphorylation of BID is Chk2-independent...........................................................55

Chapter II – Identification of proteins that interact with phosphorylated BID ..............56 Introduction..........................................................................................................................56 Results..................................................................................................................................57

BID is found as part of a 50KDa cross-linked complex in healthy cells and in cells treated with DNA damage................................................................................................57

Chapter III - ATM, mitochondria function and apoptosis ................................................64 Introduction..........................................................................................................................64 Results..................................................................................................................................67

Α−Τ Cells have higher basal ∆ψm (hyperpolarized) compared to cells expressing wt ATM..................................................................................................................................67 A-T cells are more resistant to Etop-induced mitochondrial depolarization and apoptosis compared to cells expressing wtATM..............................................................69 ROS levels are higher in Α−Τ cells compared to control cells .......................................72 Α−Τ cells grow slower compared to control cells...........................................................73

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Discussion................................................................................................................................74 BID, ATM and the DNA damage response.........................................................................74 ATM and the mitochondria..................................................................................................81

References...............................................................................................................................83 Publications ............................................................................................................................90

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List of figures Figure1 : Schematic of the extrinsic and the intrinsic apoptotic pathways. ...........................10 Figure2 : BID is important for DNA damage-induced apoptosis...........................................29 Figure3 : DNA DSBs induce the phosphorylation of BID, and this phosphorylation is

mediated by the ATM kinase. .................................................................................31 Figure4 : Mouse BID is phosphorylated on S61 and S78 whereas human BID is

phosphorylated only on S78....................................................................................34 Figure5 : Characterization of endogenous BID phosphorylation using the anti-phospho S61

and S78 Abs. ...........................................................................................................36 Figure6 : Phosphorylation of S61 and S78 is transient, rapid and occurs hours before the

onset of Etop-induced apoptosis .............................................................................37 Figure7 : Phosphorylation of S78 does not depend on the phosphorylation on S61..............38 Figure8 : Phosphorylation of BID occurs in response to non-apoptotic levels of IR and it is

dose-dependent........................................................................................................39 Figure9 : BID-S61A/S78A clones are more susceptible to Etop induced apoptosis than

wtBID clones...........................................................................................................40 Figure10 : BID-/- MEFs fail to arrest in the S phase following Etop treatment........................42 Figure11 : BID is required for S phase arrest following DNA damage. ..................................43 Figure12 : BID-/- MEFs expressing BID-S61A/S78A do not accumulate in the S phase

following Etop treatment.........................................................................................45 Figure13 : BID phosphorylation is not cell cycle dependent. ..................................................47 Figure14 : BID-/- MEFs show a delayed time course of cell death following low levels of

DNA damage...........................................................................................................48 Figure15 : Knocking down the levels of BID protein in HeLa cells partially impairs Etop-

induced S phase arrest.............................................................................................49 Figure16 : Mouse BID is partially localized to the nucleus. ....................................................51 Figure17 : BID might be involved in the immediate cellular response to DNA damage.........53 Figure18 : CDC25A degradation is more efficient in cells expressing wt BID. ......................54 Figure19 : The phosphorylation of BID is Chk2-independent .................................................55 Figure20 : Cross-linking with BS3 results in appearance of specific complexes. ....................58 Figure21 : Flag-PRX6 and BID-HA co-immunoprecipitate. ...................................................59 Figure22 : BID is found as a part of a cross-linked complex in healthy and in Etop treated

cells. ........................................................................................................................60 Figure23 : N-terminal HA tagged BID forms the 50 KDa complex. .......................................62 Figure24 : N-terminal HA tagged BID is localized to the nucleus in transfected 293 cells.....63 Figure25 : Α−Τ cells have higher ∆ψm.....................................................................................68 Figure26 : Mitochondria depolarization and cell death are lower in A-T cells post Etop

treatment..................................................................................................................70 Figure27 : IR-induced mitochondria depolarization and cell death are similar in A-T and WT

cells. ........................................................................................................................71 Figure28 : ROS levels are higher in Α−Τ cells compared to control cells. ..............................72 Figure29 : Α−Τ cells grow slower compared to control cells. .................................................73 Figure30 : Model for the dual role of BID. ..............................................................................80

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Abbreviations Ab Antibody

ATM ataxia-telangiectasia mutated

BH BCL-2 Homology

BrdU Bromodeoxyuridine (5-bromo-2-deoxyuridine)

BS3 bis(sulfosuccinimidyl) suberate

Cyt c Cytochrome c

DDR DNA damage response

DiOC6 3,3’-dihexyloxacarbocynine iodide

DSBs Double strand breaks

Etop Etoposide

FACS Fluorescence-activated cell sorter

FL-BID Full-length BID

HA Hemagglutinin

IMM Inner mitochondrial membrane

IMS Intermembrane space

IP Immunoprecipitation

IR Ionizing radiation

KD Knock down

KO Knock out

MEFs Mouse embryonic fibroblasts

MS Mass spectrometry

MTCH2 Mitochondrial carrier homolog 2

OMM Outer mitochondrial membrane

PI Propidium iodide

ROS Reactive oxygen species

SDS-PAGE Sodium dedocyl sulphate-Polyacrylamid gel electrophoresis

tBID truncated BID

TNFα Tumor necrosis factor α

∆Ψm mitochondrial membrane potential

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Abstract DNA damage leads to the activation of the ATM and ATR kinases, which in turn cause either

cell cycle arrest and DNA repair or apoptosis. In the first part of my thesis, we have

demonstrated that the pro-apoptotic BID protein is phosphorylated by ATM in response to

DNA damage, and that phosphorylation occurs in mouse BID on two ATM consensus sites

(Serine 61 and Serine 78). Interestingly, BID-/- cells failed to accumulate in the S phase of the

cell cycle following treatment with the topoisomerase II poison etoposide (Etop);

reintroducing wild-type BID restored accumulation. In contrast, introducing a non-

phosphorylatable BID mutant (BID-S61A/S78A) did not restore accumulation in the S phase,

and resulted in an increase in cellular sensitivity to Etop-induced apoptosis. These results

implicate BID as an ATM effector, and raise the possibility that pro-apoptotic BID may also

play a pro-survival role by inducing cell cycle arrest. Next, we explored the mechanism by

which BID regulates cell cycle arrest. For this, we took a biochemical approach to isolate

protein(s) that interact with BID following DNA damage. Using cross-linkers, we found that

BID is part of a 50 KDa complex in healthy and Etop-treated cells. Importantly, we found

that the phosphorylated form of BID is also detected in this complex. We are now at the stage

of scaling up the amount of the complex to identify its components.

In the second part of my thesis, we explored the connection between ATM, mitochondria and

apoptosis. At high levels of DNA damage, cells initiate an apoptotic process, which is closely

linked to mitochondrial function. However, the link between ATM and mitochondrial events,

is still largely unknown. We started to explore this link by measuring mitochondrial function

and apoptosis in cells originated from A-T patients expressing mutant ATM or ATM-/- mice.

We found that in these cells the mitochondria were hyperpolarized (higher levels of

mitochondria membrane potential). In addition, these cells were found to be less susceptible

to Etop-induced depolarization and cell death. We also found that these cells exhibited higher

levels of reactive oxygen species (ROS) and grew slower. Importantly, introducing wt ATM

into A-T cells restored the normal mitochondrial and apoptotic parameters. Thus, ATM

seems to be involved in regulating the function of mitochondria.

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In summary, this study reveals 1) that the BH3-only BID protein, a molecule that was

previously considered active only as a pro-apoptotic factor, also plays a pro-survival role as

an ATM effector and 2) a novel link between ATM, a kinase considered to be active only in

the nucleus, and mitochondrial function.

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Scientific background Apoptosis (programmed cell death - PCD) is an evolutionary conserved cell suicide

mechanism that plays a crucial role in various biological processes, including development,

maintenance of homeostasis, and removal of unwanted cells [1]. Abnormal resistance to

apoptosis can lead to disorders such as autoimmunity or cancer due to the persistence of

mutated cells [2]. In contrast, enhanced apoptosis contributes to acute diseases or chronic

pathologies such as neurodegenerative diseases [3]. Cells undergoing PCD assume

morphological features, which include membrane blabbing, chromatin condensation, and

nuclear fragmentation [4]. The genetic pathway that regulates apoptosis has been

characterized, and it appears to be conserved from the nematode C. elegans to mammals.

BCL-2 proteins are the major regulators of the apoptotic pathways [5], and caspase proteases

are the major executioners of this process [6] .

Two major apoptotic pathways have been identified in mammals: the intrinsic and extrinsic

pathways (Fig 1). In the extrinsic pathway, apoptosis is initiated through activation of certain

cell surface receptors. The best characterized are members of the TNF/Fas receptor family.

Once engaged by ligand, these receptors initiate the formation of the death inducing signaling

complex (DISC), which leads to activation of caspase-8 which then activates the downstream

caspase-3 [7]. The cell-intrinsic apoptotic pathway is triggered by death receptor-independent

stimuli such as UV-irradiation, viruses and DNA damage reagents as etoposide (Etop). This

pathway involves the activation of pro-apoptotic BCL-2 family members, which act by

inducing organelle dysfunction, of which mitochondrial dysfunction is the best characterized

[8]. At the mitochondria, pro-apoptotic BCL-2 family members induce the release of

Cytochrome c (Cyt c) and other intermembrane space (IMS) proteins to the cytosol. In the

cytosol, Cyt c together with Apaf-1 and caspase-9 form the apoptosome complex where

caspase-9 is activated. Caspase-9 activates caspase-3, which leads to apoptotic cell death [8].

Under certain circumstances, the extrinsic pathway uses the mitochondrial pathway to

amplify caspase activation. This connection is done trough BID, which is cleaved by caspase-

8 (Fig 1).

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Figure1 : Schematic of the extrinsic and the intrinsic apoptotic pathways. In the extrinsic pathway, activation of the TNF/Fas death receptor induces DISC formation that results in direct activation of caspases. In the intrinsic pathway, the BCL-2 family members are the major players. At the mitochondria, these proteins induce the release of Cyt c, resulting in the formation of the apoptosome and caspase activation. The extrinsic and intrinsic pathways are connected by BID.

The BCL-2 family

The BCL-2 family of proteins constitutes a critical intracellular checkpoint in the intrinsic

pathway of apoptosis. The founding member, BCL-2, was discovered at the chromosomal

break-point in human B-cell lymphoma [9]. Expression of BCL-2 proved to block cell death

following several apoptotic stimuli [10, 11]. By screening for BCL-2 interacting molecules,

several members of the BCL-2 family have been identified. Currently, up to 30 members of

the family were identified [12-14]. These members can be divided into three main sub-

classes, defined in part by the homology shared within four conserved regions termed BCL-2

homology (BH) 1-4 domains. These domains roughly correspond to α helices, which dictate

structure and function. The first group includes the anti-apoptotic molecules (e.g. BCL-2,

BCL-XL), which carry all four conserved domains (BH1-4). The second group consists of the

multi-domain pro-apoptotic molecules (e.g. BAX, BAK), which carry three BH domains

(BH1-3). The third group (which is a sub-group of the pro-apoptotic members) carries only

the BH3 death domain. This group includes among others BID [13, 15].

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Many of the BCL-2 family members are found at the mitochondria membrane either

constitutively or following a death signal. Most BCL-2 family members contain a C-terminal

hydrophobic stretch of amino acids that anchors them to membranes. Although anti-apoptotic

molecules such as BCL-2 appear to be exclusively membrane bound, particularly to

mitochondria, pro-apoptotic molecules are cytosolic and translocate to mitochondria during

apoptosis. Translocation of these proteins is triggered by specific post-translational

modifications such as dephosphorylation. For example, BAD is sequestered in the cytosol

bound to 14-3-3, and upon survival factor withdrawal it undergoes dephosphorylation and

translocates to the mitochondria [16]. It seems that the BCL-2 family members regulate

apoptosis mainly via the mitochondrial pathway. Mitochondria seem to play an important

role in apoptosis, since alteration of their membrane potential (∆Ψm) and the production of

reactive oxygen species (ROS) are early events in several apoptotic pathways. Furthermore,

during apoptosis, IMS proteins are released into the cytosol, among them Cyt c, which

initiates formation of the apoptosome leading to caspase-9 activation [17-19].

One of the major characteristics of the BCL-2 family members is their tendency to form

homo- as well as heterodimers. Mutagenesis studies have revealed that the BH domains are

important for these interactions. The BH1, BH2 and BH3 domains of the anti-apoptotic

members are required to heterodimerize with the pro-apoptotic members to repress cell death

[20]. On the other hand, only the BH3 domain of pro-apoptotic members is required to

heterodimerize with the anti-apoptotic molecules, and to promote cell death. Early findings

support the notion that the ratio of pro-apoptotic to anti-apoptotic molecules dictates the

susceptibility of cells to a death signal [21]. It seems that the multi domain pro-apoptotic

proteins possess two potentially independent mechanisms for promoting cell death. One

mechanism relies upon their ability to suppress the function of anti-apoptotic proteins through

heterodimerization. The other is a heterodimerization-independent function. For example,

BAX can induce cell death independent of its interaction with BCL-2 [22]. Several BH3

mutants of BAX that fail to bind BCL-2 are nevertheless still capable of inducing apoptosis

[23]. Furthermore, activation of BAX appears to involve its homodimerization [24]. A

proposed theory suggests that the BCL-2 family members form channels in the mitochondrial

membrane. This model originated from the structural similarity between the BCL-XL

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structure and structure of the pore forming region of bacterial toxins [25]. Moreover, it was

demonstrated that recombinant BAX forms channels in artificial membranes allowing the

passage of large macromolecules [26-29].

BCL-2 family members have essential roles in the mouse from early embryogenesis through

to adult tissue homeostasis. The nervous system, haematopoietic tissues and spermatogenesis

are particularly dependent on BCL-2 family protein regulation [30]. Several members of the

two pro- and anti-apoptotic classes have been knocked out in mice to reveal their

physiological roles, redundancy and interactions in vivo. In many cases there are phenotypes

of abnormal cell death, like in the case of knockout of the anti-apoptotic proteins BCL-2,

BCL-XL and BCL-W, or hyperplasia and increase in cell resistance to apoptotic stimuli, like

in the case of BAX, BIM, BID and NOXA knockouts [30].

BID

The pro-apoptotic BCL-2 family proteins can be further divided into those with multiple

BCL-2 homology (BH) domains, such as BAX and BAK, and those with only one type of BH

domain, the BH3-only proteins. The BH3-only proteins are essentially the sentinels to various

apoptotic signals [31]. For example, BAD is sensitive to growth factor deprivation; PUMA

and Noxa are sensitive to DNA damage; and BIM is sensitive to DNA damage, cytokine

deprivation and glucocorticoids. When activated by these signals, the BH3-only molecules

transmit the death signals to the mitochondria to initiate the mitochondria apoptosis pathway.

BID (BH3-interacting domain death agonist) is one of the BH3-only proteins. It is a 22 KDa

protein and it was first cloned in a screen based on its interaction with BCL-2 and BAX. BID

is phylogenetically conserved [32, 33]. Human BID shows 72.3% homology to murine BID

at the amino acid level. BID is widely expressed in various tissues, with the highest level

being in the kidney [32] and in organs of the hematopoetic system (Unpublished data, Gross

lab). In general, full length BID is quite stable and is a long-lived protein, but caspase-8

cleaved truncated BID (tBID) has a half-life of less than 1.5 h [34].

BID was initially found to be cleaved and activated by caspase-8 following death receptor

activation and thus considered specific to the death receptor pathway. Furthermore, the death

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activity of BID can be inhibited by BCL-2, suggesting that BID is acting via the

mitochondrial pathway. BID lacks a COOH-terminal membrane-anchoring segment, and

therefore is mainly found in the cytosol. Following activation of the TNF or Fas death

receptors, BID is cleaved by caspase-8 at Asp-59, to produce a p15 carboxy-terminal

fragment (truncated BID; tBID) that translocates to the mitochondria and induces Cyt c

release [13, 23, 35, 36]. Studies in recent years indicate that BID can be cleaved by other

proteases such as Granzyme B, Calpains and Cathepsins. These proteases are first activated

in response to many types of stimuli, including death receptor activation, cytotoxic T cell

attack, ischemia/reperfusion injury and lysosome damage [34]. These observations indicate

that BID is in general a sentinel to protease activation resulting from various injury stimuli.

As such, BID serves a critical role in connecting these stimuli to the mitochondria, thus

allowing the death process to be advanced and amplified.

Although most studies in the field emphasize the importance of BID cleavage in order to

activate it, there are several studies that demonstrated an apoptotic role for full-length (FL)

BID [32, 37-39]. A study that was conducted in our lab has demonstrated that a caspase-8

non-cleavable BID mutant (ncBID) is a potent inducer of apoptosis in mouse embryonic

fibroblasts (MEFs) [37]. These studies were performed with recombinant adenoviruses

carrying a tetracycline-inducible ncBID, wtBID or GFP vector and it was demonstrated that

cell death in these instances was due specifically to overexpression of nc/wtBID, since

overexpression of GFP had little effect on the viability of cells. It was also shown that both

ncBID and wtBID were much less effective than tBID in inducing Cyt c release, but only

slightly less effective in inducing apoptosis. Expression of non-apoptotic levels of both

ncBID and wtBID in BID-/- MEFs induced a similar and significant enhancement in apoptosis

in response to a variety of death signals. The most prominent cell death was with DNA-

damaging reagents such as Etop [a topoisomerase II inhibitor that forms DNA double strand

breaks (DSB)] and cisplatin [that forms covalent adducts with DNA]. In the same study from

our lab it was found that BID-/- MEFs are much less susceptible to apoptosis induced by Etop

and ionizing radiation (IR), which are two treatments known to induce DSBs in DNA, and

that ncBID was capable of restoring sensitivity to these cells. In addition, endogenous BID

was found to be rapidly phosphorylated in response to Etop and IR. Finally, BID was not

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phosphorylated in ATM-/- MEFs, indicating that phosphorylation of BID is mediated by the

ATM kinase. Based on these studies, we hypothesized that FL-BID plays a role in the DSB

DNA damage pathway.

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The objective of the research To define the role of FL-BID and specifically of BID phosphorylation in the ATM-dependent

cellular response to DSB DNA damage.

The results of the research are presented in three chapters:

Chapter I - Characterization of BID’s phosphorylation and function in cells.

Chapter II - Identification of proteins that interact with phosphorylated BID.

Chapter III - Exploring the connection between ATM and mitochondria function.

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Materials and Methods

Tissue culture

Mouse embryonic fibroblasts

BID-/- mice (originally kept on a mixed C57BL/6 x 129Sv background) had been bred to wild

type C57BL/6 mice twelve times in order to obtain animals that are F12 on a C57BL/6

background. BID-/- MEFs were generated from the F12 mice. BID+/+ and BID-/- primary

MEFs were prepared from 11-13 day-old embryos, and maintained in ISCOVE’s medium

containing 10% fetal bovine serum (MEF medium). Atm/Arf double knockout and

Atm+/+Arf-/- MEFs were obtained from Chuck J. Sherr (St. Jude Children's Research

Hospital).

hTERT transformation of primary MEFs

All the studies with BID+/+ and BID-/- MEFs described in this research were performed with

hTERT-immortalized MEFs. Immortalization of primary MEFs was performed by

transformation with hTERT (the catalytic subunit of human telomerase). PA317 packaging

cells stably producing pBABE-puro hTERT viral particles (a generous gift from Tej Pandita,

Washington University) were grown to 80% confluence, rinsed and the medium was then

replaced with complete MEF medium. The cells were incubated for 16 hrs and the medium

was collected and filtered through a 0.45 µm filter. The infecting media were stored at -80˚C

until use. Primary BID-/- and BID+/+ MEFs were grown for 3 passages and then infected at

~50% confluence with 3 ml infecting media mixed with 3 ml MEF media and 4 µg/ml

polybrene (Sigma). The cells were then incubated for 16 hrs, rinsed and incubated in fresh

medium for an additional 8 hrs. The cells were infected again as described above, rinsed and

incubated in fresh medium for an additional 48 hrs. The cells were then split 1:3 and grown

for 4 days in a selection medium containing 1 µg/ml puromycin. After selection, the cells

were washed once and incubated with MEF medium (without puromycin). Stable clones were

collected 14 to 18 days post-infection, and their propagation took 3-to-4 months.

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Generation of hTERT BID-/- stable clones expressing wtBID or the BID-S61A/S78A

mutant

ψNX cells (a 293T cell line carrying an ecotropic packaging plasmid) were seeded in a 100

mm plate at 60% confluence. The next day, the medium was replaced and cells were

incubated with a transfection cocktail containing 15 µg retroviral vector (pBABE-wtBID or

pBABE-BID-S61A/S78A) prepared using a calcium phosphate kit (Promega). Cells were

incubated for 5 hrs with the cocktail and rinsed; the medium was then replaced, and the cells

reincubated in fresh medium. The following day, the conditioned medium containing the

retroviruses was collected and filtered through a 0.45 µm filter. The viruses were divided into

aliquots and frozen at -80˚C. For infection of MEFs, cells were grown in a 60mm plate and

incubated for 4 hrs at 37˚C in 3 ml of retroviral supernatant, supplemented with 16 µg/ml

polybrene. After that, 7 ml of DMEM containing 10% FCS was added and 24 hrs later, the

medium was replaced with fresh medium containing 10% FCS and 1 µg/ml puromycin.

Puromycin was replaced every day for three days. On the fourth day, cells were seeded (100

cells per 10cm dish) and grown until single clones appeared.

Preparation of BID recombinant adenoviruses and infection of MEFs

For the expression of BID in BID-/- MEFs we have produced adenovirus vectors expressing

proteins under the control of tetracycline (tet)-regulatable promoters (“tet-on”) as previously

described [37]. Briefly, in these constructs, which rely on the reverse tet transactivator

(rtTA), the E1 region of the virus was replaced with either wild-type (wt) BID or GFP.

Viruses were grown using 293T cells. Virus preparations were made from freeze/thaw lysis

of the cells, and virus titers were done on 293T cells. In experiments, cells were generally

seeded at 70-80% confluence. Cells were infected with an MOI (multiplicity of infection) of

100 with BID containing virus and the rtTA containing virus. 1 µg/ml doxycycline (a

synthetic analog of tetracycline; Sigma) was added to the medium 12-to-15 hrs post infection

to activate gene expression from the tet-inducible promoter. Efficiency of infection was

determined using the recombinant adenovirus carrying the inducible expression vector of

GFP and was in the range of 70-to-90%.

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Human cell lines and transient transfection

293, a human embryonic kidney cell line, and HeLa, a human cervical adenocarcinoma cell

line were maintained in 10% fetal bovine serum. Transient transfections were performed by

using a calcium phosphate kit (Promega) or with lipofectamine 2000 (Gibco BRL).

HeLa BID KD cells

Human cervical adenocarcinoma cell line (HeLa) was stably transfected with BID SiRNA or

scrambled SiRNA as a control. These cells were generated in the lab of Dr. Jochen Prehn,

Dublin.

ATM KD cells

Stable ATM knocked down HeLa and 293 cells were generated by the siRNA approach using

the pRETRO-SUPER viral vector. The cells were grown under selection of 10 µg/ml

puromycin and 200 µg/ml hygromycin. These cells were generated by Prof. Yossi Shiloh,

Tel-Aviv University.

A-T Lymphoblasts

A-T lymphoblasts from an A-T patient along with lymphoblasts from a healthy individual

(obtained from Prof. Yossi Shiloh, Tel Aviv University) were cultured in RPMI medium

supplemented with 15% FCS, 100 units/ml penicillin, 100 mg/ml streptomycin and 2 mM

glutamine.

A-T Fibroblasts

SV40 transformed A-T fibroblasts stably transfected with an empty vector or an ATM vector

were obtained from Prof. Yossi Shiloh, Tel Aviv University [40]. The cells were cultured in

complete DMEM supplemented with 100 mg/ml hygromycin.

Treatments

Etop (0.1 M stock solution in dimethyl sulfoxide), Cisplatin (10 mg/ml stock), TNFα (40

ng/ml), Staurosporine ( 4 mM stock) and Thapsigargin (2 mM) were all purchased at Sigma.

19

Clonogenic survival assays

Cells were seeded at a density of 1000 cells per well (6 well plate). The next day, cells were

treated with the indicated DNA damage reagent, the medium was replaced with fresh

medium, and cells were incubated for 10 days. Once colonies were formed, cells were fixed

in 70% methanol and stained with 0.5% crystal violet. The percent of colony survival was

calculated as the ratio of the number of colonies after DNA damage to the number of colonies

in untreated cells.

Immunocytochemistry (immunofluoresence)

For immunocytochemistry, cells were grown on glass cover slips. At the designated time

points, the cells were fixed with 4% paraformaldehyde in PBS for 10 min and permeabilized

with 0.2% Triton X-100 in PBS for 5 min. For blocking, the cells were incubated in PBS

containing 0.1% Triton and 3% BSA for 1 hr at room temperature. For immunostaining, cells

were incubated overnight at 4˚C with either anti-murine BID Abs or the anti-pS61/pS78 Abs

in blocking solution. After three washes with PBS containing 0.1% Triton, the cells were

stained for 1 hr at room temperature with Alexa 488-labeled goat anti-rabbit Abs (dilution

1:120, Molecular Probes), followed by 5 min of 4’,6-diamidino-2-phenylindole

dihydrochloride (DAPI) staining (10 µg/ml). The coverslips were mounted with elvanol, and

the cells were viewed under a Nikon fluorescence microscope at a magnification of

200x/400x. Pictures were taken with a 1310 digital camera (DVC).

FACS analysis

BrdU labeling and analysis

A total of 2 x 105 cells were treated with 20 µM Etop for 2 hrs, washed twice with PBS and

incubated in fresh medium (10% FBS) for 8 hrs. The cells were then pulsed labeled with 10

µM BrdU (Sigma; added to the medium) for 30 min, washed with PBS, fixed with cold 70%

ethanol and incubated overnight at -20˚C. The next day, cells were collected and resuspended

in 2N HCl with 0.5% Triton X-100 for 30 min at room temperature followed by

neutralization with 0.1 M Na2B4O7. Cells were then collected and incubated with anti-BrdU

20

Abs (Becton-Dickinson) for 30 min in the dark at room temperature. The cells were washed

with PBS, and stained with FITC labeled goat anti-mouse Abs (Jackson) for 30 min at room

temperature in the dark. The cells were then resuspended in PBS containing PI (5 µg/ml) and

analyzed by FACScan. To evaluate cells that were in S phase, cells were gated on the BrdU+

population and DNA content was evaluated by PI.

In the BrdU pulse-chase experiments (to follow the progress of cells through S phase), cells

were treated with 20 µM Etop for 2 hrs and labeled immediately with 10 µM BrdU for 30

min. Cells were then washed, incubated in fresh medium for the indicated time points, and

fixed for BrdU analysis as described above. The percentage of BrdU positive cells in early S

phase and in late S/G2 phase was determined by PI counterstaining.

Cell viability assays

Cell viability was determined by propidium iodide (PI) dye exclusion. PI (25 µg/ml) was

added to the cells immediately prior to analysis by FACScan (Beckton Dickinson).

Cell cycle assays

3*105 cells were seeded in 6cm dish. Cells were treated with 20 µM Etop for 2 hrs, rinsed,

and then released into drug-free medium. 8 or 24 hrs after release, cells were collected for

fixation in methanol. Following fixation, cells were washed and resuspended in PBS with 25

µg/ml propidium-iodide (PI) and 50 µg/ml RNAse a half hour before FACScan analysis.

Analysis of the cell cycle results was performed using the ModFit LT program [41].

Apoptosis assays

3*105 cells were seeded in 6cm dish or in 6 well plate. Cells were treated for the indicated

times, harvested and stained as above. The percent of cells displaying sub-G1 DNA content

was determined by FACScan analysis.

Synchronization of cells in G1-S phase

4*105 cells were seeded in 10 cm dishes and grown until they reached confluency. Cells were

trypsinized and seeded into 10 µM Aphidicolin-containing medium. The cells were incubated

for 20 hrs and released into drug free medium for further treatments.

21

Determination of mitochondrial membrane potential

3*105 cells were seeded in 6cm dish. The next day cells were incubated for 15 min at 37°C

with 3,3’-dihexyloxacarbocynine iodide [DiOC6(3), 40nM, Calbiochem] followed by

FACScan analysis.

Determination of cellular ROS levels

3*105 cells were seeded in 6cm dish. The next day cells were incubated for 15 min at 37°C

with DCF-DA (2’,7’-dichlorofluorescein) or HE (Hydroethidine) (Molecular probes) to check

levels of H2O2 and superoxide, respectively. Cells were harvested and ROS levels were

determined by FACScan analysis.

Proteins analysis

Generation of phospho-specific antibodies

Anti-pS61 and anti-pS78 were generated in collaboration with Bethyl Laboratories, Inc.

(Montgomery, TX). Briefly, immunogens were phosphorylated synthetic peptides, which

represented portions of mouse BID around either serine 61 or serine 78. Antibodies that were

not phospho-specific were removed by solid phase absorption. Antibodies that were specific

for either BID pS61 or BID pS78 were affinity-purified using the phosphopeptide

immobilized on solid support.

HA-affinity chromatography

BID-/- MEFs were infected with adenoviruses containing HA-tagged BID. The next day, the

cells were either left untreated or treated with Etop, lysed (using 1% CHAPS) and incubated

for 2 hrs with beads coupled to anti-HA antibodies (Roche). The beads are then collected and

BID-HA was eluted using an HA peptide (1mg/ml; Roche). The eluted sample was

concentrated using centricon, separated by SDS-PAGE and a sample taken to Western blot

analysis. The gel was stained using gelcod (Pierce) and bands were ssent to mass

spectrometry analysis at the Smoler proteomic center of the Technion.

Cross-linking with BS3

293T cells were transfected with pcDNA3 BID-HA and treated with Etop. Cells were than

22

harvested and treated with digitonine for 10 min on ice in digitonine buffer (20 mM HEPES,

pH 7.3, 110 mM Kacetate, 5 mM NaAcetate, 2 mM MgAcetate, 1mM EGTA, 2 mM

dithiothreitol). The membrane fraction was separated from the cytosolic fraction by

centrifugations. BS3 (bis(sulfosuccinimidyl) suberate) (Pierce) was added from a 10-fold

stock solution to a final concentration of 10 mM. The cross-linker was added either to the

cytosolic/soluble fraction or to the membrane-enriched fraction. After incubation of 30 min at

room temperature, the cross-linker was quenched by addition of 1 M Tris-HCl pH 7.5 to a

final concentration of 20 mM. The cross-linked samples were lysed with RIPA (150 mM

NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 50 mM TRIS, pH 8.0) and IP experiment with anti-

HA antibodies was conducted (as described above) and Western blot analyzed with the

indicated antibodies.

Cross-linking with Formaldehyde

293 cells were transfected with pcDNA3 BID-HA and treated with Etop. Cells were

incubated with 1% formaldehyde for 10 min in RT. The cross-linker was quenched by

addition of 125 mM Glycine for 5 min and cells were harvested and lysed with RIPA buffer

(as described above). IP experiments with anti-HA antibodies were conducted as described

above and Western blot analyzed with the indicated antibodies.

Formaldehyde treatment and subcellular fractionation

Formaldehyde was added directly to the tissue culture media to a final concentration of 1%

and the cells were incubated for 10 min at room temperature. The cross-linking reaction was

stopped by adding glycine to a final concentration of 0.125 M and incubation at room

temperature for 5 min. Cells were then subfractionated as previously described [42]. Cells

were rinsed with wash buffer (125 mM KCl, 5 mM magnesium acetate, 5 mM EGTA, 1 mM

β−mercaptoethanol, 30 mM Tris-HCl, pH 7.5) at 4˚C, scraped from the plates, washed twice

with the same buffer and allowed to swell for 10 min in 0.5 ml swelling buffer [same as wash

buffer except that the KCl concentration was 10 mM and protease (set III; Calbiochem) and

phosphatase (set I and II; Sigma) inhibitor cocktails were added]. The cells were then lysed in

a 2-ml Wheaton Dounce glass homogenizer using 30 complete up and down cycles of a glass

“B”-type pestle. The homogenate obtained was overlaid on an equal volume of swelling

buffer containing 25% glycerol and centrifuged (600 x g at 4˚C for 5 min). The

23

upper layer of the supernatant was designated the cytosolic fraction. It should be noted that

all organelle membranes (besides the nuclear membrane) and the plasma membrane are

contained in this fraction. The nuclear pellet was washed once with swelling buffer

containing 25% glycerol and 0.1% Triton X-100. Nuclei were resuspended in sonication

buffer (100 mM NaCl, 2 mM MgCl2, 5 mM EGTA, 1 mM β−mercaptoethanol, 10 mM Tris,

pH 9.0). At this stage both the cytosolic and nuclear samples were incubated at 65˚C for 4-5

hrs to reverse formaldehyde cross-links. Nuclei were then disrupted by brief sonication.

Aliquots of nuclear and cytosolic fractions were separated by 12% or 15% SDS-PAGE and

transferred to PVDF membrane (Immun-blotTM, Bio-Rad).

Western blot

Proteins were size-fractionated by SDS-PAGE and then transferred to PVDF membranes

(BioRad). Western blots were developed by use of the enhanced chemiluminescence reagent

(Amersham Bioscience, Inc).

Alkaline or potato-acid phosphatase treatment

MEFs were treated with Etop for either 30 or 60 min, lysed in phosphatase buffer (150 mM

NaCl, 1% CHAPS, 10 mM HEPES, pH 7.5) and either left untreated or incubated for 30 min

at 37˚C with either alkaline phosphatase (1U/1µg protein; Roche) or with potato-acid

phosphatase (PAP; 1.5U/30µg protein; Sigma). The reaction with PAP was performed in a

phosphatase buffer adjusted to pH 5.5. At the end of the reaction, the lysates were analyzed

by Western blot using the indicated Abs.

Purification of the HA-BID cross-linked complex

100-300x10cm plates of 293T cells were transiently transfected with pcDNA3-HA-BID. 18

hrs post transfection, cells were cross-linked with BS3 or formaldehyde (as described above)

and lysed. The diluted lysate was incubated for 16 hrs with anti-HA Ab coupled to agarose

beads (Roche), followed by extensive washing of the beads with binding buffer containing

0.05% Tween 20. The material that remained bound to the beads was eluted by incubation

with 1 ml (1 mg/ml) HA peptide (Roche) for 15 min at 37ºC. Elution was repeated twice

more, and the three eluents were pooled and concentrated using a Centricon tube with a 3

KDa cutoff (Amicon). The concentrated material was loaded onto a single lane and separated

24

by SDS-PAGE followed by staining the gel with Imperial stain (Pierce).

Mass spectrometry analysis

The stained protein bands in the gel were cut with a clean razor blade and sent in separate

tubes to analysis. Mass spectrometry analysis was performed at the Smoler Protein Center,

Technion, Haifa.

25

Chapter I - Pro-apoptotic BID is an ATM effector in the DNA damage

response

Introduction

ATM and the response of cells to DNA DSBs

The genome of each cell of an organism is constantly subjected to DNA damage. DNA DSBs

are a particularly deleterious form of DNA damage and if left unrepaired can result in cancer-

causing mutations or promote aging. DNA DSBs occur as a result of oxidative metabolism,

DNA replication, or V(D)J recombination during immune system maturation; they also can

arise from exogenous agents such as IR or Etop.

Following DSBs, the cell activates a survival system that allows repair and continuation of its

normal life cycle, or it may activate the apoptotic machinery in the face of extensive or

irreparable damage [43]. The mechanism of this decision is under intense investigation. One

of the major responses associated with the survival network is the temporary arrest of cell-

cycle progression, which reflects the activation of cell cycle checkpoints [44]. The best-

documented, damage-induced cell-cycle checkpoints operate in the G1/S boundary, and at the

S and G2 phases. Upon introduction of DNA DSBs, the early events involve several proteins

that are rapidly recruited to the damaged sites, where they form prominent nuclear foci.

Among them is a trimolecular complex containing the MRE11, RAD50 and NBS1 proteins

(MRN complex) that fulfills many of the criteria for a DSB sensor [45]. The concept is that

sensor molecules are the first to sense the lesion and help convey a damage signal to

transducers, which in turn deliver it to numerous downstream effectors. A prototype

transducer is ATM, which is a nuclear serine-threonine protein kinase [46].

The ATM protein was identified as the product of the gene that is mutated (lost or

inactivated) in the human genetic disorder ataxia-telangiectasia (Α−Τ) [46]. Α−Τ is

characterized by cerebellar degeneration, which leads to neuromotor dysfunction,

immunodeficiency, genomic instability, thymic and gonadal atrophy, predisposition to

lymphoreticular malignancies and sensitivity to ionizing radiation and DSB-inducing agents.

26

Cells from Α−Τ patients exhibit a variety of abnormalities, including genomic instability,

radiosensitivity, and defective activation by DSBs of cell-cycle checkpoints. ATM is a

member of a group of conserved large proteins, most of them protein kinases involved in

mediating DNA damage responses. These proteins share several motifs, among them a

domain containing a PI3-kinase signature, which gives this group the title, "PI3-kinase-

related protein kinases" (PIKKs). Additional proteins of this family are ATR (ATM and RAD

3 related), DNA-PKCs and m-TOR.

DSBs mobilize a signaling network by activating the ATM protein kinase, which, in turn,

activates this network by phosphorylating key proteins with specificity for serine/threonine

followed by glutamine [47-49]. The activation of ATM results in a rapid intermolecular

autophosphorylation of ATM on serine 1981 that causes dimer dissociation and initiation of

ATM's kinase activity [50]. It was found that nearly the entire nuclear pool of ATM

molecules was phosphorylated on Ser1981 within minutes of cellular exposure to low doses

of IR that induced only a few DSBs [50]. The rapid and strong activation of the ATM kinase

seems to be an initiating event in cellular responses to DSBs and is required for cell cycle

arrest at the G1, S and G2 phases. Several substrates of ATM that participate in these

checkpoints include p53, Mdm2 and Chk2 in the G1 phase [51], Nbs1, Brca1 and SMC1 in

the S-phase and Brca1, Chk2 and hRad17 in the G2/M phase [52]. In addition to ATM’s

versatility as a protein kinase with numerous substrates, the ATM web contains protein

kinases that are themselves capable of targeting several downstream effectors simultaneously.

Chk1 and Chk2, a checkpoint kinases, are examples of such effectors [53].

BCL-2 family members and the response to DSBs

There are several reports that connect BCL-2 family members to the non-apoptotic response

of cells to DSBs. For example, homology-directed repair of DSBs is enhanced by the anti-

apoptotic BCL-XL protein [54], while overexpression of pro-apoptotic BAX and BID, was

found to inhibit homologous-recombination DNA repair [55]. In addition, a protein essential

for DSB repair, Ku70, was demonstrated to hold BAX in an inactive state [56]. BAX and

BCL-2 were previously reported to be localized to the nucleus in certain cells [57, 58];

however their roles in this organelle remain unknown. Recently it was published that

27

exposure of cells to IR increases the expression of BCL-2 in the nucleus, which interacts and

inhibits both Ku70 and Ku86 via its BH1 and BH4 domains [59]. Removal of the BH1 or

BH4 domain abrogated its inhibitory effect, which results in the failure to block DSB repair

as well as V(D)J recombination. In addition, it was found that BCL-XL colocalizes and binds

to cdk1(cdc2) during the G2/M cell cycle checkpoint, and its overexpression stabilizes a

G2/M arrest/senescence program in surviving cells after DNA damage [60]. With respect to

BID, BID-/- mice, as they age, have been shown to spontaneously develop a clonal

malignancy closely resembling chronic myelomonocytic leukemia (CMML), which

demonstrates consistent chromosomal abnormalities [61]. These results suggested that BID

might play an unanticipated role in regulating genomic stability. Thus, both pro- and anti-

apoptotic BCL-2 family members might also play a non-apoptotic role in the response of

cells to DSBs and other forms of DNA damage.

28

Results

Most of the results presented in this chapter were published in a manuscript entitled: “Pro-

apoptotic BID is an ATM effector in the DNA damage response”. Cell 122: 593-603 (2005)

[62].

BID is important for DNA damage-induced apoptosis

To determine whether BID is required for DNA damage-induced apoptosis, we generated

hTERT-immortalized BID+/+ and BID-/- MEFs and analyzed their response to a variety of

DNA-damaging reagents: Etop, cisplatin (Cis; forms covalent adducts with the DNA), UV

(induces thymine dimers), and IR. We found that BID-/- MEFs were less susceptible than

BID+/+ MEFs to all four treatments (Fig 2A). These DNA-damaging reagents also induced

less cell death in primary BID-/- MEFs than in primary BID+/+ MEFs (data not shown),

confirming that this decreased sensitivity is not due to hTERT immortalization.

To confirm that BID-/- MEFs are indeed less sensitive than BID+/+ MEFs to DNA damage-

induced cell death, we performed clonogenic survival assays with MEFs following DNA

damage. This assay is commonly used in the field of DNA damage and can give an indication

on the cellular response to DNA damage treatment. Our studies showed that BID-/- MEFs

have increased clonogenic survival following IR (Fig 2B). To confirm that the reduced

susceptibility of BID-/- MEFs to DNA-damaging reagents was due to the absence of BID,

BID-/- MEFs were infected with recombinant adenoviruses carrying the BID vector prior to

treatment with Etop or IR. The results show that reintroduction of BID did not induce cell

death on its own but fully restored susceptibility to Etop- (and partially to IR-) induced cell

death (Fig 2C).

29

Figure2 : BID is important for DNA damage-induced apoptosis. (A) BID-/- MEFs are less susceptible than BID+/+ MEFs to apoptosis induced by DNA-damaging reagents. Dose-response/death curves of BID+/+ and BID-/- MEFs in response to treatment with the indicated doses of Etop (24 hrs), Cis (14 hrs), UV (14 hrs), and IR (24 hrs). Cell death was monitored by FACScan using propidium-iodide (PI) dye exclusion. The data represent the means ± SEM of pooled results from three independent experiments. (B) BID-/- MEFs have increased clonogenic survival compared to BID+/+ cells following DNA damage. 1000 cells from BID+/+ and BID-/- MEFs were seeded per well and irradiated with the indicated doses of IR. Cells were then incubated for 10 days and the percent of colony survival was calculated as the ratio of the number of colonies formed after IR to the number of colonies formed in untreated cells. * represent significant differences (p < 0.05) based on Student’s t test. (C) The reduced susceptibility of BID-/- MEFs to DNA-damaging reagents is due to the absence of BID. BID+/+ or BID-/- MEFs were either left untreated (N/T) or treated with either Etop (100 �M; 24 hr) (left) or ionizing radiation (IR; 100 Gy; 24 hr) (right) and cell death was monitored by FACScan using propidium-iodide (PI) dye exclusion. Alternatively, BID-/- MEFs were infected with recombinant adenoviruses carrying a tetracycline-inducible BID vector. Two hours after the addition of doxycyclin, the cultures were washed three times and treated with either Etop or IR. Cell death was monitored as described above. The data represent the means ± SEM of pooled results from three independent experiments.

A

B

**

*

30

DSBs induce the phosphorylation of BID, and this phosphorylation is mediated by the

ATM kinase

Next, we explored whether BID was modified in response to DNA damage. Western blot

analysis using anti-BID antibodies on lysates of hTERT-immortalized BID+/+ MEFs treated

with the DNA-damaging reagents (as described in Fig 2) revealed that Etop and IR, which are

known to induce DSBs in DNA, unlike Cis or UV, induced a double electrophoretic mobility

shift in BID (Fig 3A). We also treated MEFs with several other apoptotic reagents:

thapsigargin (Thaps; stress signaling from the ER, which inhibits the Ca2+ adenosine

triphosphate pump); TNFα together with actinomycin D; or with staurosporine (STS; a

kinase inhibitor), and found that none of them affected the electrophoretic mobility of BID

(Fig 3A). Similar mobility shifts have been associated with covalent modifications of

proteins, for example, as a consequence of phosphorylation.

To define whether the double electrophoretic mobility shift in BID was due to

phosphorylation, BID+/+ MEFs were treated with Etop for 30 min, lysed and either left

untreated or incubated with alkaline phosphatase for 30 min at 37˚C. Western blot analysis

using anti-BID antibodies demonstrated that treatment with alkaline phosphatase abolished

the electrophoretic mobility shifts in BID (Fig 3B), indicating that these shifts are most likely

due to phosphorylation. Taken together, these results strongly suggested that BID is rapidly

phosphorylated in response to reagents that induce DSBs.

The ATM kinase plays a pivotal role in the immediate response of cells to DSBs. To

determine whether ATM is involved in the phosphorylation of BID, we utilized MEFs

deficient in both ATM and the p19/ARF tumor suppressor gene, since loss of ARF has been

shown to reverse premature replicative arrest of Atm-null MEFs [63]. Accordingly, Atm/Arf

double knockout MEFs, as well as ATM+/+Arf-/- MEFs, were treated with Etop or IR, and

Western-blot-analyzed using anti-BID antibodies. Figure 3C shows that following Etop or IR

treatment, the slower migrating bands of BID do not appear in the ATM-deficient cells. Thus,

the presence of the ATM kinase appears to play an essential role in the process by which

Etop and IR induce phosphorylation of BID. To corroborate these findings, we took

advantage of a stable HeLa cell line in which ATM was knocked down by siRNA (In these

31

cells, the level of ATM was reduced by ~95% [64]). Both these cells and the control cells,

which carried a siRNA against LacZ, were transfected with mouse BID, exposed to Etop, and

Western-blot-analyzed using anti-BID antibodies. Exposure of control HeLa cells to Etop

induced a double electrophoretic mobility shift in BID that was absent in the ATM knocked

down cells (Fig 3D, left and middle panels). BID-/- MEFs were used as a specificity control

(Fig 3D, right panel). These results further confirm that the presence of ATM is essential for

BID phosphorylation.

Figure3 : DNA DSBs induce the phosphorylation of BID, and this phosphorylation is mediated by the

ATM kinase. (A) Etop and IR induce a double electrophoretic mobility shift in BID. BID+/+ MEFs were either left untreated (N/T), or treated with one of the indicated cell death stimuli: Etop (100 µM), IR (50 Gy), Cis (50 µM), UV (20 J/m2), Thaps (2 mM), TNFα (4ng/ml together with 2 µg/ml actinomycin D), and STS (4 µM). Cells were collected 1 hr later, lysed, and subjected to SDS-PAGE, followed by Western blot analysis using anti-BID Abs. The blot reprobed with anti-β-actin Abs to control for loading (lower panel). * marks a cross-reactive band. The question mark marks the BID double electrophoretic mobility shift. (B) Alkaline phosphatase treatment abolishes the Etop induced double electrophoretic mobility shift in BID. BID+/+ MEFs were treated with 100 µM Etop for 30 min, lysed and either left untreated (-), or treated with alkaline phosphatase (+) for 30 min at 37˚C, followed by Western blot analysis using anti-BID Abs. BID-P marks the BID double electrophoretic mobility shift. (C) The slower migrating forms of BID do not appear in ATM-deficient MEFs. Atm/Arf double knockout MEFs (ATM-/-) and ATM+/+Arf-/- MEFs (ATM+/+) were either left untreated (N/T), or treated with 100 µM Etop or 50 Gy IR, collected after 30 min, and lysed. Samples were subjected to SDS-PAGE, followed by Western blot analysis using anti-BID Abs. In the lower panel anti-β-actin Abs to control for loading. (D) Etop induced phosphorylation of exogenous BID is detected in LacZ, but not in ATM knocked down HeLa cells. Left panel: HeLa cells were transiently transfected with pcDNA3-wtBID. 18 hrs post-transfection, cells were either left untreated (-), or treated with 100 µM Etop for 30 min, collected, lysed and Western-blot-analyzed using anti-BID Abs. Middle panel: stable LacZ knocked down and stable ATM knocked down HeLa cells were transfected with pcDNA3-wtBID, treated with Etop, and analyzed as described for the left panel. Right panel: BID-/- MEFs treated with Etop for 30 min were used as a specificity control for the anti-BID Abs.

32

Mouse and human BID are phosphorylated on PIKK consensus sites

As mentioned in the Introduction, ATM is a member of the PIKK family. The common

phosphorylation sites for PIKKs are serines or threonines followed by glutamine residues

(SQ/TQ motif) [49]. Mouse BID carries two such motifs (S61Q and S78Q), whereas human

and rat BID carry only one (S78Q) (Fig 4A).

To determine whether mouse BID is phosphorylated on one or both of these sites, we mutated

each of these serines to alanines. Our initial analysis was performed in HeLa cells transfected

with either wild-type (wt) BID or with one of the BID mutants. Western blot analysis using

anti-BID antibodies indicated that treatment of the mentioned cells with Etop resulted in a

double electrophoretic mobility shift, which was abolished in the S61A mutant (Fig 4B). In

contrast, mutating the S78 site had no effect on the appearance of the two slower-migrating

bands. Thus, BID phosphorylation on S61 is likely the cause for the electrophoretic mobility

shift.

To confirm the results presented above and to establish whether S78 is also phosphorylated in

mouse BID in response to Etop, we generated phospho-specific antibodies to S61 and S78

(see Material and methods). We initially performed Western blot analysis with these

antibodies on lysates of MEFs. To define whether these antibodies recognize the

phosphorylated form of BID, BID+/+ MEFs were either not treated or treated with Etop for 30

min, lysed, and either left untreated or incubated with potato-acid phosphatase for 30 min at

37˚C. Western blot analysis demonstrated that anti-pS61 antibodies recognized a band of the

expected size of BID in Etop treated cells, and that treatment with potato-acid phosphatase

abolished this recognition (Fig 4C, left; note that these antibodies recognize an additional

~30kD protein that shares antigenicity with pS61-BID). Western blot analysis using the anti-

pS78 antibodies demonstrated that these antibodies recognized three bands (one strong band

and two very faint bands) in Etop treated cells; treatment with potato-acid phosphatase

abolished all three bands (Fig 4C, right). The bands identified with both antibodies

corresponded to BID, since they were not identified in BID-/- MEFs (Fig 4C).

33

Next, we determined whether mutation of either S61 or S78 to alanine abolished recognition

of mouse BID by the phospho-specific antibodies. These experiments were performed in

HeLa cells transfected with either wtBID, the BID-S61A mutant, or the BID-S78A mutant.

The anti-pS61 and anti-pS78 antibodies recognized BID in cells expressing wtBID and

treated with Etop, but not in cells expressing the BID-S61A or BID-S78A mutant,

respectively (Fig 4D).

As mentioned above, human BID carries only one PIKK consensus site (S78; Fig 4A). To

determine whether endogenous human BID is phosphorylated on S78, we performed Western

blot analysis with anti-human BID and anti-pS78 antibodies on lysates of 293T cells either

not treated, or treated with Etop. The anti-pS78 antibodies recognized a band of the size of

human BID only in cells treated with Etop (Fig 4E). Thus, human BID is also phosphorylated

on S78 in response to Etop.

34

Figure4 : Mouse BID is phosphorylated on S61 and S78 whereas human BID is phosphorylated only on

S78. (A) Mouse BID carries two PIKK consensus sites (S61Q and S78Q), whereas human and rat BID carry only one (S78Q). (B) Mutation of S61 to alanine abolishes the Etop induced double electrophoretic mobility shift in BID. HeLa cells were transiently transfected with pcDNA3-wtBID, pcDNA3-BID-S61A, pcDNA3-BID-S78A, or left untransfected (-). 18 hrs post-transfection, cells were either left untreated (-), or treated with 100 µM Etop for 30 min, collected, lysed and Western-blot-analyzed using anti-BID Abs. The blot was stripped and reprobed with anti-β-actin Abs to control for loading (lower panel). (C) The phospho-specific antibodies to serine 61 and serine 78 recognize endogenous BID in MEFs treated with Etop. BID+/+ or BID-/- MEFs were either left untreated (-), or treated with 100 µM Etop for 30 min (+), lysed, and Western-blot-analyzed using the phospho-specific Abs to either S61 (left) or S78 (right). Alternatively, BID+/+ MEFs treated with 100 µM Etop for 30 min were collected, treated with potato acid phosphatase (PAP; +) for 30 min at 37˚C, lysed and Western-blot-analyzed as above. The blots were stripped and reprobed with anti-β-actin Abs to control for loading (lower panels). BID-P marks the phosphorylated form of BID. * marks a cross-reactive band. (D) The anti-pS61 and anti-pS78 antibodies do not recognize BID-S61A or BID-S78A, respectively. HeLa cells were transiently transfected with pcDNA3-wtBID, pcDNA3-BID-S61A, or pcDNA3-BID-S78A. 18 hrs post-transfection, cells were either left untreated (-), or treated with 100 µM Etop for 30 min, collected, lysed and Western-blot-analyzed using either the anti-pS61 (left) or anti-pS78 (right) Abs. In the left panel, * marks a cross-reactive band, whereas in the right panel * marks the phosphorylated form of endogenous human BID. Note that the anti-pS78 Abs also recognized the lower of the three bands in HeLa cells that were not treated with Etop, indicating a basal level of phosphorylation in healthy cells. The blots were stripped and reprobed with anti-β-actin Abs to control for loading (lower panels). (E) Human BID is phosphorylated on S78 in response to Etop. 293T cells were either left untreated (-), or treated with 100 µM Etop for 1 hr (+), lysed, and equal amounts of protein were subjected to SDS-PAGE, followed by Western blot analysis using either anti-pS78 Abs (top) or anti-human BID Abs (middle). The blot was stripped and reprobed with anti-β-actin Abs to control for loading (bottom).* mark cross-reactive bands.

35

Characterization of endogenous BID phosphorylation by using the phospho-specific BID

antibodies

To determine whether endogenous mouse BID is phosphorylated on S61 and S78 in an ATM-

dependent manner, we utilized the phospho-specific antibodies for Western blot analysis of

Atm/Arf double knockout and ATM+/+Arf-/- MEFs either not treated, or treated with Etop.

This analysis demonstrated that endogenous mouse BID is phosphorylated on S61 and S78

only in ATM+/+Arf-/- MEFs treated with Etop (Fig 5A).

To show that phosphorylation of mouse BID was specific for reagents inducing DSBs, we

treated MEFs with several DNA-damaging and other apoptotic reagents (previously

described in Fig 3). Post-treatment, cells were lysed, and the phosphorylation of endogenous

mouse BID was examined by Western blot analysis using anti-pS61 antibodies. These results

demonstrated that mouse BID is phosphorylated on S61 only in response to reagents that

induce DSBs (Fig 5B).

Finally, to define whether phosphorylation of human BID was also ATM-dependent and

occurred only in response to reagents that induce DSBs, we took advantage of a stable 293T

cell line in which ATM was knocked down by siRNA (these cells were generated like the

HeLa ATM knocked down cells) [64]. These cells and the control cells, which carried a

siRNA against LacZ, were exposed to Etop, IR, UV, or STS, and Western-blot-analyzed

using anti-pS78 antibodies. Exposure of LacZ knocked down cells to Etop or IR, but not to

UV or STS, induced phosphorylation of endogenous human BID on serine 78, which did not

occur in the ATM knocked down cells (Fig 5C).

36

Figure5 : Characterization of endogenous BID phosphorylation using the anti-phospho S61 and S78

Abs. (A) Endogenous mouse BID is phosphorylated on S61 and on S78 in an ATM-dependent manner. Atm/Arf double knockout MEFs (ATM-/-) and ATM+/+ARF-/- MEFs (ATM+/+) were either left untreated (-), or treated with 100 µM Etop for 30 min, collected, lysed, and Western-blot-analyzed using either anti-pS61 (left) or anti-pS78 (right) Abs. The blots were stripped and reprobed with anti-β-actin Abs to control for loading (lower panels). * marks a cross-reactive band. (B) Mouse BID is phosphorylated on S61 only in response to reagents that induce double-strand breaks in DNA. BID+/+ MEFs were either left untreated (N/T), or treated with the death stimuli indicated in Fig 2A. Cells were collected 1 hr later, lysed, and subjected to SDS-PAGE followed by Western blot analysis using anti-pS61 Abs (top). The blot was stripped and reprobed with anti-β-actin Abs to control for loading (bottom). * marks a cross-reactive band. (C) Phosphorylation of S78 in endogenous human BID is ATM-dependent, and occurs only in response to reagents that induce DSBs in DNA. Stable LacZ knocked down and stable ATM knocked down 293T cells were either left untreated (N/T), or treated with Etop (100 µM), IR (50 Gy), UV (20 J/m2), or STS (4 µM). Cells were collected after 1 hr, lysed, and subjected to SDS-PAGE followed by Western blot analysis using anti-pS78 Abs (top). The blots were stripped and reprobed with anti-β-actin Abs to control for loading (bottom).

37

Phosphorylation of S61 and S78 is transient, rapid and occurs many hours before the onset

of Etop- induced apoptosis

To determine the time course of endogenous mouse BID phosphorylation, BID+/+ MEFs were

treated with Etop and Western-blot-analyzed with the anti-BID, the anti-pS61, or the anti-

pS78 antibodies. Phosphorylation of S61 was detected by 15 min (the first time point

analyzed), reached a peak at 1 hr, and was reduced by 3 hrs post Etop treatment (Fig 6A).

Phosphorylation of S78 was also transient (peak at 2-3 hrs), though was somewhat delayed,

compared to phosphorylation of the S61 site. Next, we determined when apoptosis began in

MEFs treated with Etop, and found that the onset of apoptosis occurred between 8 and 12 hrs

following Etop treatment (Fig 6B). These cells were also Western-blot-analyzed with anti-

cleaved caspase-3 Abs, and as expected, the appearance of cleaved caspase-3 was detected 8

hrs post Etop treatment (Fig 6B, lower panel). Thus, BID phosphorylation occurs many hours

prior to the onset of apoptosis.

Figure6 : Phosphorylation of S61 and S78 is transient, rapid and occurs hours before the onset of Etop-

induced apoptosis (A) Time course of Etop-induced phosphorylation of endogenous mouse BID on S61 and S78. BID+/+ MEFs were either left untreated (N/T), or treated with 100 µM Etop, collected at the indicated time points, lysed, and equal amounts of protein were subjected to SDS-PAGE followed by Western blot analysis using either anti-BID (top), anti-pS61 (middle top), or anti-pS78 (middle bottom) Abs. The blot was stripped and reprobed with anti-β-actin Abs to control for loading (bottom). * marks a cross-reactive band. (B) Time course of Etop induced apoptosis of MEFs. BID+/+ MEFs were treated with 100 µM Etop, collected at the indicated time points, and cell death was monitored by PI dye exclusion assay in FACS. These samples were also Western-blot-analyzed using anti-cleaved caspase 3 Abs (lower panel).

38

Phosphorylation of S78 does not depend on phosphorylation of S61

Since the time course of S78 phosphorylation is slower then S61 phosphorylation (Fig 6A),

we decided to check whether the phosphorylation on S78 requires initial phosphorylation of

S61. BID-/- MEFs were infected with wtBID or BID-S61A. 18hrs post infection, cells were

either left untreated or exposed to Etop for different time points. Cells were lysed, collected

and Western-blot-analysed using phospho-specific Abs to S78 (Fig 7). The results clearly

show that phosphorylation of S78 occurs in cells transfected with BID-S61A (lower panel).

However, as expected, there is no electrophoretic mobility shift when using this mutant since

phosphorylation on S61 is the cause for this shift (see Fig 4B). Moreover, the time course of

phosphorylation of S78 in these cells was similar to the time course of wtBID

phosphorylation. Thus, phosphorylation of S78 does not require the initial phosphorylation of

S61.

Figure7 : Phosphorylation of S78 does not depend on the phosphorylation on S61. BID-/- MEFs were infected with WT BID or BID-S61A. Cells were treated with 100 µM Etop for the indicated time points, and lysed; equal amounts of protein were subjected to SDS-PAGE followed by Western blot analysis using anti-pS78 Abs.

39

The phosphorylation of BID occurs in response to extremely low, non-apoptotic levels of

IR and it is dose-dependent

We previously determined that phosphorylation of BID on both S61 and S78 occurs several

hours prior to the onset of apoptosis (Fig 6). We therefore speculated that phosphorylation

might also occur in response to extremely low levels of IR, which do not result in apoptosis.

Indeed, we found that a 25-fold lower dose of IR (0.2 Gy) was sufficient to induce

phosphorylation of BID (Fig 8A). The level of BID phosphorylation increased with IR levels.

Since the phosphorylation on S78 is slower, we treated cells with IR (from 1 Gy up to 20 Gy)

and checked phosphorylation on S78 after 1 hr. The results show that also the

phosphorylation on S78 is detectable at low levels of IR and is increased with the increase of

IR levels (Fig 8B). The same results were obtained with increasing levels of Etop (data not

shown). Thus, these results suggested that phosphorylated BID might play a non-

apoptotic/pro-survival role in the DNA damage response.

Figure8 : Phosphorylation of BID occurs in response to non-apoptotic levels of IR and it is dose-

dependent. (A) BID+/+ or BID-/- MEFs were either left untreated (N/T), or treated with the indicated doses of IR, collected 30 min later, lysed, and analyzed with anti-pS61 Abs. (B). BID+/+ or BID-/- MEFs were either left untreated (N/T), or treated with the indicated doses of IR, collected 1 hour later, lysed, and analyzed with anti-pS78 Abs.

40

BID-/- MEFs expressing a non-phosphorylatable BID mutant (S61A/S78A) are more

susceptible to Etop- induced apoptosis than those expressing wtBID

To explore the role of BID phosphorylation in cells, we generated wtBID and BID-

S61A/S78A stable clones by retroviral infection of BID-/- MEFs. First, we assessed the levels

of apoptosis in both type of clones either not treated, or treated with Etop. Our results

indicated that Etop induced a significantly higher rate of apoptosis in the BID-S61A/S78A

clones than in wtBID clones (Fig 9A). Of note, UV and TNFα, that do not induce

phosphorylation of BID, did not induce increased apoptosis in the mutant BID clones (Fig

9A). Western blot analysis using anti-BID antibodies indicated that the increase in apoptosis

seen in the mutant BID clones in response to Etop was not due to either higher levels of

expression of mutant BID, or to its enhanced cleavage to tBID (Fig 9B). Thus, the mutant

BID clones were found to be more susceptible to apoptosis induced solely by a reagent that

leads to DNA DSBs. This result can imply that the phosphorylation of BID either suppresses

a pro-apoptotic function or induces a pro-survival function of BID.

Figure9 : BID-S61A/S78A clones are more susceptible to Etop induced apoptosis than wtBID clones. (A) wtBID or BID-S61A/S78A clones were either left untreated (N/T), or treated with Etop (50 µM; 18 hrs), UV (20 J/m2; 24 hrs), or TNFα (4 ng/ml together 2 µg/ml with actinomycin D; 4.5 hrs). Cell death was monitored by FACS using PI. The data represent the means ± SEM of pooled results from three independent experiments. (B) The enhanced death obtained with the BID-S61A/S78A clones is not due to higher expression of mutant BID or to more cleavage to tBID. The wtBID and mutant BID clones were treated either with 50 µM Etop for 18 hrs, or with TNFα/ActD for 4.5 hrs, lysed, and subjected to SDS-PAGE, followed by Western blot analysis using anti-BID Abs. For the TNFα treatment, only clones #1 are shown. The blots were stripped and reprobed with anti-β-actin Abs to control for loading (lower panels).

A B

41

BID-/- MEFs fail to accumulate in the S and G2 phases of the cell cycle following Etop

treatment

The functional consequences of certain ATM phosphorylation events include activation of

cell cycle checkpoints, which result in temporary arrest of cell cycle progression to enable

DNA repair [44]. Since we concluded that phosphorylation of BID is not related directly to

the activation of apoptosis but might have a pro-survival role, we decided to examine the

possible involvement of BID in cell cycle arrest following DNA DSBs. First, we performed

cell cycle analyses on BID+/+ and BID-/- MEFs either not treated, or treated with a long

exposure to Etop. Surprisingly, exposure of MEFs to 20 µM Etop for 16 hrs resulted in

massive accumulation of BID+/+ MEFs in the S phase of the cell cycle, whereas such an

accumulation was not observed in the BID-/- MEFs (Fig 10A). These results suggest that BID

is important for the accumulation of cells in the S phase of the cell cycle following DSB

DNA damage. To examine the response of cells to more moderate DNA damage, we exposed

BID+/+ and BID-/- MEFs to 20 µM Etop for only 2 hrs, and released the cells into drug-free

medium after 8 and 24 hrs. We found that Etop induced accumulation of BID+/+ MEFs in the

S and G2 phases of the cell cycle after 8 hrs, whereas such an accumulation was not observed

in the BID-/- MEFs (Fig 10B). After 24 hrs, BID-/- MEFs did show a slight accumulation in S

and G2 phases.

To determine whether BID is required for S phase arrest following DNA damage, we

performed double labeling experiments with BrdU and PI to determine the level of DNA

synthesis and to follow the progress of cells through S phase after Etop treatment. The results

demonstrated that BID+/+ MEFs show a decrease in DNA synthesis (less BrdU positive cells),

whereas BID-/- MEFs fail to show a decrease in DNA synthesis (Fig 11A). Moreover, BID-/-

MEFs are not delayed in their progression form S to G2/M (Fig 11B). Taken together, these

experiments indicate that BID is required for S phase arrest following Etop treatment.

42

Figure10 : BID-/- MEFs fail to arrest in the S phase following Etop treatment (A) BID-/- MEFs do not accumulate in the S phase following long exposure to Etop. BID+/+ or BID-/- MEFs were either left untreated (NT), or treated with 20 µM Etop for 16 hrs. The DNA content was analyzed by FACS as described in the material and methods. The actual raw data from a representative experiment together with multi-line plots generated by the ModFit LT computer software program appear in the left panels. The red histograms represent the percent of cells in the G1 and G2/M phases, and the hatched histograms represent cells in S phase. The exact percentage of cells S phase is shown in the right panel. The data represent the means ± SEM os pooled results from three independent experiments. (B) BID+/+ or BID-/- MEFs were left untreated (NT), or treated with 20 µM Etop for 2 hrs, rinsed, and then released into drug-free medium. At the indicated time points, the DNA content was analyzed by FACS. The actual raw data from a representative experiment together with multi-line plots generated by the ModFit LT computer software program appear in the left panels, as describe above. The exact percentage of cells in each phase of the cell cycle is shown in the right three panels. The data represent the means ± SEM of pooled results from three independent experiments.

43

Figure11 : BID is required for S phase arrest following DNA damage. (A) BID-/- MEFs fail to decrease DNA synthesis following Etop treatment. BID+/+ or BID-/- MEFs were left untreated (NT), or treated with 20 µM Etop for 2 hrs, rinsed, and then released into drug-free medium (upper schema). 8 or 24 hrs after release, cells were pulse-labeled with BrdU for 30 min to determine DNA synthesis. The percentage of BrdU positive cells was determined by FACS. The data represent the means ± SEM os pooled results from three independent experiments. (B) BID-/- MEFs are not delayed in their progression from S to G2/M following Etop treatment. BID+/+ (left) or BID-/- MEFs (right) were either left untreated (NT), or treated with 20 µM Etop for 2 hrs, and labeled with BrdU for 30 min. Cells were then rinsed, incubated in fresh medium for the indicated time periods, and fixed. The percentage of BrdU positive cells in early S phase and in late S/G2 phase was determined by FACS. The data represent the means ± SEM of pooled results from three independent experiments.

44

BID-/- MEFs expressing BID-S61A/S78A do not accumulate in the S phase following Etop

treatment

To assess whether ATM is regulating BID’s ability to induce cell cycle arrest, we used the

wtBID and a BID-S61A/S78A stable clones described above. We initially confirmed that

BID (in the wtBID clones) was phosphorylated on S61 and S78 in response to Etop, and that

BID-S61A/S78A (in the mutant BID clones) was not (Fig 12A).

Next, we performed cell cycle analysis on two of the wtBID and two of the BID-S61A/S78A

stable clones. As shown in Figure 12B, Etop induced accumulation of the wtBID clones in

the S and G2 phases of the cell cycle (as measured 8 and 24 hrs after release into drug-free

medium), whereas the mutant BID clones bypassed accumulation in the S phase and rapidly

accumulated in the G2 phase. Thus, the mutant BID cells were found to be impaired in their

ability to temporarily arrest in S phase following DSB DNA damage.

45

Figure12 : BID-/- MEFs expressing BID-S61A/S78A do not accumulate in the S phase following Etop

treatment (A) BID-S61A/S78A expressed in BID-/- MEFs is not recognized by the phospho-specific Abs. BID-/- MEFs stably expressing either wtBID or the BID-S61A/S78A mutant were treated with 20 µM Etop for 1 hr, lysed, and subjected to SDS-PAGE, followed by Western blot analysis using either anti-BID Abs (left panel) or the phospho-specific Abs (middle and right panels). The blots were reprobed with anti-β-actin Abs to control for loading (lower panels). * mark cross-reactive bands. (B) BID-/- MEFs stably expressing BID-S61A/S78A do not accumulate in the S phase following Etop treatment. BID-/- MEFs stably expressing either wtBID or BID-S61A/S78A (two clones from each) were treated with 20 µM Etop for 2 hrs, rinsed and then released into drug-free medium. At the times indicated, the DNA content was analyzed by FACS. The actual raw data from a representative experiment together with multi-line plots generated by the ModFit LT computer software program appear in the top panel. The red histograms represent the percent of cells in the G1 and G2/M phases, and the hatched histograms represent the percent of cells in S phase. The exact percentage of cells in each phase of the cell cycle (in each of the four clones) is shown in the bottom three panels. The data represent the means ± SEM of pooled results from three independent experiments.

46

Phosphorylation of BID is not cell cycle dependent

We demonstrated that BID phosphorylation is important for S phase arrest in cells treated

with Etop (Fig 12). There are known proteins that are involved in cell cycle progression and

therefore their activity is controlled throughout the different phases of the cell cycle (e.g.,

Chk1) [65]. To assess whether phosphorylation of BID is regulated in the different stages of

the cell cycle and specifically in S phase, we synchronized cells in the G1/S boundary using

aphidicolin (a DNA polymerase α inhibitor). BID+/+ MEFs incubated with aphidicolin for 16

hrs, washed and released into drug-free medium. At the indicated time points, cell cycle

analysis was measured by FACS. As shown in Figure 13A, almost all cells were

synchronized in the G1/S boundary (time point 0) and 4 hours after release into drug-free

medium all the cells were already in S phase. To determine the effect of cell cycle

progression on BID phosphorylation, synchronized cells were treated with Etop for 1 hr, and

the level of BID phosphorylation in the different phases was determined. As shown in Figure

13B, the phosphorylation of BID on S61 and S78 did not change in the different phases.

Interestingly, synchronized cells that were not treated with Etop also showed phosphorylation

on BID, especially on S78 (Figure 13B). This phosphorylation is due to replicative stress that

is induced by aphidicolin, as previously described by Zinkel et al. [66].

47

Figure13 : BID phosphorylation is not cell cycle dependent. (A) Synchronization of cells using aphidicolin. BID+/+ MEFs were incubated with aphidicolin for 16 hrs, washed and released into drug-free medium. At the indicated time points, cell cycle analysis was done by FACS. In the upper panel histogram of DNA content as was measured with CellQuest software. The exact percentage of cells in each phase of the cell cycle is shown in the lower panel. (B) The level of BID phosphorylation on S61 and S78 does not increase during S phase. BID+/+ MEFs were incubated with aphidicolin for 16 hrs, washed and released into drug-free medium. At the indicated time points, cells were treated for 1 hr with 50 µM Etop. Cells were collected, lysed and equal amounts of protein were subjected to SDS-PAGE, followed by Western blot analysis using the phospho-specific Abs. * mark cross-reactive bands.

48

BID-/- MEFs show a delayed time course of cell death following low levels of DNA damage

Our results above demonstrate that BID is important for DNA damage-induced apoptosis on

one hand and for cell cycle arrest and inhibition of apoptosis on the other hand. Temporary

arrest of the cell cycle enables the cell to estimate and repair the damage. We assumed that

cells that do not arrest after exposure to low levels of Etop would die due to unrepaired DNA

damage. Thus, we monitored the levels of cell death in BID+/+and BID-/- MEFs several days

after exposure to lower levels of Etop and shorter duration. BID+/+ and BID-/- MEFs were

exposed to 20 µM Etop for 2 hrs, washed, and cell death was determined 2, 3, 4 and 5 days

post treatment. As expected, BID+/+ MEFs were more susceptible to Etop compared to BID-/-

MEFs (Fig 14). At first, it seemed that BID-/- MEFs are totally resistant to the treatment, but

after 5 days there was a sharp increase in cell death in the BID-/- MEFs. The levels of cell

death are relatively low, since this is the same mild treatment that we used to assess

differences in cell cycle arrest (Fig 10B). These results suggest that although BID is

important for Etop-induced apoptosis, it is also important for the arrest of cells in the S phase,

and therfore BID-/- cells eventually die. The lack of BID protein in these cells can explain the

slower kinetics in cell death compared to wt cells.

Figure14 : BID-/- MEFs show a delayed time course of cell death following low levels of DNA damage BID+/+ and BID-/- MEFs were treated with 20 µM Etop for 2 hrs, rinsed and then released into drug-free medium. Cell death was monitored by FACS using propidium-iodide (PI) dye exclusion at the indicated time points.

49

Knocking down the expression of BID in HeLa cells partially impairs Etop-induced S

phase arrest

Up until now, all our cell cycle experiments were done on hTERT-immortalized MEFs. To

assess the relevance of our results in another cell type, we used the human HeLa cell line.

These cells were stably transfected with either an SiRNA to BID or a scrambled SiRNA as a

control (Fig 15A; these cells were generated by Dr. Jochen Prehn, Dublin). We performed

cell cycle analysis on these cells that were either left untreated or treated with Etop. Etop

induced accumulation of 74% of HeLa control cells in S phase whereas only 57% of the BID

knock-down cells accumulated in the S phase (Fig 15B). There were also differences in the

G2/M phases (5% in control cells versus 15% in BID knockdown cells). These results show

that knocking down the expression of BID influences the response of HeLa cells to Etop and

that fewer cells accumulate in the S phase. The results we obtained earlier in MEFs were

more significant, probably because of the complete absence of BID.

Figure15 : Knocking down the levels of BID protein in HeLa cells partially impairs Etop-induced S phase

arrest (A) Western blot analysis showing the expression level of human BID in stable HeLa control RNAi and BID RNAi cells. (B) HeLa BID knockdown and control cells were either left untreated (NT) or treated with 100 µM Etop. 16 hrs post treatment cells were harvested for cell cycle analysis using FACS and the percentage of cells in each phase is shown. * represent significant differences (p<0.05) based on Student’s t test.

*

50

Cellular BID partially localizes to the nucleus

Next, we analyzed the cellular location of BID and its phosphorylated form. To determine the

location of BID in healthy cells, we performed immunofluoresence studies with BID+/+ and

BID-/- MEFs using anti-BID antibodies. Surprisingly, in these studies we obtained positive

staining of BID also in the nucleus (Fig 16A, left top panel). These antibodies specifically

recognized BID, since we obtained background staining in BID-/- MEFs (Fig 16A, left lower

panel). To determine whether Etop treatment leads to a change in the staining pattern of BID,

BID+/+ and BID-/- MEFs were treated with Etop for 3 hrs prior to fixation. These studies

indicated that Etop did not change the staining pattern of BID (Fig 16A, middle panel).

To assess whether BID indeed localizes to the nucleus in healthy MEFs, we performed

subcellular fractionations followed by Western blot analysis using anti-BID antibodies. In

these experiments, cellular BID was detected only in the soluble/cytoplasmic fraction (Fig

16B, left top panel). MEK and BAX (cytosolic proteins) and lamin B (a nuclear protein) were

used as markers to confirm the purity of the nuclear/cytoplasmic fractions (Fig 16B, left

bottom panel). These results together with the immunofluoresence results suggested that BID

might be loosely associated with the nuclear fraction, and that cellular disruption leads to its

dissociation from this fraction. To examine this possibility, cells were treated with

formaldehyde as a cross-linker prior to cellular disruption. These experiments demonstrated

that a small fraction of cellular BID was localized to the nuclear fraction (Fig 16B, top right

panel). These results suggest that BID is loosely associated with the nuclear fraction by

interaction with another protein(s) or with DNA, and that cross-linking is required to preserve

this interaction. To determine whether Etop treatment leads to a change in the levels of

nuclear BID, cells were treated with Etop for 1 and 3 hrs, and then treated with formaldehyde

followed by subcellular fractionation. We found that Etop did not change the levels of BID

associated with the nuclear fraction (data not shown). To determine the cellular location of

the phosphorylated forms of BID, we performed immunofluoresence studies with the

phospho-specific antibodies using BID+/+ and BID-/- MEFs either not treated or treated with

Etop for 30 min. These antibodies detected an increase in nuclear fluorescence in BID+/+

MEFs treated with Etop; however, a similar increase was detected in BID-/- MEFs (data not

51

shown). Thus, these antibodies cross-react with other phospho-S/T-Q proteins. Nonetheless,

the fact that BID partially localizes to the nucleus, and that all currently identified substrates

of ATM are nuclear proteins, suggested that BID is phosphorylated in the nucleus.

Figure16 : Mouse BID is partially localized to the nucleus. (A) Positive staining of BID in the nucleus of healthy MEFs. BID+/+ MEFs (left, top and middle panels) or BID-

/- MEFs (left, bottom) grown on glass cover slips, were either left untreated, or treated with 100 µM Etop for 3 hrs, fixed and immunostained with anti-BID Abs (green), as described in the Material and methods. The nuclei were visualized by DAPI staining (blue; right panels). (B) A small fraction of cellular BID is detected in the nuclear fraction. BID+/+ MEFs were either left untreated (-), or treated with formaldehyde (+), and subfractionated as described in the Material and methods. Aliquots of the cytosolic (C) and nuclear (N) fractions were subjected to SDS-PAGE followed by Western blot analysis using anti-BID (top), anti-MEK (middle top), anti-BAX (middle bottom), and anti-lamin B (bottom) Abs. * marks a cross-reactive band that might represent a modified form of BID.

52

BID might be involved in the immediate cellular response to DNA damage

To assess whether BID is involved in the immediate cellular response to DNA damage, we

next determined the earliest point at which BID in MEFs was phosphorylated, following

exposure to IR. Phosphorylation of S61 was detectable immediately after exposure of cells to

50 Gy IR (Fig 17A). To check other cellular markers that are known to be involved in the

immediate response to DNA damage we monitored the phosphorylation of H2AX. H2AX is

rapidly phosphorylated by ATM in the chromatin micro-environment surrounding a DNA

DSB [67, 68]. Phosphorylated H2AX, also named γH2AX, forms discrete foci at the sites of

DSBs, facilitates the recruitment of damage-responsive proteins and chromatin remodeling

complexes to the sites of DNA damage, and influences both the efficiency and fidelity of

DSB repair. We exposed BID+/+ and BID-/- MEFs to 20 µM Etop for 2 hrs, washed the cells,

and measured the levels of γH2AX 0.5, 3, 6 and 24 hrs post treatment. We found that Etop

induced the phosphorylation of H2AX in all time points that were checked and that the levels

of phosphorylated H2AX appeared to be higher in BID+/+ MEFs than in BID-/- MEFs (Fig

17B). Moreover, 24 hrs post Etop treatment there was a decrease in γH2AX levels detected in

BID-/- but not in BID+/+ MEFs. Similar differences appeared in MEFs treated with IR: 2 hrs

post irradiation, γH2AX levels appeared to be higher in BID+/+ MEFs than in BID-/- MEFs,

whereas 20 hrs post Etop wash γH2AX was still detected in BID+/+ but non in BID-/- MEFs

(Fig 17C). Since γH2AX is present at DNA break foci, we performed immunofluoresence

study with α−γH2AX Abs. BID+/+ and BID-/- MEFs were either not treated or treated with 20

µM Etop for 2 hrs, washed and analyzed for γH2AX foci formation 0.5 and 2 hrs post

treatment. As expected from the Western blot analysis results, foci formation in BID+/+ MEFs

appeared earlier and stronger than in BID-/- MEFs (Fig 17D). The most prominent difference

was 30 min post Etop wash (Fig 17D, middle panel). These results suggest that BID is also

participating in the very early stages of the DSB DNA damage response. No differences in

γH2AX foci formation were detected between BID-S61/S78A and wtBID clones (data not

shown), arguing that phosphorylation of BID is not required for this process.

53

Figure17 : BID might be involved in the immediate cellular response to DNA damage (A) Phosphorylation of BID occurs very rapidly following IR. BID+/+ MEFs were either left untreated (N/T), or treated with 50 Gy IR, collected 5 min later, and lysed. Equal amounts of protein were subjected to SDS-PAGE followed by Western blot analysis using either anti-BID Abs (top) or anti-pS61 Abs (middle). The blot was stripped and reprobed with anti-α-actin Abs to control for loading (bottom). (B) The level of γH2AX is higher in BID+/+ MEFs treated with Etop compare to BID-/- MEFs. BID+/+ MEFs (right) and BID-/- MEFs (left) were either left untreated or treated with 20 µM Etop for 2 hrs. Cells were rinsed and then released into drug-free medium for the indicated time points. Level of γH2AX was determined by western blot analysis using α γH2AX Abs. (C) The level of γH2AX is higher in BID+/+ MEFs treated with IR compare to BID-/- MEFs. BID+/+ MEFs (right) and BID-/- MEFs (left) were exposed to 50 Gy. 2 and 20 hrs post irradiation cells were lysed and the level of γH2AX was determined by western blot analysis using α γH2AX Abs. (D) γH2AX focus formation in BID+/+ MEFs is more pronounced than in BID-/- MEFs. γH2AX focus formation in BID+/+ MEFs (right) and BID-/- MEFs (left) grown on glass cover slips. Cells were either left untreated or treated with 20 µM Etop for 2 hrs. Cells were rinsed and then released into drug-free medium for 0.5 hr and 2 hrs, before fixation, permeabilization, and staining with anti-γH2AX antibody.

54

CDC25A degradation seems to be less efficient in BID-/- and BID-S61A/S78A MEFs

It is well established that to enable S phase entry CDC25A phosphatase activates the cyclin-

dependent kinase 2 (Cdk2) needed for DNA synthesis. CDC25A is degraded in response to

DNA damage or stalled replication, and loss of CDC25A prevents dephosphorylation of

Cdk2 and leads to a transient blockade of DNA replication (S phase arrest) [69]. Since we

found that BID-/- MEFs fail to accumulate in the S and G2 phases of the cell cycle following

Etop treatment, we checked whether BID is involved in regulating the levels of CDC25A.

BID+/+ and BID-/- MEFs were either left untreated or treated with 20µM Etop for 1 hr, and

CDC25A levels were monitored 0.5, 1.5 and 4 hrs post Etop wash. In BID+/+ cells, CDC25A

degradation was detected immediately post Etop exposure (Fig 18A). In BID-/- MEFs

CDC25A was degraded immediately after Etop exposure but it didn’t seem to be degraded

further as in BID+/+ cells. We also checked the degradation of CDC25A in the wtBID and

BID-S61A/S78A clones and found less degradation in the BID mutant clones (Fig 18B).

Thus, BID might be acting upstream of CDC25A and affecting its degradation. This effect

can consequently explain, at least in part, the impaired S phase arrest of the BID-/- and the

BID-S61A/S78A cells.

Figure18 : CDC25A degradation is more efficient in cells expressing wt BID. (A) Kinetics of Etop induced degradation of CDC25A in BID+/+ and BID-/- MEFs. Cells were either left untreated or treated with 20 µM Etop for 1 hr. Cells were rinsed and then released into drug-free medium for the indicated time points. Level of CDC25A was determined by western blot analysis using α CDC25A Abs. (B) BID-/- MEFs stably expressing either wtBID or BID-S61A/S78A were treated and analyzed as above.

55

The phosphorylation of BID is Chk2-independent

CDC25A degradation requires both ATM and the Chk2-mediated phosphorylation of

CDC25A on serine 123. This phosphorylation is an inhibitory phosphorylation and is

important for the arrest of cells in the S phase. To check whether the phosphorylation of BID

is also Chk2 dependent and is part of the ATM-Chk2- CDC25A -Cdk2 pathway. Chk2+/+ and

Chk2-/- MEFs were either left untreated or treated with 20 µM Etop for 5 min and up to 2 hrs.

BID phosphorylation was determined by Western blot analysis using the BID-phospho-

specific Abs. As shown in Figure 19, BID phosphorylation is not Chk2-dependent and occurs

in the same kinetics in Chk2+/+ and Chk2-/- MEFs.

Figure19 : The phosphorylation of BID is Chk2-independent Phosphorylation of BID is Chk2-independent. Chk2+/+ and Chk2-/- MEFs were either left untreated or treated with 50 µM Etop for the indicated time points. Cells were lysed and analyzed for BID phosphorylation levels by Western blot analysis using the anti pS78 Abs (upper panel) and anti-pS61 Abs (lower panel).

56

Chapter II – Identification of proteins that interact with phosphorylated BID

Introduction

It is well established that BH3-only proteins are sentinels of intracellular damage [70]. These

proteins are held at cellular locations in which they can sense a specific kind of cellular

damage, and once activated, translocate to the mitochondria to communicate the damage

signal. As presented above, a portion of BID localizes to the nucleus. In addition, all

currently identified substrates of ATM are nuclear proteins. Thus, it is most likely that BID is

phosphorylated in the nucleus. We proposed that BID functions as a nuclear effector of ATM,

and that this function(s) requires interaction with another protein(s) in the nucleus or at

another cellular location.

There are a number of reports describing proteins that interact with FL-BID. Two of them

reported that BID interacts and is phosphorylated by casein kinase I and II (CKI/II), and that

this phosphorylation decreases BID’s sensitivity to caspase-8 cleavage and protects cells

from Fas-induced apoptosis [71, 72]. Another report found that PACS-2, which is a sorting

protein that controls the ER–mitochondria axis, binds FL-BID and shuttles it to the

mitochondria [73]. In addition, this PACS-2-dependent trafficking of full-length BID to the

mitochondria seems to promote cell death by inducing the formation of tBID. Recently,

another report showed that FL-BID directly regulates the activation of BAX during TNFα-

induced apoptosis in ASTC-a-1 cells [74]. All the interactions described above relate to the

function of BID as a pro-apoptotic protein and none of them relate to its cell cycle arrest

function in the DNA damage response.

To understand the mechanism by which phosphorylated BID regulates cell cycle arrest and

its connection to apoptosis after DNA damage, we took a biochemical approach to purify

proteins that associate with BID. We used two different cellular systems in order to identify

possible interactors of BID: 1) Infection of BID-/- MEFs with adeno-BID-HA, Etop treatment

followed by IP with anti-HA antibodies; 2) 293T cells transfected with pcDNA3-BID-HA,

treated with Etop, followed by crosslinking and then IP with anti-HA antibodies.

Since the experiments using infection of BID-/- MEFs with adeno-BID-HA did not result in

57

meaningful results and the background of non-specific proteins was very high (data not

shown), we decided to use cross-linkers. The use of cross-linkers enables the capture of close

interactions in intact cells.

Results

BID is found as part of a 50KDa cross-linked complex in healthy cells and in cells treated

with DNA damage

We used the primary amine-reactive non-reversable cross linker BS3 and formaldehyde to

identify proteins that associate with BID. These cross-linkers induce nucleophilic attack of

the amino group of lysine and subsequent covalent bonding via the cross-linker. This

biochemical process allows the capture of interactors that otherwise could not be detected

under harsh lysis conditions.

1) Cross-linking using BS3- 293 cells were transfected with pcDNA3-BID-HA and treated

with Etop. Cells were than harvested and treated with digitonine for 10 min on ice. The

membrane fraction was separated from the cytosolic fraction by centrifugation and each were

incubated with BS3 for 30 min. Western blot analysis showed that most of the BID monomer

and complexes appeared in the cytosolic enriched fraction (data not shown). The anti-HA

antibodies detected a ~50 KDa band that may represent a complex of BID-HA with another

protein(s) (Fig 20A). An IP experiment in large scale with anti-HA antibodies was conducted

on the cytosolic fraction and the ~50 KDa band with several additional bands were sent to

MS analysis (Fig 20B).

58

Figure20 : Cross-linking with BS3 results in appearance of specific complexes. (A) 293 cells transfected with pcDNA3-BID-HA. 16 hrs post transfection cells were treated with 50µM Etop for 90 min. Cells were permeabilized with digitonine. BS3cross linker was added to the cytosolic enriched-fraction and IP was conducted using agarose beads coupled to α HA Abs. The beads were then collected and BID-HA and cross-linked complexes composed of BID-HA are eluted using an HA peptide. Western blot analysis of control sample (no cross-linker; -) and cross-linked sample (+) using α HA Abs. (B) 200X10cm dishes of 293 cells were transfected with pcDNA3-BID-HA. 16 hrs post transfection cells were treated with 50 µM Etop for 90 min, and permeabilized with digitonine. BS3cross linker was added to the cytosolic enriched-fraction and IP was conducted using agarose beads coupled to α HA Abs. The beads were then collected and BID-HA and cross-linked complexes composed of BID-HA were eluted using an HA peptide. The eluted sample was concentrated using centricon, and separated by SDS-PAGE (4-20%, Pierce). The gel was stained using Imperial stain (Pierce) and bands that may represent BID complexes were sent to MS.

The use of cross-linkers in a non-reverse manner, provided us with a useful tool to estimate

the size of the interacting protein(s). The MS results showed that BID-HA (25 KDa protein)

was present in the ~50 KDa band. Another protein that was identified in this band in two

separate experiments was peroxiredoxin 6 (PRX6). PRX6 is a 25 KDa protein, that reduces

H2O2 and phospholipid hydroperoxides by redox-active cysteines and is part of the

peroxiredoxin family [75]. Proteins from this family are conserved from bacteria to

mammals, and there are six subgroups of PRX enzymes (PRX1–6) in mammals. Two other

members of this family – PRX1 and PRX3, were identified also in the first experiments done

without the cross linkers, but their interaction with BID was not confirmed (data not shown).

PRX6 is mainly a cytosolic protein but is also found in the nucleus and mitochondria [76]. To

verify the interaction between BID and PRX6, we transfected 293 cells with Flag-PRX6 with

or without BID-HA and checked their interaction by cross-linking followed by co-

59

IP. Co-transfection of Flag-PRX6 and BID-HA, followed by cross-linking, IP with α HA Abs

and Western blot with anti-Flag Abs resulted in detection of Flag-PRX6 monomer (30 KDa)

and a cross-linkable higher complex (55 KDa) (Fig 21, lanes 5 and 6). As expected,

expression of each of these proteins alone gave no bands (Fig 21, lanes 1-4). These results

suggest that BID-HA, which is a 25 KDa protein and Flag-PRX6, which is a 30 KDa protein,

seem to interact and form a 55 KDa cross-linkable complex. This experiment will be repeated

with all the necessary controls to confirm this interaction.

Figure21 : Flag-PRX6 and BID-HA co-immunoprecipitate. Treatment of lysates of 293 cells expressing BID-HA or Flag-PRX6 with BS3 results in appearance of complexes. These complexes are recognized by αFlag Abs. 293 cells were transfected with BID-HA and/or Flag-PRX6. 16 hrs post transfection cells were permeabilized with digitonine. BS3cross linker was added to the cytosolic enriched-fraction and IP was conducted using α HA Abs. BID-HA and cross-linked complexes are eluted using an HA peptide. Western blot analysis using α Flag Abs was conducted.

2) Cross linking using formaldehyde – formaldehyde has been widely used to study binding

of specific proteins to DNA elements in intact cells. Formaldehyde as a cross-linker, has an

advantage because it can penetrate the membrane of living cells and it can therefore capture

protein interactions without cell disruption. In addition, it has a very short spacer (2Å) and

therefore it can cross-link proteins in close proximity.

293 cells transfected with pcDNA3-BID-HA and either treated or not with Etop, were treated

with 1% formaldehyde for 10 min at RT. The reaction was stopped using glycine for 5 min

and cells were harvested and lysed with RIPA buffer. A pull-down assay was conducted

using beads coupled to anti-HA antibodies. The bound proteins were eluted with an HA

60

peptide and separated by SDS-PAGE. Western blot analysis using α HA antibodies showed

again the appearance of a ~50 KDa band (Fig 22). This band was also detected by the anti-

phospho BID antibodies (α pS61, α pS78; Fig 22; middle and right panels) suggesting the

presence of phosphorylated BID in the complex. Of note, the relativelly high basal

phosphorylation we detected in cells that were not treated with Etop is probably due to the

transfection of cells. In addition, the ~50 KDa band appeared also in non-treated cells,

suggesting that DNA damage is not required to form this complex.

Figure22 : BID is found as a part of a cross-linked complex in healthy and in Etop treated cells. 293 cells were transfected with pcDNA3-BID-HA. Cells were left untreated or treated with 50 µM Etop. Cells were exposed to 1% formaldehyde and lysed in RIPA. A pull-down assay was conducted using beads coupled to α HA Abs. The BID-HA eluted with HA peptide and the sample was separated by a 10% SDS-PAGE. Western blot analysis using α HA, α pS61 and α pS78 Abs revealed bands that may represent complexes composed of BID and phosphorylated BID with other protein(s).

Next, we scaled up this experiment and detected the ~50 KDa band in a gel stained with

Imperial stain (data not shown). The ~50 KDa band was sent to MS analysis and the proteins

listed in the table below are the major ones detected (as determined by the % covergae of the

protein by peptides) with suitable low Mw.

61

As in previous experiments, proteins that belong to the Peroxiredoxin family were identified.

This time it was PRX1 and PRX2, compared to PRX6 that was identified in the experiments

with BS3 (Fig 21).

Another protein that drew our attention was Mitochondrial Carrier Homolog 2 (MTCH2).

MTCH2 was identified in our lab as part of a cross-linked complex with tBID in cells

signaled to die by TNFα [77, 78]. MTCH2 is a novel and previously uncharacterized protein,

which is related to a family of mitochondrial carrier proteins that catalyze the transport of

metabolites across the inner mitochondrial membrane. The function(s) of MTCH2 and its

relatives are still largely unknown.

To confirm that MTCH2 indeed interacts with FL-BID (and not with tBID, that is formed in

these cells), we constructed a new plasmid in which the HA tag is located in the N-terminus

of BID. Use of this plasmid eliminates the possibility that tBID, which is a C-terminal

fragment of BID, will be “pulled down” by the α HA Abs. Western blot analysis of cells

transfected with the C-terminal tagged BID and the N-terminal tagged BID showed that the

N-terminal tagged BID also forms the ~50 KDa complex (Fig 23). However, the pattern of

bands in the two samples was slightly different and in the C-terminal tagged BID lane two

separated bands around 50 KDa appeared (Fig 23, right panel). Taken together, it is possible

that FL-BID interacts with one or more of the proteins listed in the table and large scale

experiments using N-terminus HA-BID followed by MS will clarify this issue.

Identified proteins in cross linked band

(~50 KDa)

% Coverage of the protein KDa

BID 60% 25

MTCH2 49% 30

PRX1 33% 22

PRX2 23% 22

VDAC2 11% 33

BAX 19% 22

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Figure23 : N-terminal HA tagged BID forms the 50 KDa complex. 293 cells were transfected with BID tagged with HA in its N-terminal (N) and BID tagged in its C-terminal (C). Cells were exposed to 1% formaldehyde and lysed in RIPA. A pull-down assay was conducted using beads coupled to α HA Abs. The BID-HA eluted with HA peptide and the sample was separated by a 10% SDS-PAGE. Western blot analysis of the samples using α BID and α HA Abs revealed that in the C-terminal tagged BID there is an appearance of two separated bands around 50 KDa (two arrows), whereas in the N-terminal-tagged BID there is an appearance of only the upper band (one arrow). As expected, tBID was present only in the C-terminal tagged BID sample.

We have previously showed in MEFs that BID is partially localized to the nucleus and that

cross-linking with formaldehyde was required to detect BID in the nuclear fraction upon sub-

cellular fractionation (Fig 16). Based on these findings we suggested that BID is loosely

associated with the nuclear fraction by interaction with another protein(s) or with DNA. To

check whether BID was also localized to the nucleus in 293 cells transfected with BID-HA,

we performed immunofluoresence experiments with α HA Abs. As shown in Figure 24A,

BID-HA is also detected in the nucleus. To be sure that we are not missing proteins that

interact with BID in the nucleus (and that remained in the insoluble fraction) we sonicated the

cell lysates prior to IP, and indeed, there was an increase in yield and the appearance of

additional bands (Fig 24B). Interestingly, in cells treated with Etop there was an additional

band that appeared above the ~50KDa band. In the near future, we will add a sub-cellular

fractionation step (to analyze the nuclear fraction) and upscale the experiment for MS

analysis.

63

Figure24 : N-terminal HA tagged BID is localized to the nucleus in transfected 293 cells. (A) 293 cells transfected with BID HA were grown on glass cover slips, fixed and immunostained with anti-HA Abs as described in the Material and methods. (B) 293 cells were transfected with BID tagged with HA in its N-terminus. Cells were treated with Etop for 1 hr and exposed to 1% formaldehyde and lysed in RIPA. Cell lysates were left untreated or sonicated for 10 sec on ice. A pull-down assay was conducted using beads coupled to α HA Abs. The HA-BID eluted with HA peptide and the sample was separated by a 10% SDS-PAGE. Western blot analysis of the samples using α HA Abs revealed a higher protein yield and additional bands in the sonicated sample (arrow).

64

Chapter III - ATM, mitochondria function and apoptosis

Introduction

DNA damage activates cell cycle checkpoints that enable either cell cycle arrest followed by

DNA repair, or apoptosis (in the case of high levels of damage). The ATM kinase seems to

be a major player in this “life versus death” decision. Activation of ATM results in the

activation of Chk2 [79]. Chk2 can then phosphorylates and inhibit Cdc25C, contributing to

maintenance of G2 for cells in S or G2, and phosphorylate p53. Additional phosphorylation

of p53 by ATM, is required for activation of p53 as a transcription factor [80]. Activated p53

then induces transcription of BAX, PUMA and NOXA to initiate apoptosis in certain cell

types and induces p21 to cause inhibition of Cdk’s and G1 cell cycle arrest [80].

Cells lacking active ATM suffer abnormalities in cell cycle checkpoints after IR [81, 82]. In

addition, sensitivity to IR or radio-mimetic chemicals, and radioresistant DNA synthesis

(RDS) are hallmarks of the Α−Τ cellular phenotype [40, 83, 84]. On the contrary, in spite of

their increased radiosensitivity, it was also found that irradiated Α−Τ cells are unable to

undergo mitochondrial collapse and caspase-3 activation. These data suggest that ATM is

necessary for the initiation of molecular pathway(s) leading to IR-induced apoptosis [85].

Apoptosis is a wide signaling response that includes many cellular events. Some central

events occur at the mitochondria, including the release of caspase activators (such as

Cytochrome c), changes in electron transport, loss of mitochondrial transmembrane potential,

altered levels of reactive oxygen species (ROS) and activation of pro- and anti-apoptotic

proteins [8, 86-89]. Apart of the “apoptotic link” between ATM and the mitochondria, there

is strong evidence that mitochondria dysfunction occurs early and acts causally in

neurodegenerative diseases [90]. In addition, mitochondrial-DNA mutations and oxidative

stress contribute to aging, the greatest risk factor for neurodegenerative diseases.

In this part of my studies, we further explored the link between ATM and mitochondria by

examining several mitochondrial parameters in cells lacking ATM. The following

mitochondrial parameters were analyzed:

65

1) Mitochondrial transmembrane potential (∆ψm) - The mitochondrial respiratory chain

produces energy that is stored as an electrochemical gradient which consists of a

transmembrane electrical potential (∆ψm), negative inside of about 180-200 mV. This energy

is then able to drive the synthesis of ATP. Partial inner mitochondrial membranes

permeabilization disrupts mitochondrial ion and volume homeostasis and dissipates ∆ψm

causing depolarization of the mitochondrial membrane (reviewed in [89]). The process of

membrane permeabilization is considered as the “point-of-no-return” in numerous models of

programmed cell death. It affects the inner and outer mitochondrial membranes (IM and OM,

respectively) to a variable degree. OM permeabilization effect the release of proteins that are

normally confined to the mitochondrial intermembrane space (IMS), including caspase

activators and caspase-independent death effectors. The assessment of early mitochondrial

alterations allows for the identification of cells that are committed to die but have not

displayed yet the apoptotic phenotype [91].

2) ROS levels – The majority of ROS within eukaryotic cells is thought to be derived from

the mitochondria as by-products during the generation of ATP, through the process of

oxidative phosphorylation [89]. To generate electrochemical gradient, oxidative

phosphorylation complexes pump protons from the matrix to the intermembrane space, thus

leading to the formation of ∆ψm as well as to the generation of ROS. In healthy cells, ROS

are kept at harmless levels by the activity of both non-enzymatic and enzymatic antioxidant

systems. If not properly controlled, ROS can damage mitochondria leading to the production

of more ROS and more defective mitochondria via a vicious cycle. ROS can cause severe

damage to cellular molecules, especially DNA [92]. Since ATM is a central protein involved

in the DNA damage response, it might be involved in oxidative defense through the induction

of anti-oxidative systems mediated by catalase, peroxidases, SOD and reductases [92].

It has been found that Α−Τ patients have significantly reduced levels of total antioxidant

capacity [93] and that ATM-deficient cells are more sensitive to oxidative stress than normal

cells. It is also assumed that an increase in ROS may contribute to neurodegenration and

premature aging observed in Α−Τ patients [92]. Increase in ROS levels in Α−Τ cells puts the

cells under constant oxidative stress. ATM may have an additional role in sensing/modulating

66

redox homeostasis. The basis for the observed tissue specificity of the oxidative damage in

A-T is not clear.

67

Results

In this chapter, I will present preliminary results regarding the possible connection between

ATM and mitochondrial function.

Α−Τ Cells have higher basal ∆ψm (hyperpolarized) compared to cells expressing wt ATM

We initially assessed ∆ψm in three cell lines with impaired ATM activity using the

fluorescence dye DiOC6 (3,3-dihexyloxacarbocyanine): lymphoblasts from an Α−Τ patient

compared to healthy lymphoblasts, ATM-/- MEFs compared to ATM+/+ MEFs and Α−Τ human

fibroblasts compared to Α−Τ human fibroblasts expressing wtATM. We found that all three

lines with impaired ATM activity had higher basal ∆ψm (hyperpolarized) compared to three

controls (Fig 25A-C). This phenomenon was described earlier in the literature in several

human cancer cell lines that exhibited higher ∆ψm compared to normal cells [94]. In that

study, it was demonstrated that the hyperpolarized state of mitochondria contributed to

apoptosis resistance.

68

Figure25 : Α−Τ cells have higher ∆ψm Cells were incubated with DIOC6 for 15 min to determine the mitochondrial membrane potential. (A) On the left, overlay of FACS histogram showing the fluorescence of DIOC6 (FL-1) measured in ATM+/+ MEFs (purple) compared to ATM-/- MEFs (green). On the right, charts that represents the mean DIOC6 fluorescence that was measured in 3 repeats. (B) WT versus Α−Τ lymphoblasts. (C) Α−Τ fibroblasts expressing ATM compared to Α−Τ fibroblasts. * represent significant differences (p<0.05) based on Student’s t test.

*

*

*

69

A-T cells are more resistant to Etop-induced mitochondrial depolarization and apoptosis

compared to cells expressing wtATM

Next, we checked whether hyperpolarization is compatible with apoptosis resistance. One of

the earliest markers of apoptosis in cells is the loss of ∆ψm (depolarization), and thus we

checked whether there are any differences in the loss of ∆ψm after exposure of cells to

apoptotic stimuli. Cells were treated with 5µM Etop for 24h and incubated with DiOC6.

FACS analysis revealed that mitochondria from WT cells showed significantly stronger

depolarization (low ∆ψm) compared to Α−Τ cells (Fig 26A, top panels). The same differences

were detected in Α−Τ cells compared to Α−Τ+ATM cells (Fig 26A, bottom panels). To

detremine whether the differences in depolarization correlated with cell death post Etop

treatment, we measured PI dye-exclusion by FACS analysis. Indeed, WT cells and

Α−Τ+ATM cells showed higher levels of cell death compared to Α−Τ cells (Fig 26B). The

relative resistance of Α−Τ cells to Etop treatment was surprising since one of the hallmarks of

Α−Τ cells is increase sensitivity to DNA damage caused by IR. To explore whether Etop and

IR affected the cells in similar manner, we checked ∆ψm and cell death in response to IR.

Cells were irradiated with 10 Gy, incubated for 18 and 40 hrs, and low ∆ψm. and cell death

were determined by FACS analysis as described above. As opposed to Etop treatment, there

were no significant differences in mitochondrial depolarization (Fig 27A) and in cell death

(Fig 27B) between the lines. Thus, in these cells ATM seems to be important for Etop-

induced mitochondrial depolarization and cell death, but not for IR.

70

Figure26 : Mitochondria depolarization and cell death are lower in A-T cells post Etop treatment Cells were treated with 5µM Etop for 24 hrs. Mitochondrial membrane potential was measured by DIOC6. (A) Overlay of FACS histograms of WT lymphoblasts and Α−Τ+ATM fibroblasts (left panels, purple line) and Α−Τ lymphoblasts and A-T fibroblasts (green line) showing the distribution of DIOC6 fluorescence. On the right charts represent mitochondrial depolarization as was determined by the percentage of cells exhibiting low DIOC6 fluorescence (gated as M1 in the left histogram). * represent significant differences (p<0.05) based on Student’s t test. (C) Measurement of cell death as was determined by PI exclusion assay using FACS of the above Etop treated cells. * represent significant differences (p<0.05) based on Student’s t test.

M1

* *

*

*

71

Figure27 : IR-induced mitochondria depolarization and cell death are similar in A-T and WT cells. (A) Cells were irradiated with 10 Gy. Mitochondrial membrane potential was measured by DIOC6 and % depolarization (low ∆ψm) was measured 18 and 40 hrs post IR. On the left chart – WT lymphoblasts (black bar) and Α−Τ lymphoblasts (empty bar). On the right chart - Α−Τ+ATM fibroblasts (black bar) and A-T parent cells (empty bar) (B) Measurement of cell death by PI exclusion assay on IR treated cells (as above).

72

ROS levels are higher in Α−Τ cells compared to control cells

As mentioned previously, Α−Τ cells and tissues have increased levels of ROS. Next, we

checked whether we could detect higher ROS levels in the A-T lines. To asses the levels of

superoxide and H2O2, cells in growing phase were incubated with HE (Hydroethidine) and

DCF-DA (2’,7’-dichlorofluorescein) respectively, and taken for FACS analysis. Indeed, we

found that these lines had increased H2O2 and superoxide (Fig 28) compared to the control

lines. These results suggest that ATM plays a role in controlling cellular ROS levels or that

mitochondria of Α−Τ cells display abnormalities that can influence ROS levels in these cells.

Figure28 : ROS levels are higher in Α−Τ cells compared to control cells. Cells in growing phase were incubated with DCF-DA (upper charts) and HE (bottom charts) and analyzed by FACS to assess levels of H2O2 and superoxide, respectively. WT lymphoblasts (black bars) compared to Α−Τ lymphoblasts (empty bar). Α−Τ fibroblasts expressing WT ATM (black bars) compared to the parent Α−Τ fibroblasts (empty bars). * represent significant differences (p<0.05) based on Student’s t test.

* *

*

73

Α−Τ cells grow slower compared to control cells

Mitochondria play a central role in energy metabolism and therefore can effect cellular

growth. In addition, it was found that ATM is also required for efficient G1- to S-phase

transition during cell-cycle progression and that ATM-/- cells prematurely senesce and that

their p21 protein levels are chronically high [95]. Thus we next checked the growth rate of

the different cell lines, and found that the A-Τ lines grew slower compared to the controls

(Fig 29).

Figure29 : Α−Τ cells grow slower compared to control cells. Cell growth in WT and Α−Τ lymphoblasts (left chart) and A-T and Α−Τ+ATM fibroblasts (right chart). The data represent the means ± SEM of pooled results from three independent experiments.

74

Discussion

BID, ATM and the DNA damage response

In the present study, we demonstrated that DSB DNA-damaging reagents induced an

immediate and transient phosphorylation of BID. In addition, we showed that this

phosphorylation was mediated by the ATM kinase. Most importantly, we demonstrated that

BID-/- MEFs failed to delay the cell cycle in response to Etop, and that introduction of a non-

phosphorylatable BID mutant restored delay in the G2 but not in the S phase. Moreover, in

response to Etop, the mutant BID cells entered apoptosis more readily than the wild-type BID

cells [62].

Our current and previous findings demonstrate that in MEFs BID is important for apoptosis

induced by a variety of DNA-damaging reagents (Fig 2 and [37]). Phosphorylation of BID

seems to effect its apoptotic function since BID-/- MEFs expressing the BID-S61A/S78A

mutant are more susceptible than BID-/- MEFs expressing wild-type BID to Etop-induced

apoptosis (Fig 9). Thus, ATM-mediated BID phosphorylation might serve as a mechanism to

inhibit BID’s apoptotic activity or alternatively as a mechanism to activate a pro-survival

activity of BID.

If phosphorylation was inhibiting the apoptotic activity of BID, then what would likely be the

molecular basis of such an inhibition? The only known apoptotic function of BID lies in its

ability to induce the release of pro-apoptotic factors from the mitochondria (e.g., Cytochrome

c). tBID is much more efficient than FL-BID in inducing Cytochrome c release; it would

therefore be expected that ATM-mediated BID phosphorylation inhibits the cleavage of BID,

since casein kinase 1 and/or 2-mediated phosphorylation of BID was demonstrated to inhibit

its cleavage [71]. However, our findings suggest that ATM-mediated phosphorylation of BID

is not related to BID cleavage, for the following reasons: Phosphorylation occurs many hours

before the activation of caspases and the onset of apoptosis (Fig 6). A relatively small amount

of BID is phosphorylated (Fig 3), and phosphorylation occurs also in response to extremely

low, non-apoptotic levels of DNA damage (Fig 8). Moreover, the BID-S61A/S78A mutant

was not found to be more susceptible to cleavage than wtBID (Fig 9). Lastly, though TNFα

75

relies on the generation of tBID to induce/enhance apoptosis [96], the BID-S61A/S78A

mutant clones are not more susceptible to TNFα-induced apoptosis than the wtBID clones

(Fig 9). Thus, BID phosphorylation does not seem to serve as a mechanism to inhibit BID’s

apoptotic activity.

The results presented in Figures 10 and 11 suggest that ATM-mediated phosphorylation of

BID regulates a novel, pro-survival function of BID related to cell cycle arrest following

DNA DSBs. We first found that Etop induced accumulation of BID+/+ MEFs in the S and G2

phases of the cell cycle, whereas such an accumulation was not observed in the BID-/- MEFs

(Fig 10). Moreover, using BrdU labeling we have demonstrated that BID is specifically

required for S phase arrest (Fig 11). We then reintroduced wild-type BID into BID-/- cells,

and found that this addition restored the ability to accumulate in the S and G2 phases of the

cell cycle. Introducing the non-phosphorylatable BID mutant into BID-/- cells, however,

restored accumulation only in the G2 phase (Fig 12). These findings imply that BID

phosphorylation plays an important role in S phase arrest.

To demonstrate the BID-dependent S phase checkpoint we have used relatively high levels of

Etop, suggesting that this function of BID is associated with high levels of genotoxic stress in

fibroblasts. On the other hand, Zinkel at al. have demonstrated that in myeloid and in

activated T cells this function of BID is associated with lower levels of genotoxic stress [66].

Hematopoetic cells are primed to undergo cell cycle arrest and apoptosis following treatment

with DNA-damaging reagents, whereas fibroblasts are relatively resistant to DNA-damaging

reagents and prevent proliferation of mutations by entering into long-term G1 or G2 arrest

[51]. Thus, the activation threshold and the biological impact of the BID-dependent S phase

checkpoint function may vary largely among different cell types.

How might the involvement of BID in S phase arrest be related to apoptosis? Following DNA

DSBs, the cell may decide to activate a survival system (mainly through the ATM kinase,

which induces cell cycle arrest and DNA repair), or in the face of extensive or irreparable

damage, the cell may activate the apoptotic machinery. The results presented in Fig 9

demonstrate that BID-/- MEFs expressing the BID-S61A/S78A mutant are more susceptible

76

than BID-/- MEFs expressing wild-type BID to Etop-induced apoptosis, but not to UV- or to

TNFα-, induced apoptosis. Thus, it appears that the non-phosphorylatable BID mutant

sensitizes BID-/- MEFs to apoptosis induced only by reagents that induce DSBs in DNA.

These results, together with the cell cycle results (Figs 10 to 12) suggest that the impaired

ability of the mutant BID cells to induce cell cycle arrest results in increased sensitivity to

DSB DNA damage.

If BID is indeed capable of playing a pro-survival role in the response of cells to DNA DSBs,

then why are the BID-/- MEFs less sensitive than BID+/+ MEFs to apoptosis induced by Etop

and IR (Fig 2)? BID may play both a pro-survival and a pro-apoptotic role in this pathway.

Based on our finding that the phosphorylation of BID occurs in response to extremely low

levels of IR, and increases in an IR dose-dependent manner (Fig 8), we propose that BID acts

as a sentinel of DNA DSBs. BID might translate the damage into either cell cycle arrest/DNA

repair processes (at low levels of damage) or apoptosis (at high levels of damage). In

addition, we found that BID-/- cells that were exposed to relatively low Etop levels, eventually

die after several days (Fig 14). These results demonstrate that the lack of cell cycle arrest and

probably accumulation of unrepaired DNA damage eventually leads to death. In a parallel

study that was done by Zinkel et al., it was found that myeloid progenitor cells (MPCs)

treated with either hydroxyurea or mitomycin c, agents that induce DNA damage through

replicative stress, fail to accumulate in S phase and displayed increased genomic instability

and increased cell death [66]. These results demonstrate that the action of BID as a pro-

apoptotic or as a pro-survival protein is influenced from the cell type, the kind of treatment

and the levels of a given treatment.

To examine the role of BID phosphorylation in another cell type, we used the human HeLa

cell line. We found that knocking-down the expression of BID in HeLa cells partially impairs

Etop-induced S phase arrest (Fig 15). These results indicated that a partial reduction in the

levels of BID is sufficient to impair DNA-damage-induced S phase arrest.

Up until now, BID has been considered a cytosolic protein. Since signaling proteins involved

in cell cycle checkpoints often function in the nucleus and so are proteins that are

77

phosphorylated by ATM, we examined the subcellular location of BID, and found that it is

partially localized to the nucleus. Immunofluoresence studies using anti-BID antibodies and

subcellular fractionations of MEFs after treatment with cross-linker showed that a fraction of

BID is localized to the nucleus (Fig 16). Since cellular disruption avoided the detection of

BID in the nucleus, we suggested that BID is loosely associated with the nuclear fraction by

interaction with another protein(s) or with DNA, and that cross-linking is required to preserve

this interaction. Moreover, in a published study that was conducted by Galia Oberkovitz

(another Ph.D. student in the lab), it was found that Etop and IR induce the nuclear export of

BID and that this nucleocytoplasmic shuttling of BID is involved in regulating its activity

following DNA damage. Fusing BID to a nuclear localization signal (NLS) inhibited its

nuclear export, and impaired is ability to induce S phase arrest and cell death [97]. Zinkel et

al. also found that BID in MPCs is localized to the nuclear fraction and more specifically to

the chromatin fraction [66]. Thus, BID is most likely phosphorylated by ATM in the nucleus,

and the phosphorylated form might need to exit the nucleus to execute its function in the

DNA damage response.

The very rapid kinetics of BID phosphorylation positions it as part of a wide web of proteins

that are involved in the immediate response to DSB DNA damage (Fig 17). Another

important protein in the early response to DNA damage is H2AX. This protein is rapidly

phosphorylated and recruited to the site of DSB and forms foci. We found that H2AX

phosphorylation and foci formation in BID+/+ MEFs appeared earlier and stronger than in

BID-/- MEFs. These results suggest that BID participates in a much earlier stage of the DNA

damage response in addition to its involvement in the late response related to cell cycle

regulation.

Protein phosphorylation is widely recognized as the major mechanism that controls cell cycle

progression. In addition, the activity of proteins that regulate cell cycle progression is

controlled throughout the different phases of cell cycle. Although BID was found to regulate

the arrest of cells in S phase, the phosphorylation of BID was found to be cell cycle-

independent when using synchronized cells (Fig 13). Examination of cell cycle-dependent

downstream event such as degradation of CDC25A, which is directly effecting S phase

78

progression, revealed less degradation in BID-/- MEFs compared to BID+/+ cells (Fig 18).

Similar results were seen in BID-/- MEFs expressing the non-phosphorylatable BID mutant.

These results connect the phosphorylation of BID to one of the most well characterized

pathways in the response of cells to DNA damage: the ATM-Chk2-CDC25A pathway. Of

note, the Chk2 kinase phosphorylates many down stream substrates in this pathway, such as

p53 and CDC25A, but it is not necessary for the phosphorylation of BID (Fig 19). Therefore,

we still do not know where to position BID in this pathway. In addition, there are new ATM

substrates identified all the time that expand the web and branches of the DDR [98].

One way to define BID’s position in this expanding pathway is to identify proteins that

interact with BID upon DNA damage. We found that BID is part of a ~50 KDa complex in

both healthy cells and in cells treated with Etop. The detection of this complex was possible

due to the use of cross-linkers that stabilized this interaction prior to cell disruption (Figs 20

and 22). Importantly phosphorylated BID was detected in this complex prior and following

Etop treatment (Fig 22). MS analysis of the ~50 KDa band revealed a list of mitochondrial

proteins that included MTCH2, which was previously identified in our lab as a tBID

interactor [77]. These results suggest that either FL-BID indeed interacts with MTCH2 at the

mitochondria or that pulling down MTCH2 was a result of tBID generation in the cells. To

eliminate the involvement of tBID in the pull-down experiments, HA tag was fused to the N-

terminus of BID, and the ~50 KDa cross-linked band appeared again (Fig 23). These results

indicate that FL-BID forms a complex with another protein or that the appeared band can also

represent a BID homodimer. Future studies will determine the identity of this band. Since the

results regarding BID’s localization imply that BID may execute its function in the nucleus

possibly by interaction with a nuclear protein, we are in the process of an experiment using

formaldehyde to identify a nuclear BID complex.

Another protein that was identified by MS results as part of the ~50 KDa complex is PRX6

(Fig 21). This protein is an anti-oxidant, and our initial studies suggest that BID interacts

with this protein. It is now to be discovered whether this interaction is physiologicaly relevant

and whether it is connected to the DNA damage response and to cell cycle regulation by BID.

It is possible that we succeeded in “fishing” another interactor of BID that participates in the

79

response of cells to the elevated levels of ROS following Etop treatment.

In summary, following DNA damage, the cell activates a survival system that allows cell

cycle arrest/DNA repair and continuation of its normal life cycle, or it may activate the

apoptotic machinery in the face of extensive or irreparable damage. Our study reveals that the

BH3-only BID protein, a molecule that was previously considered to be active only as a pro-

apoptotic factor, also plays a pro-survival role as an ATM effector (see model in Fig 30). If

BID is indeed playing both a pro-apoptotic and a pro-survival function in the DNA damage

pathway, then it is an excellent candidate to link DNA repair processes and apoptosis. Indeed,

Zinkel et al.previously showed that BID-/- primary activated T cells and myeloid progenitor

cells demonstrate increased chromosomal damage in response to mitomycin c [66]. Galia

Oberkovitz, another Ph.D student in the lab, has comfirmed these finding, and further showed

that ATM-mediated BID phosphorylation is important for preserving genomic stability.

Recently, two studies regarding proteins that are known as apoptotic regulators – Apaf-1 and

Aven, showed that these proteins have a new role in cell cycle regulation. The apoptotic

protein Apaf-1 was demonstrated to regulate S phase arrest in response to DNA damage by

acting downstream of ATM/ATR and upstream of Chk1 [99] It was found that its new role

can be separated from its apoptotic activity since Apaf-1 lacking the proapoptotic CARD

domain (unable to activate caspase-9) induces S phase arrest and Chk1 phosphorylation.

Similarly, in the case of BID, ATM-mediated BID phosphorylation occurs at extremely low

levels of DNA damage and within minutes following the damage. In addition, Zinkel et al.

showed that BID lacking its BH3 death domain is capable of inducing S phase arrest [66]. In

contrast to the pro-apoptotic activities of Apaf-1 and BID, Aven is known as an apoptotic

inhibitor, and it was found to function as an ATM activator to inhibit G2/M progression

[100]. Aven was found to bind ATM and overexpression of Aven in cycling Xenopus egg

extracts prevented mitotic entry and induced phosphorylation of ATM and its substrates.

There are also similarities regarding protein localization: Aven and BID were found to

partially localized to the nucleus in healthy cells, whereas Apaf-1 was found to translocate to

the nucleus upon DNA damage. Thus BID, along with Apaf-1 and Aven demonstrate the

ability of proteins to act as double agents – in regulating apoptosis on one hand, and cell

80

cycle progression on the other hand. It is yet to be determined how these proteins function in

this “life versus death” decision.

To define the importance of ATM-mediated BID phosphorylation in-vivo, Hagit Niv (a

former post doc in the lab) generated a BID knock-in mouse, in which the endogenous BID

gene has been replaced with a gene that drives the expression of a non-phosphorylatable BID

protein (BIDS61A/S78A). Using cells from these mice it was found that BIDS61A/S78A primary T

and B cells demonstrate a defect in the intra S-phase DNA damage checkpoint, increased

chromosomal damage, and increased apoptosis in response to the DNA interstrand cross-

linking agent mitomycin c. Thus, BID's S phase arrest function seems to be critical for T and

B cells to preserve genomic stability and to survive following replication stress.

These results further strengthen the link between BID and the DNA damage response, and

future studies will determine the exact role and importance of BID phosphorylation in

mammalian organisms.

Figure30 : Model for the dual role of BID. Following Etop treatment BID is being phosphorylates in the nucleus on two SQ sites in an ATM-dependent manner. This phosphorylation is required to abrogate apoptosis and for the arrest of cells in S phase. BID was found to be loosely associated with the nuclear fraction probably by interaction with another protein(s). Indeed, we found BID as a part of a 50 KDa complex in healthy and treated cells. The interacting protein is still not known. In addition, it was found that at the mitochondria ∆ψm, ROS levels and apoptosis are regulated in an ATM-dependent manner. Whether it is regulating mitochondrial function directly or indirectly is yet to be discovered.

81

ATM and the mitochondria

Cell cycle checkpoints, which lead to survival and apoptosis, are different processes that must

be coordinated. In the case of high levels of damage, cells initiate an apoptotic pathway

which both in its physiological and pathological occurrence, is closely linked to

mitochondrial structure and function. The link between ATM, which stands at the top of the

signaling pathway, and mitochondrial events, which are downstream in the apoptotic

signaling pathway is still largely unknown. In a separate project we measured mitochondrial

function and apoptosis in cells lacking a functional ATM protein. We found that cells

expressing mutant ATM or lacking the ATM protein have hyperpolarized mitochondria (Fig

25). Hyperpolarization of mitochondria was described earlier in the literature in several

human cancer cell lines that exhibited higher ∆ψm compared to normal cells [94]. It is

assumed that hyperpolarization of mitochondria contribute to apoptosis resistance in these

cells. Indeed, we found that Α−Τ cells are less susceptible to Etop-induced depolarization of

mitochondria and this correlated with resistance to Etop-induced cell death (Fig 26).

Interestingly, the response of the cells to IR did not result in any significant differences

between Α−Τ and WT cells (Fig 27). These results suggest that the response of cells to IR is

different in these cells. Other features of these cells were higher ROS levels and slower

growth rate, which both can be the outcome of mitochondrial dysfunction. Introduction of

wtATM into Α−Τ cells restored the normal ∆ψm and the sensitivity to Etop (Fig 26). In a

recently published paper it was found that mitochondria in Α−Τ cells showed un-uniformed

dispersion in the cell and they tended to polarize at one end of the cell, a phenomena usually

exhibited by lymphoblastoid cells derived from patients suffering from known mitochondrial

diseases [101]. Another study demonstrated that Etop increases mitochondrial biogenesis in

an ATM-dependent manner [102]. In contrast to our results, these two published papers

showed that Α−Τ lymphoblastoid cells exhibit a lower ∆ψm [101, 102]. They suggested that

in the absence of ATM, DSBs induced by Etop triggers a cell death pathway involving

depolarization of mitochondria. To explore the controversies between our studies and to

further establish our findings, there is a need to investigate the differences in mitochondria

function in Α−Τ cells compared to WT cells. To do so we will compare the rate of respiration

and ATP levels in these cells. It will also be interesting to explore the possible connection

82

between high ROS levels, high ∆ψm and apoptosis in Α−Τ cells (for example by using

antioxidants). Another important aspect of this research will be to understand how ATM,

which is a nuclear protein, regulates mitochondria function (see model in Fig 30). Recently it

has been reported that ATM localizes to the microsomal fraction (containing mitochondria)

[101]. Thus, ATM might affect mitochondria parameters by being localized to mitochondria.

83

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Publications

Kamer, I., Sarig, R., Zaltsman, Y., Niv, H., Oberkovitz, G., Regev, L., Haimovich, G.,

Lerenthal, Y., Marcellus, R.C., and Gross, A. (2005). Proapoptotic BID is an ATM effector in

the DNA-damage response. Cell 122, 593-603.

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תקציר

) אפפטוזיס( שיכולים לעורר בתא תגובה של מוות תאי ATR- וATM גורם לשפעול של חלבוני הקינאז DNAנזק ל

עובר BCL-2- ה ממשפחתBIDהראנו שהחלבון האפפטוטי , בחלק הראשון של התיזה שלי. או לעצירת מחזור התא

לא עצרו -/-BIDמצאנו שתאי . 78S-ו S61 על שני אתרי זירחוןDNA- בתגובה לנזק לATMי "פוספורילציה ע

. DNAגדיליים ב - שגורם לשברים דוEtop שלהם בתגובה לטיפול בחומרDNA-את מחזור התא ואת שיכפול ה

בעוד שהחזרה ) S-שלב ה (DNA-ב סינטזת ה לתאים אלו הביאה לעצירת מחזור התא בשלBIDהחזרה של החלבון

. לא הביאה לעצירת מחזור התא אלא למוות אפפטוטי של התאים, ATMי "שאינו עובר זירחון ע, מוטנטBIDשל

הינו בעל תפקיד חיוני בשרידות של תאים ובבקרה , שידוע כחלבון אפפטוטיBID-התוצאות הללו מצביעות על כך ש

. מבקר את התקדמות מחזור התאBIDבחנו את המכניזם בו , בשלב הבא. DNA-זק לשל עצירת מחזור התא אחרי נ

ומאפשרים את תפקודו BIDלשם כך נקטנו בגישה ביוכימית על מנת לבודד חלבונים שעוברים אינטרקציה עם

50 - מהווה חלק מקומפלקס בגודל שלBID-מצאנו ש, cross-linkers -תוך שימוש ב. DNA-בתגובה לנזק ל

KDaבתאים לא מטופלים ובתאים שטופלו ב -Etop . כמו כן מצאנו שהחלבון המזורחן מהווה גם חלק מהקומפלקס

על מנת לזהות את החלבונים המצויים בקומפלקס בכוונתנו לערוך ניסוי בקנה מידה גדול ולשלוח את הקומפלקס . ל"הנ

. י ספקטרומטריה"לזיהוי ע

הקשר בין . פעילות מיטוכונדריאלית ואפפטוזיס, ATMהאפשרי בין בחלק השני של העבודה חקרנו את הקשר

אינו אקטיבי או בתאים מעכברים בהם אין כלל ATMבהם , T-Aהגורמים השונים נבדק בתאים שמקורם בחולי

ATM . מצאנו שממברנת המיטוכונדריה בתאים אלו הינה בעלת פוטנציאל חשמלי גבוה יותר מתאי הביקורת המכילים

תאים אלו נמצאו כרגישים יותר לנפילה של פוטנציאל ממברנת המיטוכונדריה אחרי , בנוסף. אקטיביATMחלבון

מצאנו שתאים אלו היו פחות רגישים למוות מושרה , בהתאמה. תהליך המתרחש בזמן אפפטוזיס, Etop-טיפול ב

Etop .ראדיקלים חופשיים(מצאנו שבתאים אלו קיימת רמה גבוהה יותר של מולקולות חמצן ריאקטיביות , בנוסף( ,

. תאים אלו הראו גם קצב גדילה איטי יותר. שמקורן העיקרי בתא הוא בפעילות הנשימה המתבצעת במיטוכונדריה

רמטרים המיטוכנדריאלים אקטיבי הראה שהפATM המקודד לחלבון DNA אליהם הוחדר T-Aשימוש בתאי

שידוע כחלבון גרעיני משפיע על תפקוד ATM-לכן נראה ש. חזרו לרמות נורמליותEtop -ורגישות התאים ל

. המיטוכונדריה בתא

ישנה פעילות בה הוא , חלבון שהיה ידוע כבעל פעילות אפפטוטית בלבד, BID -ל מצא של"המחקר הנ, לסיכום

בפעילות זו . DNA-גדיליים ב-יוני להישרדות תאים אחרי נזק הגורם לשברים דומשמש בתא בתפקיד של חלבון הח

BIDחשוב לעצירת מחזור התא בשלב ה -S ולעיכוב Apoptosis.