neuronal uv-initiated apoptosis is prevented by 5- bromo … · neuronal uv-initiated apoptosis is...

Neuronal UV-Initiated Apoptosis Is Prevented By 5-Bromo-2’-Deoxyuridine (BrdU) Or A Deficiency in

Cockayne Syndrome B Or Xeroderma Pigmentosum A

by

Nishani Rajakulendran

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Pharmacology and Toxicology University of Toronto

© Copyright by Nishani Rajakulendran 2012

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Neuronal UV-Initiated Apoptosis is Prevented by 5-Bromo-2’-

Deoxyuridine (BrdU) or a Deficiency in Cockayne Syndrome

B or Xeroderma Pigmentosum A

Nishani Rajakulendran

Master of Science

Graduate Department of Pharmacology and Toxicology University of Toronto

2012

ABSTRACT

This project addressed mechanisms of the neuronal DNA damage response after

treatment with the model DNA damaging agent ultraviolet light (UV). The thymidine

analogue, 5-bromo-2’-deoxyuridine (BrdU) protected against UV-initiated neuronal

apoptosis in a concentration-dependent manner (p<0.001). BrdU did not protect

proliferating mouse embryonic fibroblasts from UV-induced apoptosis. We assessed

whether the mechanism of BrdU neuroprotection was through a modification in the

neuronal DNA damage response. BrdU neuroprotection was independent of BrdU

incorporation into DNA, neuronal DNA repair, p53 activation or cell cycle re-entry, a

neuronal DNA damage response. Neurons deficient in Cockayne Syndrome B (CSB) or

Xeroderma Pigmentosum A (XPA) were paradoxically resistant to UV-initiated

apoptosis. Therefore, CSB and XPA play essential roles in the neuronal DNA damage

response.

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ACKNOWLEDGEMENTS

This thesis was made possible by the guidance and encouragement I received

from an incredible group of people. I would like to express my gratitude to everyone who

has been extremely supportive during the past two years.

First, I would like to thank my supervisor and mentor, Dr. Rebecca Laposa, for

her inspiration and support. Her enthusiasm and positive outlook on science has helped

shape my appreciation for scientific research. Also, her endless encouragement has

been instrumental to my success as a graduate student.

I would also like to thank past and present members of the Laposa lab for

creating a wonderful work environment. I am very grateful to Laura Tamblyn who has

trained me in most of the techniques I used in this project and has been incredibly

patient with my numerous questions. I would also like to thank Fernando Bralha who

was extremely helpful, especially on those days when I was working on too many

experiments, and for our engaging discussions. Sharanya and Temi have also become

good friends and it was a joy working with them.

I would also like to thank my advisor Dr. McPherson whose feedback has been

invaluable. I am also grateful to the McPherson lab members, who have allowed us to

use their equipment and for their assistance in technical problems.

Finally, I have to thank my family and friends. I am extremely grateful to my

brother and sister for their encouragement and motivation during my ups and downs.

Most importantly, I am deeply grateful to my parents for their unconditional support in

my academic pursuits and career goals. They are incredible people who have taught

me the value and importance of education and I would not have come this far without

their love and inspiration.

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TABLE OF CONTENTS

ABSTRACT .................................................................................................................... II

ACKNOWLEDGEMENTS ............................................................................................. III

TABLE OF CONTENTS ................................................................................................ IV

LIST OF TABLES ......................................................................................................... VII

LIST OF FIGURES ...................................................................................................... VIII

ABREVIATIONS ............................................................................................................. X

1. INTRODUCTION ....................................................................................................... 1

1.1 DNA damage in eukaryotic cells ............................................................................ 1

1.1.1 Endogenous and exogenous sources of DNA damage ...................................... 1

1.1.2 DNA damage response signaling ....................................................................... 1

1.1.3 UV DNA lesions .................................................................................................. 8

1.2 Nucleotide excision repair ................................................................................... 11

1.2.1 Overview of nucleotide excision repair ............................................................. 11

1.2.2 Diseases caused by defective nucleotide excision repair ................................. 14

1.2.3 Nucleotide excision repair in neurons ............................................................... 15

1.3 Neuronal cell cycle re-entry ................................................................................. 16

1.3.1 Overview of the cell cycle ................................................................................. 16

1.3.2 In vivo evidence of neuronal cell cycle re-entry ................................................ 19

1.2.3 In vitro evidence of neuronal cell cycle re-entry ................................................ 20

1.4 5-bromo-2’-deoxyuridine (BrdU) .......................................................................... 24

1.4.1 Overview of BrdU ............................................................................................. 24

1.4.2. The role of BrdU in DNA repair ........................................................................ 26

1.4.3. The role of BrdU in the cell cycle ..................................................................... 29

2. OVERVIEW............................................................................................................... 33

2.1 Hypothesis............................................................................................................. 34

2.2 Aims ....................................................................................................................... 34

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3. MATERIALS AND METHODS ................................................................................. 35

3.1 Cortical neuron culture ........................................................................................ 35

3.2 Embryonic genotyping ......................................................................................... 36

3.3 Mouse embryonic fibroblast (MEF) isolation ...................................................... 36

3.4 Cortical neuron survival assays .......................................................................... 37

3.4.1 Neuronal immunocytochemistry ....................................................................... 37

3.4.2 Quantification of apoptosis with flow cytometry ................................................ 38

3.4.3 Quantification of apoptosis with immunocytochemistry .................................... 38

3.5 Mouse embryonic fibroblast survival assay ....................................................... 39

3.6 BrdU labeling using immunocytochemistry ....................................................... 39

3.7 Quantification of UV DNA lesions ....................................................................... 40

3.8 DNA content quantification using immunocytochemistry ................................ 41

3.9 Quantification of cell cycle distribution using flow cytometry ......................... 42

3.10 Cell cycle marker expression ............................................................................. 42

3.11 Quantification of global DNA methylation ........................................................ 43

3.12 Statistical analyses ............................................................................................. 43

4. RESULTS ................................................................................................................. 44

4.1 BrdU prevents mouse cortical neurons from UV-induced death ...................... 44

4.1.1 BrdU reduces UV-induced cleaved caspase-3 in cortical neurons ................... 47

4.2 Absence of detectable BrdU labeling in cortical neurons ................................. 49

4.3 Cell type specificity of BrdU protection .............................................................. 51

4.4 Chemical specificity of BrdU protection ............................................................. 53

4.5 The UV-initiated increase of p53 is unaltered by BrdU ...................................... 55

4.6 BrdU does not alter UV DNA lesion removal ...................................................... 57

4.7 Xpa-/- and Csb-/- neurons are resistant to UV-initiated death ............................. 59

4.7.1 BrdU does not alter caspase-3 in Xpa-/- and Csb-/- neurons ............................ 60

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4.8 Influence of BrdU on neuronal cell cycle ............................................................ 63

4.8.1 BrdU does not alter neuronal DNA content ...................................................... 63

4.8.2 Neither UV-irradiation nor BrdU incubation alter cell cycle distribution ............. 65

4.8.3 UV does not induce cell cycle markers in neurons ........................................... 65

4.9 BrdU does not alter neuronal global DNA methylation ..................................... 68

5. DISCUSSION............................................................................................................ 70

5.1 Characterization of BrdU protection ................................................................... 70

5.2 UV-induced cell signaling .................................................................................... 71

5.3 UV-induced DNA repair ........................................................................................ 71

5.4 UV-induced cell signaling in Xpa-/- and Csb-/- neurons ...................................... 73

5.5 Neuronal cell cycle re-entry ................................................................................. 75

5.6 Neuronal DNA methylation................................................................................... 78

6. CONCLUSIONS AND FUTURE DIRECTIONS ........................................................ 79

7. REFERENCES ......................................................................................................... 82

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LIST OF TABLES

INTRODUCTION:

TABLE 1.1 Evidence of neuronal cell cycle re-entry after DNA damage ....................... 23

TABLE 1.2 Evidence of cell cycle modulation by thymidine analogues ........................ 30

MATERIALS AND METHODS:

TABLE 3.1 Summary of Xpa primers, cycling conditions and band sizes ..................... 36

TABLE 3.2 Summary of Csb primers, cycling conditions and band sizes ..................... 36

RESULTS:

TABLE 4.1 Flow cytometric analysis of cell cycle phase in DNA-damaged cortical

neurons .......................................................................................................................... 65

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LIST OF FIGURES

INTRODUCTION:

FIGURE 1.1 The role of ATM and ATR in the DNA damage response ............................ 3

FIGURE 1.2 Downstream targets of p53 mediated apoptosis ......................................... 7

FIGURE 1.3 UV DNA lesions ......................................................................................... 10

FIGURE 1.4 The two sub-pathways of nucleotide excision repair (NER): global genome

repair (GGR) and transcription-coupled repair (TCR) .................................................... 13

FIGURE 1.5 Chemical structures of thymidine and 5-bromo-2’-deoxyuridine (BrdU) .... 25

RESULTS:

FIGURE 4.1 BrdU protects cortical neurons from UV-induced apoptosis ...................... 46

FIGURE 4.2 BrdU reduces UV-induced cleaved caspase-3 in neurons ........................ 48

FIGURE 4.3 Absence of detectable BrdU labeling in UV-irradiated and control cortical

neurons .......................................................................................................................... 50

FIGURE 4.4 In fibroblasts, BrdU does not reduce UV-induced apoptosis ..................... 52

FIGURE 4.5 Nucleosides and halogenated thymidine analogues prevent UV-initiated

apoptosis ....................................................................................................................... 54

FIGURE 4.6 UV-induced p53 up-regulation is unaltered by BrdU ................................. 56

FIGURE 4.7 Time course of disappearance of UV DNA lesions in cortical neurons ...... 58

FIGURE 4.8 Xpa-/- and Csb-/- cortical neurons are resistant to UV-initiated apoptosis ... 61

FIGURE 4.9 BrdU does not alter the proportion of cleaved caspase-3 + neurons after

UV in Xpa-/- and Csb-/- ................................................................................................... 62

FIGURE 4.10 DNA content of cortical neurons is unaltered by UV or BrdU .................. 64

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FIGURE 4.11 Neither UV nor BrdU alter neuronal Cdk4, Cdk6 or Ki67 expression ...... 67

FIGURE 4.12 Neither UV-irradiation nor BrdU alter neuronal global DNA methylation .. 69

x

ABREVIATIONS 6-4PP pyrimidine 6-4 pyrimidone photoproducts Apaf-1 apoptotic protease activating factor 1 APV 2-Amino-5-phosphonopentannoic acid ATM ataxia telangiectasia mutated ATR ATM and Rad3-related protein ATRIP ATR interacting protein βIIITub βIIITubulin Bak Bcl-2 homologous antagonist/killer Bax Bcl-2 associated X protein Bcl-2 B-cell lymphoma 2 Bxl-XL B-cell lymphoma-extra large BrdU 5-bromo-2’-deoxyuridine Cdk cyclin dependent kinase CDKI cyclin dependent kinase inhibitor Chk1 checkpoint kinase 1 Chk2 checkpoint kinase 2 CldU chlorodeoxyuridine CPD cyclobutane pyrimidine dimers cPu cyclopurines CS Cockayne Syndrome CSB Cockayne Syndrome B DAR domain associated repair DDR DNA damage response DNA-PK DNA-dependent serine/threonine protein kinase dNTP Deoxyribonucleotide triphosphates DS dissecting solution DSB double-stranded bread dTTP thymidine triphosphate EdU 5-ethynyl-2’-deoxyuridine FdU fluorodeoxyuridine GGR global genome repair IdU iododeoxyuridine i.p. intraperitoneal injection IR ionizing radiation MEF mouse embryonic fibroblast MRN Mre11, Rad50 and Nbs1 NER nucleotide excision repair NPC neural precursor NSC neural stem cell PAN-118 3-aminopyridine-2-carboxaldehyde thiosemicarbazone PARP poly (ADP-ribose) polymerase PI propidium iodide PIKK phosphatidylinositol 3-kinase-like protein kinase RPA replication protein A SSB single-stranded break s.q. subcutaneous injection

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TCR transcription-coupled repair TFIIH multi-subunit transcription factor-IIH TTD trichothiodystrophy ROS reactive oxygen species RR ribonucleotide reductase UV ultraviolet XP Xeroderma Pigmentosum XPA Xeroderma Pigmentosum A

1

1. INTRODUCTION

1.1 DNA damage in eukaryotic cells

1.1.1 Endogenous and exogenous sources of DNA damage

DNA damage can be caused by exogenous or endogenous sources.

Approximately 105 DNA adducts are estimated to be generated in every cell each day

(Hoeijmakers, 2009). Exogenous DNA damage can be caused by physical sources

such as ultraviolet (UV) light or ionizing radiation (IR) or by chemical sources such as

chemotherapeutic agents or cigarette smoke (Ciccia and Elledge, 2010). Different types

of damage can be caused by chemotherapeutic agents depending on their mode of

action. For example some drugs are alkylating agents (i.e. temozolomide) which attach

alkyl groups to DNA bases whereas other drugs are crosslinking agents (i.e. cisplatin).

which can induce the formation of covalent links between DNA bases (Ciccia and

Elledge, 2010). Endogenous DNA damage can be caused by reactive oxygen species

(ROS) which are produced during regular cellular metabolism. ROS can oxidize DNA

bases and cause DNA breaks (Ciccia and Elledge, 2010).

1.1.2 DNA damage response signaling

The ability of a cell to detect DNA damage and respond is a signal transduction

pathway that is referred to as the DNA damage response (DDR). At the molecular level,

key proteins involved in DDR are members of the phosphatidylinositol 3-kinase-like

protein kinase (PIKKs) family and of the poly (ADP-ribose) polymerase (PARP) family.

Members of PIKKs family include ATM (ataxia telangiectasia mutated), ATR (ATM and

Rad3 related) and DNA-PK (DNA-dependent serine/threonine protein kinase) (Ciccia

and Elledge, 2010).

ATM is primarily activated by DNA strand breaks that can be formed by ionizing

radiation (Caporali et al., 2004) whereas ATR signals after DNA replication fork stalling

by DNA lesions (Fig. 1.1) including but not limited to those caused by UV irradiation

(Caporali et al., 2004; Myers and Cortez, 2006; Vrouwe et al., 2011). Double-stranded

break (DSB) detection and downstream signaling following DSBs involves the

interaction between the MRN (Mre11, Rad50 and Nbs1) complex and ATM (Roos and

Kaina, 2012). The MRN complex recruits and activates ATM at the site of DSBs

(Bakkenist and Kastan, 2003; Bartek and Lukas, 2003; Sengupta and Harris, 2005).

ATM generally exists as a dimer but following its recruitment to the DSB, it

autophosphorylates which results in the dissociation of the dimer into two active

monomers (Bakkenist and Kastan, 2003). Once ATM is activated, it phosphorylates

downstream signaling cascades which may lead to DNA repair, cell cycle arrest and/or

apoptosis (Bartek and Lukas, 2003). The ATR-ATRIP (ATR-interactive protein) complex

is recruited to the site of DNA damage that causes replication stress by RPA (replication

protein A) (Byun et al., 2005). Several other proteins are also recruited to form a

complex which results in the phosphorylation of ATR (Kurz and Lees-Miller, 2004).

Similar to ATM, the activation of ATR by phosphorylation initiates phosphorylation of

downstream targets and can also at times involve ATM (Kurz and Lees-Miller, 2004).

Targets of ATR and ATM include checkpoint kinase 1 (Chk1) and checkpoint kinase 2

(Chk2) which are involved in the phosphorylation of p53 (tumor suppressor) (Latonen

and Laiho, 2005). Activation of p53 can result in several downstream effects including

DNA repair, cell cycle arrest and/or apoptosis.

2

At the cellular level there are several consequences following DNA damage

including DNA repair, DNA lesion tolerance, cell cycle arrest and/or apoptosis. Not all

cell types are able to execute all of these functions. These four consequences will be

described briefly.

1.1.2.1 DNA repair

Several DNA repair pathways have evolved to remove a variety of DNA lesions.

When DNA bases are incorporated into DNA incorrectly during DNA replication, the

mispaired bases are excised and are replaced with the correct bases during mismatch

repair (Ciccia and Elledge, 2010; Friedberg, 2003). DNA lesions caused by hydrolytic

deamination, alkylating agents, ionizing radiation or intracellular metabolites are

repaired by base excision repair and involves DNA glycosylases (Rastogi et al., 2010).

A wide range of DNA lesions that result in the helical distortion of DNA, including lesions

caused by UV light, bulky chemical adducts and DNA intrastrand crosslinks, are

repaired by nucleotide excision repair (NER) (Hess et al., 1997). During NER, incisions

are created on either side of the lesion and a fragment, about 30 nucleotides, is

removed (Ciccia and Elledge, 2010; Friedberg, 2003). NER is described in more detail

in section 2.2. Double strand breaks are repaired by homologous recombination or non-

homologous end joining (Rastogi et al., 2010).

1.1.2.2 DNA lesion tolerance

Occasionally the DNA lesion is not repaired and the DNA strand break or adduct

remains but the cell tolerates the damage. For example, translesion DNA synthesis may

occur where the damaged DNA strand is passed over during DNA replication and gene

expression by low-fidelity DNA polymerases (Goodman, 2002). These DNA

4

polymerases are less stringent and can continue DNA replication in the presence of

DNA lesions which will often stall high-fidelity polymerases. Although these low-fidelity

polymerases are able to add nucleotides during replication of a region of DNA

containing a DNA lesion, mutations are more likely to occur in the recently synthesized

DNA strand since the inserted nucleotide may not be accurate (Goodman, 2002).

1.1.2.3 Cell cycle arrest

DNA damage may arrest the advancement of the cell cycle in order to provide

the cell an opportunity to repair the damage prior to DNA replication or the separation of

a damaged chromosome (Hartwell and Weinert, 1989). DSBs activate ATM and DNA-

PK whereas single-stranded breaks (SSBs) activates ATR. Once ATM and ATR are

activated, Chk2 and Chk1 are phosphorylated respectively. The DNA damage signal is

then passed to proteins involved in the cell cycle such as Cdc25 that is phosphorylated

by Chk1/Chk2. When Cdc25 is phosphorylated it is degraded by an ubiquitin-mediated

pathway resulting in G1-S phase arrest. It has also been suggested that the activation

of ATM/ATR may directly, or indirectly through Chk2, phosphorylate p53 which results in

the arrest of the G1/S or G2/M cell-cycle checkpoint (Harris and Levine, 2005; Lukas et

al., 2004; Rastogi et al., 2010).

1.1.2.4 Apoptosis

If a cell does not repair damaged DNA it is theorized that programmed cell death

(apoptosis) may be activated. Apoptosis is a controlled death mechanism in cells where

cellular characteristics include blebbing, cell shrinkage, chromatin condensation and

DNA and nuclear fragmentation (Munoz-Pinedo, 2012). Apoptosis can prevent

carcinogenesis following DNA damage (Rastogi et al., 2010). The extrinsic death

5

receptor apoptosis pathway and/or the intrinsic mitochondrial apoptosis pathways are

the most frequent programmed cell death pathways that are activated following DNA

damage (Fig. 1.2) (Roos and Kaina, 2006). p53 initiates the extrinsic apoptotic pathway

by regulating several genes including the Fas ligand (Harris and Levine, 2005). The

caspase cascade is then activated which results in the degradation of DNA by caspase-

activated DNase and caspases 3 and 7 inactivating cellular proteins (Roos and Kaina,

2012). p53 also activates the intrinsic apoptotic pathway including Bax (Bcl-2-

associated X protein) (Harris and Levine, 2005). Bax as well as pro-apoptotic factor Bak

(Bcl-2 homologous antagonist/killer) and anti-apoptotic factors such as Bcl-2 (B-cell

lymphoma 2) and Bxl-XL (B-cell lymphoma-extra-large) regulate the release of

cytochrome c from mitochondria. Cytochrome c interacts with Apaf-1 (Apoptotic

protease activating factor 1) to initiate a protease cascade which results in the activation

of caspase-9 followed by caspase-3 (Harris and Levine, 2005). After caspase-3

activation the anionic phospholipid phosphatidylserine is exposed on the cell surface

and apoptosis occurs (Niu and Chen, 2010).

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1.1.3 UV DNA lesions

Oxidative DNA damage is likely the most common form of DNA damage found in

neurons (McMurray, 2005). NER can remove oxidized DNA bases but the majority of

DNA lesions caused by ROS is repaired by base excision repair (Bjelland and Seeberg,

2003). Severe neurodegeneration is found in patients without functional NER (Brooks,

2002). Since neurons are not exposed to UV light which produces DNA adducts

repaired by NER, there may be other types of endogenous DNA damage in neurons

which is primarily repaired by NER (Andrews et al., 1978; Robbins et al., 1983). There is

no conclusive evidence regarding an endogenous form of DNA damage found in

neurons which is primarily repaired by NER but there are several candidate DNA

lesions which may be involved. For example, it has been proposed that cyclopurines, a

class of oxidative lesions, may be repaired by NER (Brooks, 2002). Cyclopurine is a

substrate for NER and not measurably repaired by base excision repair or direct repair

(Brooks et al., 2000; Kuraoka et al., 2000).

UV light was utilized in the current study as the DNA damaging agent since it

induces the formation of DNA lesions that are substrates for NER. UV light was utilized

for its reliability, simplicity, and efficacy, lack of requirement for bioactivation and lack of

metabolism. UV light can be categorized by wavelength. UV light with short

wavelengths (high energy) correlates with DNA lesions such as cross-links, while UV

light with long wavelengths (low energy) correlates with DNA modifications, strand-

breaks and sites of base loss (Kielbassa et al., 1997). UVA (wavelength: 315-400nm)

has low energy and is inefficient in inducing DNA damage since it is not absorbed by

native DNA (Rastogi et al., 2010). UVA often causes the production of ROS which result

in oxidative DNA lesions (i.e. 8-oxoguanine) or strand breaks (Ananthaswamy and

8

Pierceall, 1990). UVB (280-315nm) and UVC (<280nm) have higher energy and can

induce chemical modifications in DNA and alter the molecular structure of DNA by

causing the formation of pyrimidine dimers (Rastogi et al., 2010). We used UVC as our

DNA damaging agent since it produces lesions that are substrates for NER.

The most frequent DNA lesions induced by UVC are cyclobutane pyrimidine

dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts (6-4PPs) (Fig. 1.3)

(Rastogi et al., 2010). The structure of CPDs is a four-member ring structure which

includes two carbon atoms of neighboring pyrimidine bases. 6-4PPs are noncyclic

structures with a bond between adjacent pyrimidine bases (Rastogi et al., 2010).

Adjacent thymine-thymine or thymine-cytosine bases are more photoreactive than

cytosine-thymine or cytosine-cytosine bases (Rastogi et al., 2010). CPDs are more

frequent and are often 75% of UV-induced DNA adducts whereas 6-4PPs are 25% of

lesions (Sinha and Hader, 2002). Even though CPDs and 6-4PPs are both repaired by

NER, 6-4PPs are repaired at least 5-fold faster than CPDs (de Laat et al., 1999; van

Hoffen et al., 1995).

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1.2 Nucleotide excision repair

1.2.1 Overview of nucleotide excision repair

The nucleotide excision DNA repair (NER) pathway, removes bulky DNA adducts

which distort the DNA helix. These lesions include intrastrand crosslinks, bulky chemical

lesions, cyclodeoxyadenosine, thymine glycol, cyclopurines and sometimes 8-oxo-G

(Friedberg, 2003). Bulky DNA adducts can arise from UV irradiation or the metabolism

of endogenous or exogenous compounds. During NER, over 30 proteins are involved in

the recognition, incision, and excision of an approximately 30 nucleotide long

oligonucleotide which is replaced and resealed by DNA polymerase and DNA ligase

respectively (Le May et al., 2010).

NER can be divided into two sub-pathways depending on how the DNA adduct is

initially recognized, global genome repair (GGR) and transcription-coupled repair (TCR)

(Fig.1.4). In GGR, DNA adducts are recognized by the XPC-hHR23B complex that

recruits other NER factors. The rate of GGR is dependent on the type of DNA lesion, for

example, 6-4PPs are repaired at a faster rate than CPDs even though both are UV

lesions. The XPC protein is restricted to GGR and not required in TCR (van Hoffen et

al., 1995). In TCR, DNA adducts are recognized by a stalled RNA polymerase which

recruits two proteins CSA and CSB. Following this initial DNA damage recognition,

GGR and TCR are very similar (Rastogi et al., 2010). The DNA at the location of the

damage is unwound by components of multi-subunit transcription factor-IIH (TFIIH).

Two helicases that are subunits of TFIIH, XPB and XPD, open the DNA double helix

(Tirode et al., 1999). Next, RPA and XPA are recruited to verify the damage (Hitomi et

al., 2007), XPG and ERCC1-XPF cleave the DNA strand on 3’ and 5’ ends of the

damage and an approximately 30 base oligonucleotide fragment is removed (Houtgraaf

11

et al., 2006). DNA polymerase δ and ε fill in the gap and DNA ligase seals it (Moser et

al., 2007).

12

1.2.2 Diseases caused by defective nucleotide excision repair

Inherited deficiency in NER has been linked to at least three diseases in humans:

xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy

(TTD). Eleven out of the 30 proteins discovered to date to be involved in the NER

pathways have been connected to one or more of these three diseases. XP is caused

by mutations in one or more of the 7 complementation groups of XP, XPA to G,

whereas CS is caused by mutations in CSA or CSB. TTD is caused by mutations in

TFIIH subunits (XPB, XPD or TTDA) which results in dysfunction of the transcription

factor.

The frequency of XP is estimated to be approximately 1 in 105 and is autosomal

recessive. XP is characterized by photosensitivity, hyperpigmentation, dry scaly skin

caused by sun exposure and a 1000 fold increased risk of carcinomas and melanomas

of the skin and eyes (Kraemer et al., 1987). The symptoms vary depending on the

mutation and the degree to which the mutation disrupts NER. For example, patients

with defective XPC have a milder form of the disorder than patients with defective XPA

since XPC is only required for GGR while XPA is required for GGR and TCR

(Niedernhofer, 2008). Neurological symptoms occur in approximately 20% of XP

patients (Robbins et al., 1991) and characterized by mental retardation, microcephaly,

growth retardation, spasticity and ataxia (Brooks, 2002). The majority of neurological

symptoms are found in patients with defective XPA (Hentati et al., 1992). The source of

these neurological symptoms is due to progressive neurodegeneration (Niedernhofer,

2008).

Similarly to XP, CS is also an autosomal recessive condition. Although CS is less

common than XP it is a more severe disorder and a majority of the patients exhibit

14

progressive postnatal growth failure, neurological dysfunction and accelerated aging

(Nance and Berry, 1992). Neuropathological findings in the central nervous system

include atrophy of the white matter, enlarged ventricles and atherosclerosis

(Niedernhofer, 2008). Similar to XP, apoptosis in granule cells and loss of Purkinje cells

were found in several CS patients (Kohji et al., 1998).

1.2.3 Nucleotide excision repair in neurons

Apoptotic cell death of neurons found in XP and CS patients is suggested to be

caused by the lack of repair of endogenous DNA damage. As DNA adducts accumulate

in the neuron, defective proteins may be transcribed which may ultimately lead to cell

death (Lu et al., 2004; Taddei et al., 1997). Thus, functional NER appears to be

important in neurons.

NER is attenuated in neurons relative to proliferating cells (Nouspikel, 2007). In

human neuroblastoma cells (Jensen and Linn, 1988), NT2 neuroteratoma cells

(Nouspikel and Hanawalt, 2000) and primary neurons (Nouspikel and Hanawalt, 2002),

NER has been shown to be attenuated. For instance, when NT2 human neuroteratoma

cells were differentiated into neuron-like cells there was a loss of NER of CPD lesions.

CPDs were removed in active genes but were not removed from silent genes indicating

a lack of GGR (Nouspikel and Hanawalt, 2000). NER has been observed to be

attenuated in other differentiated cells such as striated muscle, macrophages and

keratinocytes (Nouspikel, 2008). A decrease in repair of silent genes was also seen in

other types of differentiated cells like myoblasts. In rat L8 myoblast cell line, UV-induced

CPD adducts were repaired in active genes but not in silent genes (Ho and Hanawalt,

1991). An explanation for functional TCR but attenuation of GGR in differentiated cells

is that since these cells do not divide and do not need to replicate DNA, they do not

15

need to expend energy in repairing DNA damage. However, repair of active genes

should be maintained since these genes are transcribed (Nouspikel, 2007). When DNA

repair of active genes was examined in detail, it was discovered that both the

transcribed and non-transcribed strands of transcribed genes are both repaired in

human neurons (Nouspikel and Hanawalt, 2000) and macrophages in a process called

domain associated repair (DAR) (Hsu et al., 2007). The lack of GGR in neurons has

been hypothesized to be connected to neurodegenerative diseases such as Alzheimer’s

and Parkinson’s.

1.3 Neuronal cell cycle re-entry

Neurons are considered to be post-mitotic cells. One stress response observed

in neurons is re-entry into the cell cycle, an event that is followed by cell death (Herrup

et al., 2004). The precise molecular mechanisms that initiate cell cycle re-entry and

couple it to a death response remain poorly understood and areas of active research.

1.3.1 Overview of the cell cycle

Cell proliferation is a tightly regulated process controlled by the cell cycle. For a

brief review, the cell cycle can be divided into four phases: the first gap phase (G1), the

DNA synthesis phase (S phase), the second gap phase (G2) and mitosis (M). Cells that

have exited the cell cycle are in a quiescent state (G0). The cell cycle manages the

transition of cells from G1 through M with several checkpoints to assess damage and

ensure successful completion of the previous phases. The progression of a cell through

the cell cycle is regulated by two classes of proteins: cyclins and cyclin-dependent

kinases (Cdks) (Malumbres and Barbacid, 2001). Cyclins can be divided into two main

families, mitotic cyclins (cyclin A and cyclin B) and G1 cyclins (cyclin C, cyclin D and

16

17

cyclin E) (Hess et al., 1997; Kurz and Lees-Miller, 2004). Cdks are a group of

serine/threonine kinases that bind to cyclins (Malumbres and Barbacid, 2001) and form

active heterodimer complexes and drive the cell through the cell cycle (Malumbres and

Barbacid, 2001). Nine structurally related Cdks (Cdk1-Cdk9) have been identified

(Schwartz and Shah, 2005). Several Cdks function together, primarily Cdk4, Cdk6,

Cdk2, Cdk1 and perhaps Cdk3, to ensure successful progression through the cell cycle

(Malumbres and Barbacid, 2001).

Cdks need to form a complex with cyclins in order for kinase activity to be

activated (Malumbres and Barbacid, 2001). The transition from G1 to S phase referred

to as the restriction point is controlled by growth factors (Coller, 2007). During G1

progression, levels of cyclin D isoforms (cyclin D1, D2 and D3) increase in the presence

of extracellular mitogens. Cyclin D1 interacts with Cdk2, 4 and 6 and phosphorylates

downstream targets (Coller, 2007; Schwartz and Shah, 2005). Cyclin E interacts with

Cdk2 in late G1 (Schwartz and Shah, 2005). S phase progression and the transition

from S phase to G2 are regulated by the cyclin A/Cdk2 complex (Schwartz and Shah,

2005). After the G2 phase, the cyclin B/Cdk1 complex shuttles from the cytoplasm into

the nucleus in order to initiate mitosis (Malumbres and Barbacid, 2001). For the cell to

exit mitosis, cyclin B needs to be degraded (Malumbres and Barbacid, 2001).

During G1 progression, several members of the retinoblastoma protein (Rb)

family are phosphorylated by cyclin D/Cdk4/6 followed by cyclin E/Cdk2 (Schwartz and

Shah, 2005). Rb is tumor suppressor gene and is hypophosphorylated in its active

state. An active Rb protein interacts with a group of transcription factors, E2F-DP

(E2F1, E2F2 and E2F3) to form an inhibitory complex that prevents the transcription of

S-phase proteins (Malumbres and Barbacid, 2001). The consecutive phosphorylation of

Rb by cyclin D/Cdk4/6 followed by cyclin E/Cdk2 alters Rb activity. First, Cyclin

D/Cdk4/6 partially phosphorylates Rb. While Rb continues to be bound to E2F-DP, it

does not completely inhibit E2F-DP and E2F-DP transcribes several genes including

cyclin E. Cyclin E, in turn, binds to Cdk2 and this complex (hyper)phosphorylates Rb

(pRb) (Malumbres and Barbacid, 2001). pRb releases E2F-DP and the E2F

transcription factors transcribe many S phase proteins (Schwartz and Shah, 2005).

Once the cell has entered the S phase, cyclins D and E are degraded and cyclin A/Cdk2

regulates the S phase. Several proteins required for DNA synthesis such as histones

and proliferating cell nuclear antigen (PCNA) are produced. At the same time, cyclin

A/Cdk2 phosphorylates E2F; this results in the inactivation and degradation of E2F

(Schwartz and Shah, 2005).

Cdk inhibitors (CDKIs) are able to control the cell cycle at several checkpoints by

preventing the formation or inhibiting the function of cyclin/Cdk complexes (Schwartz

and Shah, 2005). CDKIs can be divided into two families INK4 and Cip/Kip. The INK4

family of CDKIs prevents Cyclin/Cdk complexes from forming whereas the kinase

activity of already formed cyclin/Cdk complexes is inhibited by the Cip/Kip family (Sherr

and Roberts, 1999).

The differentiation of neurons occurs in the ventricular zone (VZ) and

subventricular zone (SVZ) of the central nervous system (CNS) where neuroblasts

become mature neurons and exit the cell cycle, becoming permanently post-mitotic

(Lennington et al., 2003; Yang and Herrup, 2007). Neurons cannot proliferate once they

have exited the cell cycle (Liu and Greene, 2001). However, recent evidence has

indicated that after neurons have entered the G0 phase they can still re-enter the cell

18

cycle but contrary to expectations, they undergo apoptosis instead of completing the cell

cycle (Kruman, 2004).

1.3.2 In vivo evidence of neuronal cell cycle re-entry

Increasing experimental evidence demonstrates a correlation between cell cycle

re-entry and neuronal loss in neurodegenerative diseases (Wang et al., 2009). Cell

cycle protein expression has been shown in several neurodegenerative disorders

including Alzheimer’s disease (AD), Parkinson’s disease, amyotrophic lateral sclerosis,

stroke and ataxia-telangiectasia. In addition, several animal models of diseases, such

as AD, amyotrophic lateral sclerosis, traumatic brain injury and cerebral hypoxia-

ischemia, have shown neuronal cell cycle re-entry (Copani et al., 2007; Wang et al.,

2009).

Alzheimer’s disease is a well-studied area of research in neuronal cell cycle re-

entry. One of the first pieces of evidence of neuronal cell cycle re-entry in AD was the

presence of active cell cycle molecules in AD postmortem brains shown by Vincent and

Davies, 1996. Neuronal cell cycle re-entry was primarily shown indirectly using

immunocytochemistry. In AD but not control postmortem brains, several cell cycle

proteins such as cyclin D, E, Cdk4, PCNA, cyclin B, cdc2, and Ki67 were found. CDKIs,

p16, p21 and p105 were also shown to be present in AD postmortem brains but not in

controls (Herrup et al., 2004). However, these findings did not confirm neuronal cell

cycle re-entry since these proteins may be involved in other unknown cellular functions.

One of the strongest pieces of direct evidence of neuronal cell cycle re-entry was

conducted by Yang et al who used fluorescent in situ hybridization (FISH) to illustrate

that DNA replication had actually occurred in neurons of AD brains. These findings

suggest that neurons may initiate the S phase of the cell cycle. Yang et al also showed

19

that neuronal cell cycle re-entry occurred in early stages of AD by looking at individuals

who died with mild cognition impairment (MCI) (Yang et al., 2001). MCI is considered to

be an early stage of AD since a large percentage of individuals with MCI are diagnosed

with AD several years later (Herrup et al., 2004).

Abnormal neuronal cell cycle may be an early indication of neuronal stress in

human and mouse AD (Herrup et al., 2004).There are also several transgenic mouse

models of AD. In four of these models the presence of abnormal cell cycle re-entry of

neurons in susceptible areas of the brain was shown (Herrup et al., 2004). Although

there is no significant loss of neurons in these models, amyloid plaques are formed

(Herrup et al., 2004). Interestingly, in one of the models, R1.40, Yang et al. showed that

neuronal cell cycle re-entry can be seen as early as 6 months, several months before

amyloid plaques begin forming (Herrup et al., 2004). This finding along with the

discovery from patients with MCI described above suggests that abnormal neuronal cell

cycle re-entry may be an early indication of neuronal stress in human and mouse AD

(Herrup et al., 2004)

1.2.3 In vitro evidence of neuronal cell cycle re-entry

Similar to the above in vivo findings, in vitro findings also noted neuronal cell

cycle re-entry (Table 1.1). There is indirect evidence suggesting that cerebellar granule

neurons, sympathetic neurons and cortical neurons re-enter the cell cycle after

conditions of stress and ultimately undergo apoptosis (Yang and Herrup, 2007). Many

DNA damaging agents including UV, etoposide, and camptothecin also cause abnormal

neuronal cell cycle re-entry (Table 1.1). The degree of neuronal progression through the

cell cycle prior to apoptosis depends on the stressor. For instance, neurons treated with

thrombin undergo apoptosis as soon as they transition from G0 to G1 (Liu et al., 2010).

20

However, neurons treated with etoposide proceed as far as the S phase before they

undergo apoptosis (Kruman et al., 2004). Interestingly, the furthest the neurons have

progressed through the cell cycle before they die is the S phase. To date, there has

been no evidence of neurons in the G2 phase or M (Liu et al., 2010).

Since cell cycle proteins appear in neurons prior to apoptosis, several

investigators have tested whether inhibitors of these cell cycle proteins prevent neuronal

apoptosis. Previous studies using caspase inhibitors to prevent apoptosis slowed down

neuronal death but did not prevent it. Cdk inhibitors (CDKIs), on the other hand,

protected neurons longer than caspase inhibitors (Wang et al., 2009). By inhibiting

Cdks, there was not any cytochrome c release, loss of mitochondria transmembrane

potential and caspase activation. These findings indicated that cell cycle re-entry is

upstream of other apoptotic pathways (Wang et al., 2009). Pharmacological inhibition of

Cdks prevents the G1/S transition and protects cultured neurons from death initiated by

several DNA damaging agents: UV, camptothecin and cytosine arabinoside (Park et al.,

1998a; Park et al., 1998b). Furthermore, dominant-negative kinase-inactive forms of

Cdk4 and Cdk6 reduced UV-induced death (Park et al., 1998a; Park et al., 1998b).

Thus, there has been some indirect evidence of neuronal cell cycle re-entry in vitro and

correlation between DNA damage and neuronal cell cycle regulation (Kruman et al.,

2004). There has been relatively little direct evidence of cell cycle re-entry in neurons.

For example, one study reported a significant increase of S phase neurons from

cultures treated with genotoxic chemicals (etoposide, homocysteine, methotrexate and

AB1-42) using flow cytometry to measure DNA content (Kruman et al., 2004) which

indicate that neurons can initiate the S phase prior to apoptosis. The majority of studies

conducted suggest neuronal cell cycle re-entry based on evidence of cell cycle marker

21

expression or prevention of DNA-damage induced apoptosis using Cdk inhibitors

however these proteins may have cellular functions independent of the cell cycle in

neurons.

22

Table 1.1 Evidence of neuronal cell cycle re-entry after DNA damage DNA Damage Experimental evidence of cell cycle entry Experimental

Model Reference

UV flavopiridol (inhibits Cdk1/2/4) rescues neurons from death

sympathetic or cortical neurons

(Park et al., 1998b)

olomoucine (inhibits Cdk1/2/5 and ERK-1/MAP-1 kinases) reduces death

expression of Cdk inhibitors (p16, p21 or p27) reduces UV-induced death

rat sympathetic neurons culture

(Park et al., 1998a)

expression of dominant-negative kinase-inactive form of Cdk4 or 6 reduces UV-induced death

expression of dominant-negative DP1 suppresses death

sympathetic neuron culture

(Park et al., 2000)

Camptothecin flavopiridol (Cdk1/2 inhibitor) or olomoucine (Cdk1/2/5 inhibitor) rescue death


(Morris et al., 2001; Park et al., 1997)

Increased cyclin D kinase activity rat cortical neuron culture, pRb kinase assay

(Park et al., 1998a)

rapid increase in Cdc25A activity cortical neurons

(Zhang et al., 2006)

rapid pRb and p107 phosphorylation, then loss of Rb and p107

(Park et al., 2000)

pRb with mutated phosphorylation sites inhibited death

Pim-1 induction (Zhang et al., 2010) Pim-1 inhibition activates Cdc25A, cyclin D1-

associated kinases and blocks camptothecin-induced neuron death

cytosine arabinoside

flavopiridol (Cdk1/2/4 inhibitor) or olomoucine (Cdk1/2/5 inhibitor) rescue death


(Park et al., 1997)

Etoposide increased proportion of S-phase neurons assessed by flow cytometry

35% neurons, PI to assess S phase

(Kruman et al., 2004)

hydrogen peroxide

significant increase in BrdU positive neurons measured by flow cytometry, indicated of S phase entry

cortical neurons

(Schwartz et al., 2007)

RNA interference against Cdks (Cdk4/6 or Cdk3) reduces death

increase in Cyclin E (marker of G1 to S transition)

hydrogen peroxide plus ATM inhibition

increased DNA content by EdU cytometry (EdU pulse 15 min before fix)

differentiated human neural stem cells

(Hitomi and Stacey, 2010) increased cyclin D1

Homocysteine increased proportion of S phase neurons assessed by flow cytometry

20% of neurons, PI to assess S phase


increased expression of cell cycle marker Cdc25A (a phosphatase involved in cell cycle progression by activating Cdks, regulator of G1-to-S transition)

cortical neurons

reducing cyclin D1 expression protects from death cortical neurons

(Ye and Blain, 2010)

inhibiting from Cdk2/4 protects from death

immunoreactivity of cell cycle proteins after homocysteine

23

DNA Damage Experimental evidence of cell cycle entry Experimental Model

Reference

ionizing radiation

increase in proportion of neurons double positive for DNA synthesis and apoptosis

cortical neurons


Methotrexate increased proportion of S-phase neurons assessed by flow cytometry

20% of neurons, PI to assess S phase

1.4 5-bromo-2’-deoxyuridine (BrdU)

1.4.1 Overview of BrdU BrdU is an analogue of the nucleoside thymidine, where a methyl group is

replaced by bromine (Fig. 1.5). Since it is structurally similar to thymidine, BrdU can be

incorporated into DNA during DNA repair or DNA replication during the S-phase of the

cell cycle (Beisker and Hittelman, 1988; Gratzner, 1982; Nowakowski et al., 1989; Ross

et al., 2011). When BrdU enters cells it is phosphorylated into BrdU triphosphate and

this precursor is incorporated into DNA in the place of deoxythymidine triphosphate

(Jackson and Cook, 2008). Numerous studies suggest negative effects of BrdU

incorporation in DNA (Littlefield and Gould, 1960; Stellwagen and Tomkins, 1971), but

since BrdU is not believed to have any significant detrimental effects if cells are treated

for a short period of time, BrdU is often used to label cycling cells in vitro and in vivo

(Cameron and McKay, 2001; Lehner et al., 2011; Levkoff et al., 2008). In vitro,

mammalian cells are often supplemented with BrdU for one hour and BrdU

incorporation is measured using immunocytochemistry or flow cytometry. In vivo,

animals are injected with BrdU and immunohistochemistry is conducted. Using these

techniques BrdU can be used for cell birth dating, tracking cells following transplantation

and understanding cells which proliferate in the brain such as neural stem cells

(Cooper-Kuhn and Kuhn, 2002; Kuhn and Cooper-Kuhn, 2007; Lehner et al., 2011).

24

BrdU incorporation has been suggested to change DNA stability and increase the

possibility of sister-chromatid exchanges, mutations and double stranded breaks when

cells are also exposed to other stresses. For instance, BrdU has been used as a

radiosensitizer since it can cause chromosomal breakage and cause cells to become

more sensitive to ionizing radiation (Djordjevic and Szybalski, 1960; Erikson and

Szybalski, 1963; Hsu and Somers, 1961). Therefore, BrdU has been used in

conjunction with chemotherapy and radiation for treatment of different cancers in clinical

trials (Groves et al., 1999; Kinsella et al., 1984; Phillips et al., 1991; Prados et al., 2004;

Robertson et al., 1997a; Robertson et al., 1997b). The clinical benefits of BrdU as a

radiosensitizer has not been great but there may be other uses for BrdU (Levkoff et al.,

2008). The following two sub-sections will discuss the possible role of BrdU in DNA

repair and cell cycle progression.

1.4.2. The role of BrdU in DNA repair

Deoxyribonucleotide triphosphates (dNTPs) are required for DNA synthesis and

repair. As a cell transitions from the G1 phase to the S phase of the cell cycle dNTP

pools are increased approximately 5 to 10 fold and maintained at this level until DNA

replication is finished. When cells progress pass the S phase dNTP levels are reduced

(Reichard, 1988). Even though the dNTP pool sizes of neurons have not been

determined, examination of G0 phase 3T3 mouse fibroblasts indicated that thymidine

triphosphate (dTTP) pools are reduced by 20-fold in comparison with S phase cells.

Furthermore, dTTP turnover was 200-fold faster in S phase cells than G0 cells (Spyrou

and Reichard, 1988). Since G0 cells are not replicating their DNA, a small dNTP pool

may be sufficient for DNA repair and mitochondrial DNA synthesis. Yet, if there is a

great deal of DNA damage, neurons may not have enough dNTPs for repair. Thus,

26

increasing neuronal dNTP pool size may improve DNA repair. Thymidine supplement

does increase intracellular dTTP pool size in hepatocytes (Rottgen and Rabes, 1989)

and BrdU exposure to hamster melanoma cells formed a large BrdU triphosphate pool

which remained at a high concentration for several days (Ashman et al., 1981). At the

same time, deoxycytidine triphosphate levels dropped drastically after the addition of

BrdU (Ashman and Davidson, 1981). This finding supports the idea that increasing

extracellular concentrations of pyrimidines may increase intracellular concentrations of

dNTPs.

Terminally differentiated cells such as neurons are often relatively deficient in

DNA repair. During cellular differentiation, DNA repair is gradually reduced and cells

may be more vulnerable to DNA damage (Kruman, 2004; McCombe et al., 1976). In rat

neurons there is evidence of low NER (Subrahmanyam and Rao, 1991) and double-

strand break repair (Gobbel et al., 1998; Wang and Wheeler, 1978). NER was also

shown to be decreased in human neuroblastoma and NT2 neuroteratoma (Jensen and

Linn, 1988; Nouspikel and Hanawalt, 2000). DNA repair consumes a great deal of ATP

(Roca and Cox, 1997). Since neurons are metabolically active cells that require a large

amount of energy, it has been suggested that DNA repair is down-regulated in neurons

to save energy (Kruman, 2004). NER can be divided into two pathways, TCR which

repairs DNA lesions in transcribed genes and GGR which repairs DNA lesions

throughout the entire genome. Although repair of active genes in neurons by TCR has

been maintained in neurons (Kruman, 2004), GGR is significantly lower in neurons than

fibroblasts (Kruman, 2004; Nouspikel and Hanawalt, 2000; Yamamoto et al., 2007).

Removal of DNA adducts in transcribed genes is important in order for the cell to

maintain proper cellular function whereas GGR may not be essential since neurons

27

have exited the cell cycle and do not divide (Kruman, 2004). The attenuation of GGR

may be responsible for the higher sensitivity of neurons to DNA damage-initiated

apoptosis. Improving neuronal DNA repair could theoretically improve neuronal survival

following DNA damage.

NER is inducible. DNA repair in general was first discovered to be inducible in

1975 in bacteria and is referred to as the SOS response (Radman, 1975). NER has also

been shown to be inducible in mammalian cells as well (Gilchrest and Eller, 1999). For

example, single stranded DNA fragments such as thymidine dinucleotide (pTT) induce

protective responses in skin cells as well as intact guinea pig skin (Eller et al., 1994).

Furthermore, pTT partly increases repair of UV lesions by activating p53 (tumor

suppressor protein and transcription factor) and up-regulating several proteins involved

in DNA repair (Gilchrest and Eller, 1999). Interestingly, in vitro, pTT enhanced skin cells

ability to repair UV lesions by activating p53 and improving endogenous DNA repair

(Goukassian et al., 2004). pTT did not only improve removal of UV lesions but also

benzo(α)pyrene adducts which are also repaired by NER (Maeda et al., 1999).

Oligonucleotides similar to the mammalian telomere repeat sequence 5’-TTAGGG-3’ (T-

oligos) also improved DNA repair similar to pTT (Eller et al., 2002). Other compounds,

in addition to pTT, also improve DNA repair. For example, treatment with green tea

polyphenols improved the removal and/or repair of CPDs (DNA adducts caused by UV)

in mice skin exposed to UVB (Katiyar et al., 2010). The improvement in DNA repair of

DNA adducts in mouse skin could be due to increased levels of NER proteins XPA and

XPC. In order for green tea polyphenols to improve repair of UV-induced DNA adducts,

functional NER is required indicating that the effect of green tea polyphenols is through

the NER pathway (Katiyar et al., 2010).

28

1.4.3. The role of BrdU in the cell cycle

Although BrdU has been used to label proliferating cells and assumed that it

incorporates into DNA, there are other cellular effects of BrdU. Around the time BrdU

was initially introduced, cellular effects of BrdU were studied and it was discovered that

BrdU does not benignly substitute for thymidine. For instance, BrdU causes mouse

neuroblastoma C1300 cells to differentiate and structurally look like mature neurons

even without DNA synthesis (Schubert and Jacob, 1970). Thus, halogenated

pyrimidines do not need to be incorporated into DNA in order to change the function of

cells (Schubert and Jacob, 1970). While it has not been shown that BrdU alters cell

cycle re-entry in neurons, BrdU does alter cell cycle progression in proliferating cells

(Table 1.2). A single BrdU exposure to cancer cells impairs cell cycle progression.

Interestingly, these cells slowly accumulate in the G1 phase of the cell cycle and do not

die (Levkoff et al., 2008). BrdU treatment has also slowed down cell growth in vivo (Gyi

and Wrathall, 1983). Similarly, other thymidine analogues, CldU, IdU, FdU and EdU,

have all been shown to inhibit cell proliferation (Levkoff et al., 2008; Ross et al., 2011).

29

Table 1.2 Evidence of cell cycle modulation by thymidine analogues Nucleoside analogue

Cell type Effect of nucleoside analogue Experimental conditions

Reference

BrdU B-cell lines Synergistically cytostatic with cisplatin, bleomycin and chlorambucil

100-500 µM BrdU

(Poot et al., 1991)

Melanoma cells

Reduced growth in vivo 3 µg/mL BrdU (Gyi and Wrathall, 1983)

Neurogenic astrocytes

Caused rapid increase in proportion of cells in G1 and decrease in proportion of cells in S

Single 24h pulse of 50 µM BrdU;

(Ross et al., 2008)

Breast and bladder cancer lines

Reduced cell cycle progression but no increase in death

3.5, 5 and 60 µM BrdU

(Diermeier et al., 2004)

Gastric carcinoma lines

Caused G1 arrest and S phase delay 20 µM BrdU (Peng et al., 2001)

A549 lung cancer cells

Reduced proliferation 50 µM BrdU for 24 h, cells are p16-null

(Masterson and O'Dea, 2007)

MG-63 osteosarcoma cell line

Reduced proliferation 50 µM BrdU for 18 h

(Levkoff et al., 2008)

H9 T-cell lymphoma cell line

50 µM BrdU for 24 h

Saos-2 human osteosarcoma cell line

TT human thyroid tumour cell line

BJ human fibroblasts

RG2 rat glioma cell line

Reduced proliferation in a dose-response manner

1 – 50 µM BrdU


Cells pretreated with BrdU then injected into mice showed suppressed proliferation in vivo relative to vehicle controls

50 µM BrdU for 24 h


Mice inoculated with glioma cells and treated with BrdU showed improved survival relative to vehicle controls

6 i.p. injections of 300 mg/kg BrdU over 2 days


Mice inoculated with glioma cells and treated with BrdU showed improved survival relative to vehicle controls

BrdU 0.8 mg/mL in drinking water

CldU H9 (human lymphoma cells)

Reduced proliferation in a dose-response manner similar to BrdU

1 – 50 µM CldU for 18 h

IdU H9 Reduced proliferation in a dose-response manner similar to BrdU

1 – 50 µM IdU for 18 h

FdU H9 Transiently reduced proliferation in a dose-response manner, but not as effective as BrdU

1 – 50 µM FdU for 18 h

30

Nucleoside analogue

Cell type Effect of nucleoside analogue Experimental conditions

Reference

EdU hGBM, primary human glioblastoma cell lines

Suppressed proliferation in culture better than BrdU

1 – 50 µM EdU for 24 h

(Ross et al., 2011)

MG63 osteosarcoma cell line



Mouse embryonic fibroblasts



hGBM glioblastoma cells

Reduced tumour volume in vivo in xenograft model

50 mg/kg/day EdU i.p. or s.q. long term (up to 100 days)

hGBM glioblastoma cells

Improved survival of xenografted mice 50 mg/kg/day EdU i.p. or s.q. long term (up to 100 days)

BrdU inhibits ribonucleotide reductase (RR). RR catalyzes the rate-limiting step

of the synthesis of deoxyribonucleotides (dNTPs) from ribonucleotide precursors (Cory

and Sato, 1983; Jiang et al., 2006; Thelander and Reichard, 1979). In the 1970s, BrdU

was discovered to be an allosteric inhibitor RR (Meuth and Green, 1974). Thus,

functional RR is required for sustaining dNTP supply necessary for DNA repair and

synthesis (Finch et al., 2000). 3-aminopyridine-2-carboxaldehyde thiosemicarbazone

(PAN-118) is an inhibitor of RR and was developed for cancer treatment (Jiang et al.,

2006). PAN-811 has some neuroprotective effects and prevents ischemic neurotoxicity

as well as hypoxic toxicity and also prevents neuronal death from neurotoxic agents

such as staurosporine, veratridine and glutamate (Jiang et al., 2006). Although Jiang et

al suggest that PAN-118 may be neuroprotective by altering excitatory neurotoxic

pathways (Jiang et al., 2006), PAN-118 may also be neuroprotective by inhibiting RR.

Recently, the effect of BrdU on neural stem cells (NSCs) has also been studied

in the mammalian brain. NSCs are found in the subependymal zone of the lateral

31

ventricles and in the hippocampus and can differentiate into neurons and glia

(astrocytes and oligodendrocytes) (Doetsch, 2003). BrdU prevents NSC cell cycle

progression, induces them to differentiate into astrocytes and causes DNA

demethylation (Schneider and d'Adda di Fagagna, 2012). Methylation (adding a methyl

group) to cytosines in DNA occurs in regions called CpG islands and is highly regulated

in stem cells (Hermann et al., 2004; Wu and Zhang, 2010). Particularly, DNA

methylation of specific genes affects differentiation (Meissner et al., 2008). DNA

methyltransferases (DNMT) methylate either de novo (DNMT3a and DNMT3b) or newly

replicated DNA (DNMT1) (Hermann et al., 2004). DNMT1 localizes to replication foci

during S phase of the cell cycle and is down-regulated in proliferating cells whereas

DNMT3a and DNMT3b may be independent of the cell cycle (Hermann et al., 2004;

Schneider and d'Adda di Fagagna, 2012). Demethylation of DNA can either occur by

removing methylated cytosines or inhibition of DNMTs (Wu and Zhang, 2010). Other

thymidine analogues, CldU and IdU, also have effects similar to BrdU in neural stem

cells (Schneider and d'Adda di Fagagna, 2012). Studies on NSC have also shown that

BrdU treatment of neurosphere culture caused cellular senescence (Lehner et al., 2011;

Ross et al., 2008).

32

2. OVERVIEW The limited capacity to replace neurons stresses the importance of the neuronal

DNA damage response. Neuronal DNA damage can lead to apoptosis. To prevent

neuronal DNA damage-induced apoptosis, understanding the neuronal DNA damage

response is essential.

In this project, it was observed that BrdU prevented UV-induced neuronal death.

This is the first report of the neuroprotective action of BrdU and the mechanism of BrdU

neuroprotection is unknown. Therefore, we investigated dose-response relationships as

well as determined whether proliferating cells were also protected by BrdU. Since BrdU

is a thymidine analogue, we determined whether other thymidine analogues and DNA

nucleosides protect neurons. p53 is a central stress response protein involved in many

neuronal death pathways (Harris and Levine, 2005). To determine whether BrdU altered

the UV-induced neuronal DNA-damage response we investigated p53 expression after

UV and its potential modulation by BrdU. Theoretically, the repair of DNA damage

should be neuroprotective, thus we also interrogated whether BrdU improves neuronal

DNA repair. Indirect evidence indicates that UV induces neuronal cell cycle re-entry

which ultimately leads to apoptosis (Park et al., 1998a). Since BrdU has been shown to

impair cell cycle progression in proliferating cells (Levkoff et al., 2008; Schneider and

d'Adda di Fagagna, 2012) we examined whether BrdU prevented neuronal cell cycle re-

entry.

33

2.1 Hypothesis

We hypothesized that BrdU prevented UV-induced neuronal apoptosis by

improving the DNA damage response.

2.2 Aims

This work has four specific aims:

1. Characterize dose-response relationships, cell type specificity and chemical

specificity of BrdU protection.

2. Determine whether BrdU modulates UV-induced cell signaling by p53

expression.

3. Assess the role of nucleotide excision repair in UV-initiated neuronal apoptosis.

4. Determine whether UV or BrdU modulate neuronal cell cycle re-entry.

34

3. MATERIALS AND METHODS

3.1 Cortical neuron culture

CD1 males and females were mated and the plug date used was E0.5. The

cortex was dissected in cold dissecting solution (DS) from embryo brains (E16.5). DS

contains 137 mM NaCl, 5.4 mM KCl, 0.17 mM Na2HPO4, 0.22 mM KH2PO4, 9.9 mM

Hepes, 33.3 mM D (+)-Glucose and 43.8 mM sucrose in ultra-filtered water. The freshly

dissected embryonic cortices were digested in DS solution containing 9 units/ml Papain

(Worthington), 10 ng/ml 2-Amino-5-phosphonopentannoic acid (APV, Sigma) and 20

ng/ml L-cysteine at 37oC for 30 mins. The tissue was then transferred to an enzyme

inhibitor solution containing 20 mg/ml BSA (Bovine Albumin, Sigma), 20 mg/ml Trypsin

Inhibitor and 10 ng/ml of APV in DS using a Pasteur pipette. The tissue was rinsed once

with cortical neuron media before dissociation using micropipetting to single cell

suspensions in cortical neuron media. Cortical neuron media consisted of neurobasal

medium, 2% B-27 serum-free supplement, 0.25% glutaMAXTM-I Supplement and 0.2%

Penicillin-Streptomycin from Life Technologies. Culture ware (BD) was coated with 50

µg/ml poly-L-lysine (Sigma). 2.3 x 102 cells/mm2 were plated on 6 cm dishes and 2.9 x

102 cells/mm2 on 18 by 18 mm coverslips. For coverslips, cells were diluted to a

concentration of 1.36 x 105 and 700 ul was seeded on the coverslip. The media was

replaced in coverslips and dishes after approximately 3 hours after the cells had

adhered to the surface. The cultures were maintained at 37oC and 5% CO2.

For Xpa-/- or Csb-/- cortical neuron isolation, Xpa+/- or Csb+/- males and females

were mated respectively. Similar to CD1 cultures, the date used was E16.5 and the

cortex was dissected in cold DS. Embryonic tails were also removed for genotyping of

each embryo. The tissue was stored in Hibernate-E with 2% B-27 serum-free

35

supplement (Life Technologies) for approximately 3 hours while genotyping was

completed. The processing of the tissue was identical to the above protocol.

3.2 Embryonic genotyping

Embryonic tails were removed from each embryo from mixed Xpa+/- and Csb+/-

litters during dissections and labeled to correspond to the culture samples from that

embryo. The tails were digested, neutralized and amplified using the REDExtract-N-

AmpTM Tissue PCR (XNAT) kit (Sigma) according to manufacturer’s instructions. Table

3.1 and 3.2 summarizes the primer sequences, PCR conditions and band sizes of XPA

and CSB respectively. PCR products were run on a 2% agarose gel at 120 Volts for 1 h

and visualized using ethidium bromide and a UV transilluminator.

Table 3.1 Summary of Xpa primers, cycling conditions and band sizes Primers Cycling

Conditions Band Sizes

XP26: 5’-GTGTCAGGCATAAGATCTATGACAA-3’

1. 95oC for 5min 2. 94oC for 1min 3. 58oC for 1min 4. 72oC for 2min Repeat 2-4 34x 5. 72oC for 10min

WT: 300bp Xpa-/-: 200bp

XP47: 5’-AGGCAAGCACCTGCAGCTGT-3’ PGK2: 5’-GGCCACTTGTGTAGCGCCAA-3’

Table 3.2 Summary of Csb primers, cycling conditions and band sizes Primers Cycling

Conditions Band Sizes

CSB4: 5’-GCTGCTTATAATAATCCTCATC-3’

1. 95oC for 5min 2. 94oC for 30s 3. 58oC for 45s 4. 72oC for 1min Repeat 2-4 34x 5. 72oC for 10min

WT: 200bp Csb-/-: 500bp

CSB5: 5’-ATCTGGGTGTTCGAATTCGCCAATG-3’ CSB6: 5’-GTCTTCTGATGACGTTAGCTATGAG-3’

3.3 Mouse embryonic fibroblast (MEF) isolation

CD1 males and females were mated and the plug date used was E0.5. Embryos

were collected at E14.5 and the head and all internal organs were removed. The

36

embryo body was transferred to microfuge tube on ice. The embryo was cut into small

pieces in the microfuge tube and then transferred to a 6-well plate. The tissue was

incubated at 37oC with 0.05% Trypsin-EDTA (Gibco) for 30 mins. The tissue was

dissociated with a pipette and incubated again with 0.05% Trypsin-EDTA for 20mins.

The trypsin was inactivated with fetal bovine serum (FBS, Life Technologies). The cells

were centrifuged at 524 x g at room temperature for 10 mins and the top layer was

removed. The cultures were maintained in Dulbecco’s Modified Eagle Medium (DMEM)

containing 25 mM glucose, 10% FBS, 1% L-glutamine, 1% Penicillin-Streptomycin and

55 µM B-mercaptoethanol (all reagents from Life Technologies). The cultures were

maintained at 37oC and 5% CO2.

3.4 Cortical neuron survival assays

3.4.1 Neuronal immunocytochemistry

CD1 cortical neurons were seeded on coverslips. At 5 days post isolation, media

was removed from coverslips and using the CL-1000 UVP Crosslinker, wells were

individually treated with 0, 10 or 50 J/m2 of UVC at a wavelength of 254 nm. Cortical

neuron media was added with vehicle or BrdU (30 µM, Sigma) and cells were fixed

using 4% paraformaldehyde (PFA) from Sigma 24 hours later. Cells were washed with

PBS, permeabilized with 0.4% TritonX-100 in PBS for 15 mins and blocked with 1%

goat serum and 0.2% TritonX-100 in PBS (blocking solution) for 30 mins. The cells were

incubated with the primary antibody, mouse anti-tubulin, beta III isoform (1:1000,

StemCell Technologies, 01409) overnight at 4oC in blocking solution. This antibody

reacts with the neurons specific beta III isoform of tubulin (βIIITub) and is commonly

used as a neuronal marker. The cells were washed the following day with PBS and

37

incubated with the secondary antibody goat anti-mouse Alexa-568 (Invitrogen) for an

hour and then mounted onto slides using Vectashield Mouting Media (Vector)

containing DAPI (nuclear stain). Cells were imaged using AxioImager.M2

Epifluorescence Microscope from Zeiss.

3.4.2 Quantification of apoptosis with flow cytometry

CD1 cortical neurons were seeded on 6 cm dishes at the density 2.3 x 102

cell/mm2. At 5 days post isolation, media was removed from dishes and UVC-irradiated

using the CL-1000 UVP Crosslinker (0, 5 or 10 J/m2) and media was added with vehicle

or BrdU (30 µM) for the UV dose-response. For the BrdU dose-response, cells were

irradiated with 0 or 10J/m2 and supplemented with media containing 0 to 10 µM BrdU.

24 hours post-irradiation, media was collected (to ensure dead cells were harvested as

well), and the cells were incubated with 0.05% trypsin-EDTA (Gibco) for 7 mins at 370C.

Trypsin was deactivated with FBS (Life Technologies) and cells were dissociated and

harvested into 15 ml falcon tubes (BD). Cells were centrifuged at 524 x g; supernatant

aspirated, washed with PBS and transferred to 5 ml round-bottom tubes (BD). The cells

were centrifuged at 524 x g for 5mins, supernatant removed and cells resuspended in

150ul binding buffer containing 5 µl of FITC Annexin V (apoptotic marker) and 50ng/ml

propidium iodide (PI). The binding buffer and FITC Annexin V were from BD

Pharmingen and PI was from Sigma. The cells were incubated at room temperature and

analyzed using BD FACSCalibur flow cytometer (BD) and FLOJO software (Tree Star).

3.4.3 Quantification of apoptosis with immunocytochemistry

Cortical neurons were UVC-irradiated (0 or 10J/m2) using the CL-1000 UVP

Crosslinker 5 days post isolation and supplemented with media containing vehicle or

38

BrdU as before. CD1 cortical neurons were treated with 0 or 10 J/m2 UV, Xpa-/- and

Csb-/- cortical neurons were treated with 0, 2, 10 or 50 J/m2 UV and Xpa+/+ and Csb+/+

(wild type controls) were treated 0, 10 or 50 J/m2 UV. Cells were fixed using 4% PFA

and the staining was identical to 2.2.1 but with different antibodies. The primary

antibodies were mouse βIIITub (1:1000) and rabbit cleaved caspase-3 (1:100, Cell

signaling, Asp175) as an apoptotic marker. The secondary antibodies used were goat

anti-mouse Alexa-568 and goat anti-rabbit Alexa 488 (Invitrogen). AxioImager.M2

Epifluorescence Microscope (Zeiss) was used to take sequential individual channel

images at 10x magnification using the same exposure times. Metamorph Software

(Olympus) was used to quantify the percentage of apoptotic neurons (cells that were

positive for cleaved caspase-3 and βIIITub) and the total number of neurons (cells that

were positive for βIIITub).

3.5 Mouse embryonic fibroblast survival assay

CD1 passage 0 MEFs were seeded at the density 1.12 x 102 cells/mm2. 24 hours

later the media was removed and the cells were UVC-irradiated (0 or 25J/m2) using the

CL-1000 UVP Crosslinker and supplemented with media containing vehicle or BrdU (0-

30 µM). The cells were harvested 24 hours later for flow cytometry with FITC Annexin V

(BD Pharmingen) and PI (Sigma) following the same protocol that was used for cortical

neurons described above.

3.6 BrdU labeling using immunocytochemistry

Cortical neurons were UVC-irradiated (0 or 50J/m2) using the CL-1000 UVP

Crosslinker 5 days post isolation and supplemented with media containing 10 µM BrdU

and fixed with 4% PFA at 24 h. MEFs were used as a positive control and incubated

39

with 30 µM BrdU for 1 h and fixed with 4% PFA. Cells were washed with PBS and

permeabilized with 0.4% TritonX-100 in PBS for 15mins. The cells were than washed

with PBS and incubated with 10U/ml DNase (Thermo Scientific) in 10mM Tris, 0.1mM

CaCl2 and 2.5mM MgCl2 for 1h at 37oC. The cells were than washed with PBS and

blocked similar to above and incubated with the primary antibodies mouse βIIITub

(1:1000) and rat BrdU (1:500, Abcam, ab6326) and then secondary antibodies goat

anti-mouse 568 and goat anti-rat Alexa-488 (Invitrogen) for an hour and then mounted

onto slides using Vectashield Mounting Media (Vector) containing DAPI (nuclear stain).

Cells were imaged using AxioImager.M2 Epifluorescence Microscope from Zeiss.

3.7 Quantification of UV DNA lesions

UV DNA lesions were quantified using an enzyme-linked immunosorbant assay

(ELISA). CD1 cortical neurons were seeded on 10cm dishes and UVC-irradiated (0, 50

or 100J/m2) using the CL-1000 UVP Crosslinker 5 days post isolation. Cortical neuron

media with vehicle or BrdU (10 µM) supplemented was added to the dishes and cells

were harvested 0, 2, 4 or 8 h after UV-irradiation similar to cells harvested for flow

cytometry described above. The pellet was washed with PBS and flash frozen. Genomic

DNA was extracted using GenEluteTM Mammalian Genomic DNA Miniprep kit (Sigma)

according to manufacturer’s instructions and stored at 4oC. The DNA was quantified

using Nanodrop 1000 (Thermo Scientific) and diluted to a concentration of 4.0 ng/µl for

the 6-4PP lesion and 0.2 ng/µl for the CPD lesion. In order to denature the DNA, the

DNA solution was incubated at 100oC for 10mins and then rapidly chilled on ice.

In order for the DNA to adhere to the plate, 96 well microtiter plates were coated

with 50 µl of 0.003% protamine sulfate solution overnight at 37oC in order to dry

completely and washed with distilled water the following day. 50 µl of the diluted DNA

40

was added per well and dried completely overnight at 37oC. The following day, the

DNA-coated plates were washed with PBS-T (0.05% Tween-20) and blocked with 2%

FBS in PBS for 30 mins. The wells were washed again with PBS-T and either the 64M-2

antibody (1:1500, Cosmo Bio Co.) for the 6-4PP lesion or the TDM-2 antibody (1:1000,

Cosmo Bio Co.) for the CPD lesion diluted in PBS was added to each well and the plate

was incubated at 37oC for 30mins. The plates were then washed with PBS-T and then

incubated with Biotin-F(ab’)2 fragment of anti-mouse IgG (H+L) (1:2000) in PBS for

30mins at 37oC. The plates were washed again with PBS-T and incubated with 1:10000

Peroxidase-Streptavidin in PBS for 30mins at 37oC. The wells were washed with PBS-T

and then washed with Citrate-phosphate buffer at a pH of 5. Then 100 µl of o-

Phenylene diamine and H2O2 in Citrate-phosphate buffer was added to each well for 2-3

mins. In order to stop the enzyme reaction 50 µl of 2M H2SO4 was added to each well

and the absorbance at 492 nm was measured using a plate reader.

3.8 DNA content quantification using immunocytochemistry

CD1 cortical neurons were UVC-irradiated (0 or 50J/m2) using the CL-1000 UVP

Crosslinker 5 days post isolation and supplemented with media with vehicle or BrdU (10

µM). Cells were fixed with 4% PFA at 12 or 18 h after UV-irradiation. Similar to 3.4.1

immunocytochemistry protocol, cells were washed with PBS, permeabilized with 0.4%

TritonX-100 and blocked with 0.2% TritonX-100 and 1% goat serum in PBS. Neurons

were incubated with mouse βIIITub (1:1000) overnight at 4oC and washed with PBS the

following day. The cells were then incubated with the secondary antibody goat anti-

mouse Alexa-568 (Invitrogen) for one hour. The cells were washed with PBS and

incubated with Hoechst 33342 (1 µg/ml) for 15 mins at room temperature and washed

with PBS five times. The coverslips were then mounted onto slides using Vectashield

41

Mounting Media (Vector) without DAPI. The cells were imaged using AxioImager.M2

Epifluorescence Microscope from Zeiss.

3.9 Quantification of cell cycle distribution using flow cytometry

CD1 cortical neurons were UVC-irradiated (0 or 50J/m2) using the CL-1000 UVP

Crosslinker 5 days post isolation and supplemented with vehicle or BrdU (10 µM).

Control neurons were incubated with 1 µM etoposide. 12 hours post-irradiation cells

were trypsinized (0.05% Trypsin-EDTA, Gibco) for 7 mins at 37oC and collected as a

single cell suspension in a 15 ml falcon tube (BD). The cells centrifuged at 524 x g for 5

mins at room temperature and the supernatant was removed. The cells were

resuspended and fixed with ice cold 70% ethanol and stored at -20oC for at least 20

mins. The cells were transferred to 5 ml round-bottom tubes (BD), centrifuged and the

supernatant removed. The cells were then washed with PBS and permeabilized with

0.4% TritonX-100 in PBS for 15 mins. The cells were washed again with PBS and then

incubated with the primary antibody βIIITub (1:200, StemCell Technologies) with 0.2%

TritonX-100 and 1% donkey serum for 3 h. The cells were washed with PBS and

incubated with the secondary antibody, donkey anti-mouse-FITC (1:200), in PBS for 30

mins at room temperature. The cells were finally washed with PBS and resuspended

with PI (50ng/ml, Sigma) and analyzed using BD FACSCalibur flow cytometer (BD) and

FLOJO software (Tree Star).

3.10 Cell cycle marker expression

Immunocytochemistry was similar to the protocol described above for βIIITub

only. The cells were incubated with primary antibodies, mouse anti-tubulin, beta III

isoform (1:1000, StemCell Technologies) and one of the following cell cycle markers:

42

rabbit anti-Cdk4 (1:100, Santa Cruz Technology, sc-260), rabbit anti-Cdk6 (1:100, Santa

Cruz Technology, sc-177) or Ki67 (1:100, abcam, ab66155). The secondary antibodies

were goat anti-mouse Alexa-568 (Invitrogen) and anti-rabbit Alexa-568 (Invitrogen).

Cells were imaged using AxioImager.M2 Epifluorescence Microscope from Zeiss.

3.11 Quantification of global DNA methylation

CD1 cortical neurons were seeded at a density of 2.3 x 102 in 10 cm dishes and

UVC-irradiated (0 or 50 J/m2) using the CL-1000 UVP Crosslinker 5 days post isolation.

Cortical neuron media with vehicle or BrdU (10 µM) supplemented was added to the

dishes and cells were harvested 0, 6, 12 or 18 h after UV-irradiation. The cells were

harvested and genomic DNA extracted was similar to protocol described above for

quantification of UV lesions. Global DNA methylation was quantified using the

MethylampTM Global DNA Methylation Quantification Kit (Epigentek) according to

manufacturer’s instructions. The assay consists of binding the genomic DNA to assay

well. The methylated portion of DNA was quantified using an anti-5-methylcytosine

antibody with an enzyme with an ELISA-like reaction. The following formula was used to

calculate the percent methylation.

% Methylation = (OD (sample-blank)/2)/ (OD (positive control-blank)) x 100

3.12 Statistical analyses

All statistical analysis was performed with GraphPad Prism software where data

was expressed with error bars indicating standard deviation of the mean (+/- S.D.). Two

way analysis of variance (two-way ANOVA) where p<0.05 was considered statistically

significant was used to determine the effect of BrdU on UV-induced apoptosis followed

by Bonferroni analysis.

43

4. RESULTS

4.1 BrdU prevents mouse cortical neurons from UV-induced death

Since BrdU labeling is a common technique to examine the S phase of the cell

cycle we decided to treat cortical neurons with UV and supplement the cells with BrdU

to determine if these neurons entered the S phase following DNA damage. DNA repair

and DNA replication involve DNA synthesis but DNA replication likely represents the

majority of BrdU labeling (Cooper-Kuhn and Kuhn, 2002). In proliferating cells, BrdU

supplementation for an hour is often sufficient for BrdU to become phosphorylated,

incorporated into DNA and label S phase (Cavanagh et al., 2011; Taupin, 2007).

However, since neurons are post-mitotic and it is unknown at what time neurons might

enter the S phase following UV treatment, neurons were UV treated and immediately

supplemented with BrdU (30 µM) for a full 24 hours. This BrdU concentration is routinely

used to label dividing cells in culture (Tamblyn et al., 2009). If neurons entered the S

phase at any point during this 24 hour time period, they should incorporate BrdU. The

approach of utilizing BrdU to mark neurons entering the S phase after stress has been

shown in other studies (Kruman et al., 2004). 50 J/m2 UVC-irradiation generates up to 5

DNA lesions per 10 kB DNA (van Hoffen et al., 1995). UV treated neurons died by

apoptosis. Surprisingly, UV treated neurons with BrdU for 24 h survived. UV irradiation

at 10 and 50 J/m2 caused extensive neuronal death by 24 h (Fig. 4.1A-C). UV-irradiated

neurons cultured with BrdU immediately after UV did not die. Instead, qualitative

assessment of neuronal morphology using the structural marker βIIITubulin revealed

that BrdU-treated neurons (Fig. 4.1D-F) appeared healthy and similar to unirradiated

neurons (Fig. 4.1A).

44

In order to assess whether BrdU protected neurons from UV-initiated apoptosis,

neurons were stained with the apoptotic marker Annexin V (Niu and Chen, 2010) and

the fraction of Annexin V positive cells was assessed by flow cytometry 24 h after

treatment. UV (5 and 10 J/m2) caused a significant increase in Annexin V positive cells

(p<0.05, Fig. 4.1G). Treatment with 30 µM BrdU immediately after UV significantly

reduced the proportion of Annexin V positive cells. Therefore, BrdU can prevent UV-

induced apoptosis in cortical neurons confirming our qualitative observations.

In order to determine whether BrdU concentrations lower than the 30 µM utilized

above were neuroprotective, the effect of 0.1 – 10 µM BrdU on UV-initiated neuronal

apoptosis was assessed (Fig. 4.1H). A concentration dependent neuroprotective effect

of BrdU was observed, with BrdU concentrations as low as 0.1 µM significantly reducing

UV-initiated neuronal apoptosis.

In order to determine the toxicity of BrdU on its own, unirradiated neurons were

incubated with a range of BrdU concentrations (0.1-10 µM) for 24 hours and apoptosis

was quantified using flow cytometry with Annexin V (Fig. 4.1H). BrdU (0-10 µM) did not

significantly increase the basal levels of neuronal apoptosis. Since 10 µM BrdU reduced

UV-induced apoptosis but was not toxic, 10 µM BrdU was used for future experiments.

45

4.1.1 BrdU reduces UV-induced cleaved caspase-3 in cortical neurons

The Annexin V flow cytometry experiments described above assess apoptosis in

all cell types present in our cultures. Since cortical neuron cultures derived from

embryonic mice produce predominantly but not exclusively neurons, cleaved caspse-3

staining (marker of late apoptosis) was conducted on neurons (immunocytochemistry)

taking advantage of double labeling with a neuronal marker. βIIITub was used as a

neuronal marker to restrict the analysis to neurons. Approximately 1500 neurons were

scored for each treatment group. Cortical neurons were UV-irradiated (0 or 10 J/m2) and

incubated with vehicle or BrdU (10 µM) for 18 h (Fig. 4.2A-L). Among βIIITub positive

neurons, cells were classified as cleaved caspase-3 positive using automated image

analysis. BrdU significantly reduced UV-induced neuronal apoptosis (Fig. 4.2M).

47

4.2 Absence of detectable BrdU labeling in cortical neurons

BrdU was initially utilized as a DNA synthesis marker. To assess whether the

neuroprotective effects of BrdU resulted from BrdU incorporation into newly synthesized

DNA, immunocytochemistry with an anti-BrdU antibody was conducted. Proliferating

mouse embryonic fibroblasts (MEFs) were used as a positive control. BrdU labeling was

readily detectable in MEFs (Fig. 4.3A). MEFs were labeled with BrdU (30 µM) for one

hour. The intensity (green) of labeling varied among MEFs indicating that these cells

were at different phases of the cell cycle. Absence of BrdU labeling is seen in control

MEFs (Fig. 4.3B) not incubated with BrdU. Detectable BrdU labeling was absent in UV-

irradiated (50 J/m2) neurons incubated with BrdU (30 µM) for 24 hours (Fig. 4.3C) as

well as negative control neurons: in the absence of BrdU (Fig. 4.3D) and unirradiated

neurons incubated with BrdU (Fig. 4.3E). βIIITub (red) was used as a neuronal marker.

BrdU neuroprotection is independent of global DNA synthesis and UV-irradiation did not

appear to trigger cell cycle re-entry in our model system. However, this methodology

does not have the sensitivity to detect BrdU incorporation during DNA repair when small

patches of new DNA are synthesized.

49

4.3 Cell type specificity of BrdU protection

To determine whether BrdU protection was specific to neurons the cell type

specificity of BrdU protection was analyzed. MEFs were utilized to determine whether

BrdU reduces UV-induced apoptosis in proliferating cells. MEFs were UV-irradiated (25

J/m2) and incubated with 0 – 30 µM BrdU for 24 hours. Apoptosis was quantified using

flow cytometry with Annexin V. UV-irradiation significantly increased apoptosis in MEFs

and BrdU did not reduce UV-induced apoptosis (Fig. 4.4).

51

4.4 Chemical specificity of BrdU protection

To determine if the protective effect of BrdU could be extended to structurally or

functionally related molecules the potential neuroprotective effect of DNA nucleosides

and halogenated thymidine analogues was assessed. In order to evaluate the chemical

specificity of BrdU protection, UV-irradiated neurons (10 J/m2) were incubated with

equimolar concentrations (30 µM) of thymidine or its halogenated analogues (CldU or

IdU) for 24 h. Immunocytochemistry with TUNEL and βIIITub was used to evaluate

neuronal apoptosis. Qualitatively, there appears to be a reduction in TUNEL positive

neurons when UV-irradiated cells were incubated with thymidine or its analogues, BrdU,

CldU or IdU (Fig. 4.5A-E). Since thymidine is a DNA nucleoside, UV-irradiated neurons

were also incubated with equimolar concentrations (30 µM) of the other DNA

nucleosides (adenosine, cytidine or guanosine) as well as a mix of the four DNA

nucleosides (dNT, 30 µM each). Qualitatively, the other nucleosides as well as a mix of

the four nucleosides also appeared to reduce UV-induced apoptosis to varying degrees

(Fig. 4.5F-I) but none were as potent as thymidine and its analogues. Although Fig.

4.5H indicates very few apoptotic neurons when UV-irradiated neurons are incubated

with guanosine, the number of neurons at 24 h of guanosine treatment is lower

suggesting that some neurons have lost adherence and washed away which may occur

when neurons are undergoing cell death.

53

4.5 The UV-initiated increase of p53 is unaltered by BrdU

A common cellular response to stress including DNA damage is an up-regulation

of p53 (Lanni et al., 2012). UV-induced cell signaling in cortical neurons was determined

by examining p53 expression using immunocytochemistry. There was no p53

expression in unirradiated neurons (Fig. 4.6A) and 1h after UV-irradiation in the

absence or presence of BrdU (10 µM, Fig. 4.6B-C) but there was an increase in p53

expression as early as 4 hours following UV-irradiation (Fig. 4.6D-E) in both the

absence and presence of BrdU. p53 expression continued to increase 8 (Fig. 4.6F-G)

and 12 (Fig. 4.6H-I) hours in UV-irradiated neurons regardless of BrdU incubation.

There appears to be no difference in p53 expression following UV-irradiation with or

without BrdU. Thus, the neuroprotective activation of BrdU is not due to a change in p53

accumulation.

55

4.6 BrdU does not alter UV DNA lesion removal

The removal of UV DNA lesions over time was examined in cortical neurons to

determine whether BrdU incubation alters DNA repair. DNA lesions (6-4PP and CPD)

were measured using an ELISA. Figure 4.7A and B show the removal of the 6-4PP

lesion over time following 50 or 100 J/m2 UV-irradiation respectively whereas figures

4.7C and D show the removal of the CPD lesion over time following 50 or 100 J/m2 UV-

irradiation respectively. There was a significant reduction in 6-4PP lesions 4 hours after

UV-irradiation. Although BrdU appears to delay or stop repair of 6-4PP and CPD

lesions, the removal of 6-4PP and CPD lesions following UV-irradiation was not

statistically altered with BrdU incubation. Thus, BrdU does not protect UV-irradiated

neurons by improving the global genome repair (GGR) of UV lesions.

57

4.7 Xpa-/- and Csb-/- neurons are resistant to UV-initiated death

DNA lesions created by UVC-irradiation are primarily repaired by the NER

pathway. To determine the role of NER in UV-initiated neuronal death, two mouse

models of NER were used. Xpa-/- neurons are deficient in NER throughout the genome

while Csb-/- neurons lack NER of transcribed genes. Since MEFs from Xpa-/- and Csb-/-

mice and pluripotent embryonic stem cells from Xpa-/- mice are hypersensitive to UV (de

Vries et al., 1995; de Waard et al., 2008; Nakane et al., 1995), we hypothesized that

Xpa-/- and Csb-/- neurons would also be hypersensitive to UV. Wild type, Xpa-/- and Csb-

/- neurons were UV-irradiated at several doses and neuronal apoptosis was quantified

18 hours later using immunocytochemistry with βIIITub and cleaved caspase-3. Xpa-/-

and Csb-/- neurons were UV-irradiated with a lower dose (2 J/m2 UV) as well as higher

doses since other cell types of Xpa-/- and Csb-/- mice are hypersensitive to DNA damage

(de Waard et al., 2008). Immunocytochemistry images of UV-irradiated (0, 2, 10 and 50

J/m2) Xpa-/- and Csb-/- neurons are shown in figures 4.8A-D and 4.8E-H respectively.

Cells were classified as apoptotic neurons if they were positive for both cleaved

caspase-3 and βIIITub staining using automated image analysis software Metamorph.

As shown previously, UV-irradiation (50 J/m2) significantly increased apoptosis in wild

type neurons (Fig. 4.8I). In stark contrast to wild type, UV did not increase apoptosis in

Xpa-/- or Csb-/- neurons. Neurons deficient in NER, the DNA repair pathway that repairs

UV lesions are paradoxically protected from UV-initiated apoptosis at this 18 h time

point.

As a complementary measure of neuronal death following UV-irradiation, the

number of neurons remaining per unit area 18 h after UV was calculated since neurons

lose their adherence to coverslips when they undergo cell death. Figure 4.8J represents

59

the number of neurons remaining per mm2 18 hours following UV treatment for wild

type, Xpa-/- and Csb-/- neurons. The number of wild type neurons per unit area

significantly decreased following UV-irradiation (10 and 50 J/m2). However, the number

of Xpa-/- neurons per unit area following UV-irradiation is unaltered and the number of

Csb-/- neurons is significantly decreased at the highest UV dose (50J/m2) in comparison

to unirradiated control. When the number of neurons per unit area is compared among

the three genotypes, there was a significant reduction in the number of wild type

neurons but not Xpa-/- and Csb-/- neurons remaining at both 10 and 50 J/m2 UV. These

results further support the resistance of Xpa-/- and Csb-/- neurons to UV-induced

apoptosis.

4.7.1 BrdU does not alter caspase-3 in Xpa-/- and Csb-/- neurons

In wild type neurons, BrdU protected against UV-induced apoptosis (Fig. 4.9A-

B). BrdU incubation (10 µM) did not change the fraction of cleaved caspase-3 positive

neurons or the number of cells per unit area for both Xpa-/- and Csb-/- neurons (Fig.

4.9C-F) following UV irradiation. Therefore, BrdU prevents UV-induced apoptosis in wild

type neurons but is ineffective in Xpa-/- and Csb-/- neurons at this time point since they

are already UV-resistant.

60

4.8 Influence of BrdU on neuronal cell cycle

4.8.1 BrdU does not alter neuronal DNA content

To study whether UV-irradiation induces neuronal cell cycle re-entry, neuronal

DNA content was quantified. UV-irradiated cortical neurons were incubated with or

without BrdU and harvested at 0, 8 and 12 h. Neurons were stained with βIIITub and

Hoechst (DNA stain), imaged at identical exposure times and Hoechst intensity was

quantified using Metamorph. There was a statistically significant increase in Hoechst

intensity of cortical neurons 12 hours following UV-irradiation (50 J/m2) in the absence

and presence of BrdU (10 µM) and a significant increase 18 hours following UV-

irradiation without BrdU in comparison to unirradiated neurons (Fig. 4.10). However,

there was a significant reduction in the Hoechst intensity of UV-irradiated neurons

incubated with BrdU for 18 hours in comparison to unirradiated neurons as well as

neurons 12 hours following UV-irradiation (with and without BrdU) and neurons 18

hours after UV-irradiation. Furthermore, Hoechst intensity was variable following UV-

irradiation and there was a significant reduction in the coefficient of variation of Hoechst

staining 18 hours after UV-irradiation with BrdU in comparison to 12 hours after UV-

irradiation. Although there are statistically significant changes in DNA intensity of

cortical neurons after UV-irradiation the increase is only 5% which may not be

biologically significant.

63

4.8.2 Neither UV-irradiation nor BrdU incubation alter cell cycle distribution

As an alternative measure of neuronal cell cycle distribution, neuronal DNA

content was measured using flow cytometry with propidium iodide (DNA content) and

βIIITub. Cortical neurons were harvested 12 hours following treatment and analyzed

using flow cytometry. DNA content of unirradiated neurons, etoposide treated neurons

(1 µM, positive control), and UV-irradiated neurons (50 J/m2) in the absence and

presence of BrdU (10 µM) was analyzed. The percentage of neurons in the G0/G1

phase, S phase and G2 phase of the cell cycle was determined using the Watson

Pragmatic model (Table 4.1). The majority of the neurons in all treatments were in the

G0/G1 phase and only a very small percentage (less than 3%) were in the S phase or G2

phase. There was no significant difference in the percentage of neurons in G0/G1 phase,

S phase or G2 phase in comparison to unirradiated control neurons. These results

indicate that cortical neurons do not re-enter the S phase of the cell cycle following UV-

irradiation in our model.

Table 4.1: Flow cytometric analysis of cell cycle phase in DNA-damaged cortical neurons Sample G0/G1 mean (S.D.) S phase mean

(S.D.) G2 mean (S.D.)

Unirradiated 92% (3.1) 2.2% (1.0) 2.8% (0.4) Etoposide (1µM) 89% (1.2) 1.8% (0.4) 1.7% (0.5) 50J/m2 UV 97% (2.0) 1.5% (0.5) 1.3% (1.0) 50J/m2 UV + BrdU 98% (1.9) 1.1% (0.1) 1.3% (0.2)

4.8.3 UV does not induce cell cycle markers in neurons

UV-induced neuronal death can be rescued by using the Cdk inhibitors

flavopiridol and olomoucine (Roos and Kaina, 2012). Flavopiridol inhibits Cdk1/2/4

whereas olomoucine inhibits Cdk1/2/5 and ERK-1/MAP-1 kinases (extracellular signal-

regulated-1/mutagen-activated protein kinases) (Bakkenist and Kastan, 2003; Bartek 65

and Lukas, 2003; Byun et al., 2005; Roos and Kaina, 2012). In addition, the dominant-

negative kinase-inactive forms of both Cdk4 and Cdk6 reduced UV-induced neuronal

death (Park et al., 1998a). Since Cdk4 and Cdk6 are involved in the cell cycle

progression through the G1 phase (Liu et al., 2010), we analyzed Cdk4 and Cdk6

expression following UV-irradiation in the presence and absence of UV. Ki67 expression

was also analyzed since it is expressed during G1, S, G2 and mitosis but is absent

during G0 (Sinha and Hader, 2002). Furthermore, Ki67 expression has been used to

verify neuronal cell cycle re-entry after hydrogen peroxide treatment (Schwartz et al.,

2007). In order to verify that UV-irradiation does not induce neuronal cell cycle re-entry

in our model, we determined whether UV-irradiation or BrdU incubation changes cell

cycle marker expression. MEFs were used as a positive control to determine the quality

of Cdk4 and Cdk6 antibodies (Fig. 4.11A and B respectively). Neither UV-irradiation nor

BrdU altered Cdk4 (Fig. 4.11C-H), Cdk6 (Fig. 4.11I-N) and Ki67 (Fig. 4.11O-T)

expression. These results support our previous findings that neither UV-irradiation nor

BrdU induce neuronal cell cycle re-entry in our model.

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4.9 BrdU does not alter neuronal global DNA methylation

BrdU can cause global DNA demethylation in neural stem cells (Schneider and

d'Adda di Fagagna, 2012). To investigate whether BrdU caused neuronal DNA

demethylation, global DNA methylation, using a 5-methylcytosine antibody against

isolated genomic DNA, was measured at 6, 8 and 12 h. The positive control is

proprietary and was provided by the manufacturer of the global DNA methylation kit.

There was no change in neuronal global DNA methylation levels in UV-irradiated

neurons in the absence or presence of BrdU (Fig. 4.12).

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5. DISCUSSION

5.1 Characterization of BrdU protection

The initial goal of this project was to study the neuronal DNA damage response,

in particular the DNA damage response to bulky DNA adducts that are primarily

repaired by the nucleotide excision repair pathway. An interesting neuronal death

mechanism and response to other forms of DNA damage (e.g. double stranded breaks)

involves neuronal cell cycle re-entry (Kruman et al., 2004) that precedes apoptosis. In

order to determine whether bulky DNA adducts trigger neuronal cell cycle re-entry,

cortical neurons were UVC-irradiated and incubated with the cell cycle marker BrdU for

24 hours. Since BrdU is a thymidine analogue, it is incorporated into DNA during

replication and can be used as a maker for the S phase of the cell cycle. Interestingly,

UV-irradiated neurons incubated with BrdU as low as 100 nM had significantly lower

levels of apoptosis than UV-irradiated neurons. The most common application of BrdU

is as an S phase marker but the absence of detectable BrdU labeling in UV-irradiated

cortical neurons suggested that the protective mechanism of BrdU is not due to its

incorporation into new DNA during DNA replication. Thus, we concluded that BrdU

prevents UV-induced apoptosis through an alternative mechanism.

Neurons are post-mitotic cells that have exited the cell cycle. In order to

determine whether BrdU protection was specific to neurons or could also be extended

to proliferating cell types, we assessed BrdU protection in primary MEFs and observed

a lack of protection. These observations support the concept that BrdU protection is

specific to post-mitotic cells and thus the mechanism of protection may involve features

of the DNA damage response that are unique to post-mitotic cells. Qualitative

observations indicated that UV induced neuronal apoptosis could be reduced by

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thymidine or halogenated thymidine analogues and not only BrdU. Thus, it appears that

several chemical structures similar to thymidine may reduce UV-induced neuronal

apoptosis, which may provide clues to the mechanism of BrdU neuroprotection.

5.2 UV-induced cell signaling

One of the initial questions regarding the protective effect of BrdU was whether

BrdU prevented neurons from responding to UV-induced DNA damage through

canonical signaling. p53 expression was analyzed since it is generally required for

neuronal apoptosis following DNA damage (Lanni et al., 2012). Although p53 is

expressed at low levels in unstressed cells, when cells are stressed, p53 degradation is

reduced and p53 accumulates (Essmann and Schulze-Osthoff, 2012). Qualitative

observations indicated that after UV, BrdU-treated neurons increased p53 levels after

UV similar to vehicle-treated neurons. This suggests that the mechanism of BrdU

protection occurs after p53 activation or independent of p53 activation. p53 is activated

by one or more post-translational modifications including phosphorylation, acetylation,

methylation, ubiquitination or sumolation depending on the stress response (Harris and

Levine, 2005). However, our methodology looked at total p53 levels and did not

distinguish between post-translationally modified forms of p53.

5.3 UV-induced DNA repair

A rational approach to preventing UV-initiated apoptosis is to repair UV-initiated

DNA damage. Interestingly, a molecule related to thymidine, thymidine dinucleotide

(pTT) has been shown to improve the repair of UV lesions in part by activating p53 and

up-regulating gene products involved in DNA repair (Gilchrest and Eller, 1999). Since

the chemical structure of pTT is related to that of thymidine and BrdU, we postulated

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that the neuroprotective action of BrdU may be similar to pTT. To measure the impact of

BrdU on NER, an ELISA was used to measure the abundance of UV DNA damage in

the form of 6-4PP and CPD lesions in DNA extracted from UV-irradiated neurons. A

limitation of this methodology is that a decrease in 6-4PP or CPD lesion level only

provides information about initial steps in the multistep NER pathway (Fig. 2.4).

Furthermore, the ELISA used to measure the removal of UV lesions does not

distinguish between UV lesions on transcribed genes and non-transcribed genes. Since

the transcribed genes are a small percentage of the genome (1-2%) (Elgar and Vavouri,

2008), GGR is measured by this methodology. If GGR is silenced due to differentiation,

there is a possibility that this assay may be reporting the repair of transcribed genes.

Whether, GGR is attenuated in our cultures and the sensitivity of this assay to report

TCR is unknown.

There was a significant reduction in 6-4PP lesions 4 hours following UV-

irradiation compared to the amount of lesions immediately after UV-irradiation indicating

that post-mitotic neurons can repair this form of damage. This is in agreement with

previous results that showed a 40% removal of 6-4PP lesions in rat neurons four hours

after UV-irradiation using immunofluorescence (Yamamoto et al., 2007). Our results

were also consistent with these results and there was about a 40% reduction in 6-4PP

lesions in cortical neurons 4 hours after UV-irradiation. However, the level of CPD

lesions did not decline during the 8 hour time period. These findings are consistent with

previous reports that CPDs are repaired at a slower rate than 6-4PPs (de Laat et al.,

1999; van Hoffen et al., 1995). Although both UV lesions are repaired at the same rate

by TCR, 6-4PPs are repaired faster than CPDs by GGR in human skin (Nijhof et al.,

2007). In addition, GGR of CDPs is very low or absent in rodent epidermal cells (Nijhof

72

et al., 2007). Our results also confirmed that CPD lesions were repaired at a slower rate

or not repaired at all in murine cells.

There was no significant difference in the removal of UV lesions (6-4PP and

CPD) at 8 hours following UV-irradiation in the absence or presence of BrdU. However,

there appears to be trend toward inhibition or slowing of repair with BrdU. BrdU may be

preventing UV-induced apoptosis by inhibiting repair. Experiments with higher sensitivity

are required to determine whether BrdU inhibits DNA repair and if BrdU alters the repair

of transcribed genes. We concluded from these findings that BrdU does not improve

DNA repair in neurons and thus BrdU is preventing UV-induced neuronal apoptosis

through an alternative mechanism.

5.4 UV-induced cell signaling in Xpa-/- and Csb-/- neurons

To determine the role of NER in BrdU neuroprotection we utilized NER-deficient

neurons. We hypothesized that Xpa-/- and Csb-/- cortical neurons would be

hypersensitive to UV compared to wild type neurons since neurological dysfunction is

characteristic of patients with defective XPA or CSB (Nance and Berry, 1992; Robbins

et al., 1991). Furthermore, XPA and CSB patient fibroblasts, Xpa-/- MEFs and

pluripotent embryonic stem cells as well as Csb-/- MEFs and Xpa-/- and Csb-/- neural

stem cells are hypersensitive to UV in comparison to wild type controls (de Vries et al.,

1995; de Waard et al., 2008; Nakane et al., 1995; Sacco et al., (submitted)). Xpa-/-

cerebellar neurons are also more vulnerable to UV than wild type at 48 h (Enokido et

al., 1997). Surprisingly, our findings show that Xpa-/- and Csb-/- cortical neurons were

less sensitive to UV than their wild type counterparts at 18h. In light of the

neurodegeneration phenotype of patients with defective NER, our results suggest that

Xpa-/- neurons may have a delayed response to UV compared to wild type neurons.

73

Differences that might account for the contrasting findings between our study and

Enokido et al are the age of mice (embryonic vs. postnatal), brain region (cortical vs.

cerebellar neurons), media conditions and assessment of UV-induced apoptosis (18h

vs. 48h). A longer time course is necessary to determine whether Xpa-/- cortical neurons

have a delayed response to UV-irradiation relative to wild type neurons.

One possible explanation for the UV resistance observed in Xpa-/- neurons is that

these cells have defective DNA damage response signaling. UV-irradiation activates

ATR (Paulsen and Cimprich, 2007) which in turn phosphorylates Chk1 and p53 (Guo et

al., 2000; Hsu et al., 2007; Nouspikel, 2007). Expression of p53 increases after UV DNA

damage (Lanni et al., 2012) as we observed. In support of the importance of functional

NER in signaling, NER is required for UV-induced Chk1 and p53 phosphorylation in

non-proliferating fibroblasts (Marini et al., 2006). Chk1 and p53 are phosphorylated after

UV in fibroblasts from normal donors and CS patients (defective CSA or CSB) but are

not phosphorylated in fibroblasts from patients defective in XPA (Marini et al., 2006). A

limitation of this study is that phosphorylated Chk1 and p53 were only measured at one

time point (1 h) after UV in non-proliferating Xpa-/- human fibroblasts (Marini et al.,

2006). These results suggest that non-proliferating cells require NER to recognize and

start processing the UV DNA lesion in order to activate a cellular stress response that

results in the activation of Chk1 and p53 (Marini et al., 2006). Similarly, it is possible that

in Xpa-/- cortical neurons, Chk1 and p53 are not phosphorylated after UV, resulting in a

delay or absence of UV-induced apoptosis and this is currently being assessed

experimentally. It is also possible that UV-induced Chk1 and p53 activation could occur

at a later time point through an alternative pathway that is NER-independent.

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Another explanation for the UV resistance of Xpa-/- neurons could be an

alternative role of NER factors. Recent studies have shown that XPA, XPC, XPG and

XPF are recruited to initiate gene expression by RNA polymerases I and II-dependent

genes in the absence of DNA damage (Le May et al., 2010). Therefore, the absence of

gene expression in NER-null neurons that requires XPA may prevent UV-induced

apoptosis.

5.5 Neuronal cell cycle re-entry

CDK inhibitors have been shown to prevent UV-induced death suggesting that

UV-irradiation may cause neuronal cell cycle re-entry (Park et al., 1998a). BrdU inhibits

ribonucleotide reductase (Meuth and Green, 1974) which may prevent the production of

dNTPs that may be necessary for neuronal cell cycle re-entry. Since BrdU has been

shown to impair cell cycle progression in proliferating cells (Levkoff et al., 2008), BrdU

may be preventing UV-induced neuronal apoptosis by inhibiting neuronal cell cycle re-

entry. In contrast to previous studies which showed that DNA damage increased

neuronal DNA content (Kruman et al., 2004), UV-irradiation did not increase neuronal

DNA content in our cultures. We used two methodologies to analyze neuronal DNA

content. In the first methodology we analyzed neuronal DNA intensity using Hoechst

staining with immunocytochemistry. Hoechst is a DNA stain that binds to the minor

groove of the DNA helix (Garner, 2009). Although there was a significant increase in

neuronal Hoechst intensity 12 to 18 hours after UV-irradiation the increase was

approximately 5% which may not be biologically significant. However, the change in

neuronal Hoechst intensity could suggest chromatin remodeling after UV-irradiation.

Chromatin remodeling involving decondensation occurs early during the DNA damage

response. DNA damage appears to initiate chromatin decondensation by decreasing

75

the DNA double helix torsional strain (Kielbassa et al., 1997). On the other hand, UV-

induced chromatin condensation is a late DNA damage response and results in

apoptosis. For example, UV-induced chromatin condensation resulting in apoptosis was

found in both mouse microglial cells and human keratinocyte cell line (Bjelland and

Seeberg, 2003; Breen and Murphy, 1995; Friedberg, 2003). Chromatin condensation

results in higher intensity Hoechst staining and is often used to examine chromatin

structure. (Cheng et al., 1978; Kuraoka et al., 2000; Robbins et al., 1983).

UV-induced chromatin remodeling is important for DNA repair enzymes to

access the lesions that need to be repaired (Jiang et al., 2010). In chromatin, DNA is

wrapped around an octomer of histones which blocks DNA repair enzymes from UV-

induced DNA lesions. There are several mechanisms in place to make DNA more

accessible including ATP-dependent chromatin remodeling complexes, incorporation of

histone variants into the nucleosome and covalent histone modifications (Bjelland and

Seeberg, 2003). There is a possibility that the change in neuronal DNA intensity we

observed is caused by UV-induced chromatin remodeling. However, the changes we

observed were small and additional experiments are required to determine whether UV-

irradiation and/or BrdU changes chromatin remodeling.

In the second methodology we examined neuronal cell cycle distribution using

flow cytometry with PI. We examined the cell cycle distribution 12 hours after UV-

irradiation because the Hoechst methodology indicated that changes in DNA content

may occur as early as 12 hours after UV and remains consistent 18 hours after UV-

irradiation. Over 97% of the neurons were in the G0/G1 phase of the cell cycle and there

was no significant difference between any of our treatments. Etoposide was used as a

positive control because it was shown to increase the percentage of S-phase cortical

76

neurons (from ~3% to over 30%) (Kruman et al., 2004). Surprisingly, etoposide

treatment did not induce neuronal cell cycle re-entry and increase the number of

neurons in the S phase of the cell the cycle in our cultures. Although we used the same

concentration of etoposide (1 µM) as the previous study, we analyzed the cells after 12

hours whereas the previous study analyzed the cells 20 hours after treatment.

Furthermore, the previous study used rat cortical neurons whereas we used mouse

cortical neurons. Thus, additional experiments are required to analyze cell cycle re-entry

in neurons 20 hours after etoposide treatment. Nevertheless, these results further

confirm that neither UV-irradiation nor BrdU incubation alters neuronal DNA content and

that in our cultures, UV-irradiation does not induce neurons to enter the S phase of the

cell cycle.

Cell cycle markers were unaltered in cortical neurons following UV-irradiation

suggesting that UV-irradiation does not induce neuronal cell cycle re-entry in our

cultures. Ki67 is a nuclear antigen that is expressed during G1, S, G2 and mitosis but is

absent during G0 (Sinha and Hader, 2002). Neuronal cell cycle re-entry has been

verified using Ki67 in previous studies where neurons were treated with hydrogen

peroxide (Schwartz et al., 2007; Tomashevski et al., 2010). Although there were several

Ki67 positive neurons in all treatments, the majority of the neurons were Ki67 negative

at several time points following UV-irradiation. Cdk4 and Cdk6 expression in cortical

neurons was also analyzed because they are involved in the progression of the G1

phase in the cell cycle (Caporali et al., 2004). In addition, dominant-negative kinase-

inactive forms of Cdk4 and Cdk6 expression have reduced UV-induced neuronal death

as well as B-amyloid induced neuronal death (Giovanni et al., 1999; Park et al., 1998a).

Similarly to Ki67 findings, there were no differences in expression of Cdk4 or Cdk6 with

77

UV-irradiation or with BrdU incubation. These findings confirmed that neuronal cell cycle

re-entry does not occur following UV-irradiation in our cultures.

5.6 Neuronal DNA methylation

A recent study showed that proliferation of neural precursors (NPCs) decreases

when NPCs are incubated with BrdU (Schneider and d'Adda di Fagagna, 2012). BrdU-

treated neural stem cells (NSCs) lose expression of stem cell markers including Nestin,

Sox2 and Pax6. Furthermore, after BrdU treatment there was an up-regulation of GFAP,

an astrocytic marker (Schneider and d'Adda di Fagagna, 2012). Interestingly, the

increase in GFAP expression coincided with a decrease in DNA methyltransferases as

well as a reduction in global DNA CpG methylation (Schneider and d'Adda di Fagagna,

2012). However, in our cultures, global DNA methylation was unaltered after UV-

irradiation as well as with BrdU incubation. Since differentiation has been associated

with DNA demethylation (Singh et al., 2009), cortical neurons may already have low

levels of DNA methylation in comparison to stem cells. There is a possibility that BrdU

incubation after UV-irradiation does alter DNA methylation in cortical neurons but the

change may be small and/or gene-specific that it is undetectable with the methodology

we used. Therefore, a more in depth study is required to determine if BrdU changes

neuronal DNA methylation as well as whether it changes expression of DNA

methyltransferases.

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6. CONCLUSIONS AND FUTURE DIRECTIONS UV damage causes neuronal apoptosis. Although neurons are not exposed to

UV radiation, UV-irradiation creates bulky DNA lesions that are representative of a

wide-range of lesions including chemotherapeutic agents. UV-induced neuronal

apoptosis can be prevented with BrdU concentrations as low as 100 nM, two orders of

magnitude below concentrations routinely used to label DNA synthesis. However, BrdU

does not protect proliferating cells such as fibroblasts from UV-induced apoptosis.

Therefore, BrdU likely protects cells from DNA damage-induced death through a

mechanism that is specific to neurons and perhaps other post-mitotic cells.

Furthermore, thymidine and other thymidine analogues may also be neuroprotective

suggesting that other structures similar to thymidine may also be neuroprotective. Since

thymidine may also be protective, the neuroprotective mechanism of BrdU may be an

interaction with a protein or a pathway that interacts with endogenous thymidine; the

bromine in BrdU is not essential for neuroprotection. In the current study, UV-irradiation

did not cause mouse cortical neurons to re-enter the cell cycle and the majority of

neurons remained in the G0 phase of the cell cycle regardless of the absence or

presence of BrdU.

BrdU reduces cleaved caspase-3 as well as phosphatidylserine exposure on the

cell surface in neurons after UV-irradiation. Since phosphatidylserine is exposed on the

cell surface after caspase-3 is cleaved (Niu and Chen, 2010), BrdU protects neurons

from apoptosis upstream of caspase-3 cleavage. UV-induced p53 expression occurs

after UV-irradiation in the absence and presence of BrdU. Therefore, BrdU likely

protects neurons downstream of p53 accumulation and up stream of caspase-3

activation.

79

Many aspects of BrdU neuroprotection remain to be clarified. Future investigation

is required to determine if BrdU following UV-irradiation delays neuronal apoptosis or if

neurons are protected indefinitely. Furthermore, the mechanism of BrdU protection

remains to be elucidated. Further examination of UV-induced cell signaling pathways,

specifically expression of proteins downstream of p53 and upstream of caspase-3 such

as activation of Bax and release of cytochrome c from the mitochondria, should provide

insight into the mechanism of BrdU protection.

The most surprising and definitive finding in the current study is that NER-null

neurons are resistant to UV-initiated apoptosis. UV-induced apoptosis occurs by UV

DNA damage activating ATR (Paulsen and Cimprich, 2007) which phosphorylates Chk1

and p53 (Guo et al., 2000) resulting in apoptosis. In non-proliferating fibroblasts,

functional NER is required for Chk1 and p53 phosphorylation (Marini et al., 2006). It has

been suggested that in proliferating cells the stalling of the replication fork and RPA on

single-stranded DNA recruits ATR and is an early trigger for the DNA damage response

(Byun et al., 2005). In neurons and post-mitotic cells, replication fork stalling cannot be

the trigger for ATR. Instead functional NER may be required to activate the DNA

damage response. Additional experiments are required to investigate the protective

mechanism of UV-resistance in NER-null neurons. Specifically, whether Chk1 and p53

are phosphorylated in NER-null neurons is an important area of future study.

About 20% percent of patients without functional NER have neurological

symptoms. Therefore, functional NER is important in neurons. Most of the neurological

symptoms in patients can be credited to progressive neurodegeneration (Rapin et al.,

2000). Thus, additional experiments are required to determine whether UV-irradiated

80

neurons without functional NER have a delayed response to DNA damage and undergo

death through an alternative mechanism.

Understanding the neuroprotective mechanism of BrdU can provide insight into

designing drugs to prevent neuronal DNA-damage induced apoptosis. Contrary to

expectations, NER deficient neurons are resistant to UV-induced apoptosis.

Understanding the role of NER in UV-induced apoptosis should provide insight into the

neuronal DNA damage response.

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