Cell Cycle Effects Associated with Human Adenovirus
E4orf4 Protein-Induced Tumour Cell Death
Neera Sriskandarajah
BSc Biochemistry
Department of Biochemistry
and Rosalind and Morris Goodman Cancer Research Centre
McGill University
Montreal, Quebec, Canada
September 2014
A thesis submitted to McGill University in partial fulfillment of
the requirements of the degree of Master of Science
© Neera Sriskandarajah, 2014
Neera Sriskandarajah
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Table of Contents
Abstract…………………………………………………………………………………... 5
Abrégé………………..…………………………………………………….……………...6
Acknowledgements ………………………..……………………………………………...7
Preface …………………………..……………………………………………………….. 8
Contribution of Co-Authors ………………..…………………………………………......8
List of Figures……………………………………..………………………………………9
List of Tables……………………………….…………………………………………......9
List of Abbreviations………………………..………………………………………..10-11
Original Contributions to Knowledge……………………………..……………………..12
Publications………………………………………………………………………………12
I. Introduction..................................................................................................................13
1. Human Adenoviruses and their Classification……...……………………………..13-14
1.1. Adenovirus structure…...……...………………………………………...14-15
1.1.2 Adenovirus Infection……...………….…………………...……16-17
1.2. Adenovirus genome and gene expression.……….……………………...18-20
1.2.1. The Adenovirus E1A gene.…….…...………………………....21-22
1.2.2. The E4 transcription unit.……...………………………………….23
1.2.3. Late gene expression. ……...……………………………………...24
1.2.4. Host Shut-off. ……...……………………………………….……..25
1.2.5. Cytopathic Effect (CPE).….…………...………………………….25
2. The Adenovirus E4orf4 protein.……...………………………………….……………26
2.1. The structure of E4orf4…..……….………………………………..…….26-27
2.1.2. The function of E4orf4. ……...……………………………….…...27
2.1.2.1. E4orf4 interacting partners.……...……………….……..28
2.1.2.1.1 Protein Phosphatase 2A………………………..28
2.1.2.1.1.2. PP2A the Cell Cycle……………….………..29
2.1.2.1.1.3. E4orf4-PP2A Interaction……………….. 29-31
2.1.2.2 E4orf4 and splicing..……...……………………………...31
2.2. E4orf4 mediated cell killing and the Cell Cycle………………….....………..….32-36
3. Experimental Rationale ………………………..……………………………………...37
II. Materials and Methods.........................................................................................38-41
III. Results.......................................................................................................................42
3.1. G0G1 Synchronization................................................................................42-47
3.2. G0G1 Cell Synchronization and Release....................................................48-51
3.3. Time Lapse Microscopy of H1299 cells expressing E4orf4 ……...…….52-60
3.4. E4orf4 induces cell death at both G1 diploid and G1 tetraploid states….. 61-64
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IV. Discussion...................................................................................................................65
4.1. Development of a novel method of G0/G1 synchronization in p53-/- H1299
Human Lung Carcinoma cells……………………………………………………65
4.2. Cellular morphology of E4orf4 expressing cells………………………...65-66
4.3. Cell fate induced by E4orf4……………………………………………...66-67
4.4.Concluding Remarks…………………………….…………………………...69
V. Supplementary Videos…………………..…………………………………………..69
VI. References............................................................................................................ 70-78
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Abstract
The adenovirus E4orf4 protein induces cell death independently of p53 in human
cancer cells but not normal primary cells through a mechanism that remains to be
elucidated. Previous studies have suggested that E4orf4 induces an arrest or a delay in
mitosis, a fate that is heavily dependent on E4orf4’s interaction with the Bα regulatory
subunit of protein phosphatase 2A.
In this study, a protocol using 0.01% FBS media was developed to synchronize
human lung carcinoma (H1299) cells in G0/G1. Time-lapse microscopy was performed
using this G0/G1 synchronization as well as S phase arrest and release in mCherry-H2B
H1299 cells to look at both morphological changes by phase contrast and cell cycle
progression by immunofluorescence. It was found that cells expressing E4orf4 display an
overall increased mobility, rounding up, and demonstrate a slow transit through mitosis, a
delay and often failure in cytokinesis, and those cells that survive take a prolonged period
of time to re-enter another round of the cell cycle. In fact, when E4orf4 was expressed in
G0/G1 arrested cells, most cells failed to initiate DNA synthesis, as shown in parallel flow
cytometry studies by our group (Cabon, Sriskandarajah et al, 2013). Studies in this thesis
using time-lapse microscopy confirmed that in such cells no further nuclear division was
evident. However, when E4orf4 was expressed in cells blocked in S phase and then
released, cells completed DNA synthesis but often remained tetraploid and by time-lapse
microscopy, demonstrated difficulties completing cytokinesis successfully. Studies in this
thesis also addressed the issue of whether the cell death induced by E4orf4 occurs in G1
arrested diploid or tetraploid cells. Cell sorting experiments followed by flow cytometry
revealed that E4orf4 induced cell death occurred in both tetraploid and diploid cells. Thus
my work has confirmed that E4orf4 toxicity is characterized by difficulty in transiting
though mitosis to successful completion of cytokinesis.
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Abrégé
Il a été démontré que la protéine adénovirale E4orf4 induit la mort cellulaire
indépendamment de p53 dans des cellules cancéreuses humaines mais non dans des
cellules primaires normales par un mécanisme d'action qui cependant reste largement
inconnu. Des études ont suggéré que l'intéraction entre E4orf4 et la sousunité régulatrice Bα de la protéine phosphatase 2A résulte en l'induction d'un arrêt ou un
délai dans la progression mitotique.
Dans cette thèse, un protocole de synchronisation de cellules de carcinome
pulmonaire humaines (H1299, en culture à 0.01%FBS) ) en G0/G1 a été
développé. Grâce à cette synchronisation combinée à un arrêt puis libération de la phase
S du cycle cellulaire de cellules H1299 exprimant un mCherry-H2B, une étude
microscopique par immunofluorescence ainsi que par contraste de phase a permis
d'investiguer les changements morphologiques ainsi que la progression à travers le cycle
cellulaire.
Il a été constaté que les cellules exprimant E4orf4 affichaient une mobilité globale
accrue ansi qu'une morphologie arrondie. Ces cellules ont également démontré une
progression mitotique plus lente, un retard et même souvent l'échec dans la
cytokinèse. De surcroît, les cellules qui survivent prennent une période de temps plus
longue pour amorcer un autre cycle cellulaire. En effet, notre groupe a démontré par des
études de cytométrie par flux (Cabon, Sriskandarajah et al, 2013) que lorsque E4orf4 est
exprimé dans les cellules en arrêt G0/G1, la majorité de ces cellules n'initient pas de
synthèse d'ADN, ce qui a été également confirmé par les études dans cette
thèse. Cependant, lorsque E4orf4 est exprimé dans des cellules bloquées en phase S puis
libérées, ces cellules complètent la synthèse d'ADN, mais demeurent souvent tétraploïdes
et ont des difficultées à compléter la cytokinèse. Les études par cytométrie de flux de
cette thèse ont également déterminé que la mort cellulaire induite par E4orf4 se produit
dans des cellules tétraploïdes ainsi que diploïdes. Ainsi, mon travail a confirmé que la
toxicité de E4orf4 se caractérise par une difficulté à transiter de la mitose à la réussite de
la cytokinèse.
Neera Sriskandarajah
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Acknowledgements
This thesis summarizes my research work carried out in the laboratories of
Professors Philip E. Branton and Jose Teodoro. My studies here at McGill University
would not be possible without many people and as such I would like to thank each and
every one of them.
To Dr. Phil Branton who saw something in me when I was a second year
Biochemistry student during my Undergraduate degree. Thank you for being the kind
person that you are and an extraordinarily passionate and patient scientist. Thank you for
lighting this passion for research in me, back when I was not sure where my path in
academics would be. I am so very grateful to have been given the chance be your very
last graduate student in your long and successful research career. To Dr. Jose Teodoro
who equally gave me the opportunity to pursue my interest in biochemical research.
Thank you for your attentiveness, and innovative ideas.
I also want to thank the present and past members of Phil and Joe’s laboratories,
Dr. Paola Blanchette, Dr. Theresa ChiYing Cheng, Dr. Zarina J. D'Costa, Dr. Timra
Gilson, Dr. Melissa Z. Mui, Dr. Peter Wimmer, Dr. Dennis Takayesu, Frédéric Dallaire,
Thomas Kucharski, David Sharon, Jieyi Yang, Dr. Wissal El-Assaad, Isabelle Gamache,
Amro Mohammed, Mohamed Moustafa, Linda Smolders, Dr. Rachid Zagani and YiQing
Lü for your all your support and smiles.
I would like to give a special thanks to Thomas Kucharski who gave me a head
start in a lot of the techniques that I have learned over the past two years but also for the
friendly and scientific conversations. Thank you to Ken MacDonald and Dr. Abba Malina
for their help teaching me world of flow cytometry. Thank you to Meena and Sophia for
the early morning conversations and all the smiles, your work is always appreciated. I
also want to thank my Research Advisory Committee, Dr. Gordon Shore, Dr. Anne
Roulston and Dr. Jacques Archambault for all the valuable advice, suggestions and
critical questions that can only make me improve as a scientist. Specifically, I would like
to thank Dr. Archambault for kindly accepting me as a student in his lab for my PhD
studies this Fall.
My thanks also go to each of my friends for all our conversations both at work
and our ventures outside. I would like to give a special thanks to Dr. Sami K. Boualia for
giving me his constant loving support, as well as his lending hand in partial reviewing of
the manuscript and abstract translation.
To my great-uncle Dr. Rajendram Saddanathar who was such an inspiration
growing up, and continues to be one of the greatest role models in my life. I remember
you every day I’m at the bench. Most importantly, I would like to thank my wonderful
parents for teaching me how important education is, and never to give up on your dreams.
Thank you for letting me grow into the person I am today, and for loving me and
supporting me in every way that you do.
Neera Sriskandarajah
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Preface
In compliance with the Guidelines concerning Thesis Preparation of the Faculty of
Graduate and Postdoctoral Studies of McGill University; Section 3.3 of this thesis
contains parts of a paper that will eventually be submitted for publications; Section 3.4 of
this thesis represents parts of the text of a paper published in Journal of Virology 2013
Dec; 87(24): 13168-78.
Contribution of Co-Authors
All of the figures presented in this thesis were generated by the author, except the
following:
Figure I-1: from Nemerow, Pache et al. 2009.
Figure I-5/6: from Cabon, Sriskandarajah et al. 2013.
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List of Figures
Figure I-1: Schematic diagram of the HAdV structure…………………………………15
Figure I-2: Schematic Adenovirus infection……………………………………………17
Figure I-3: Transcriptional map and gene products of adenovirus type 5 (Ad5)…….…20
Figure I-4: APC and the cell cycle…………………………………………………...…33
Figure I-5: Cell cycle analysis by flow cytometry post-synchronization in S phase...…35
Figure I-6: Cell cycle analysis by flow cytometry post-synchronization in G0/G1……..36
Figure III-1: G0/G1 Synchronization by 1% (low) serum conditions………………...…42
Figure III-2: G0/G1 Synchronization by Nutrient depleted serum starvation………...…43
Figure III-3: G0/G1 Synchronization by Amino Acid (aa) free media+/- serum………..44
Figure III-4: H1299 G0/G1 Synchronization using 0.01% FBS media……………….…45
Figure III-5: Varying seeding amounts with 0.01% serum……………………………..46
Figure III-6: G0/G1 Cell Synchronization and Release……………………………….…48
Figure III-7: Validation of G0/G1 release by a BrdU assay………………………….50-51
Figure III-8: Cell cycle fate of Asynchronous mCherry-H2B H1299 cells in the presence
of E4orf4…………………………………………………………………………………53
Figure III-9: Aggregation of G0/G1 arrested mCherry-H2B H1299 cells………………55
Figure III-10: Cell Cycle fate of mCherry-H2B H1299 cells post G0/G1 Synchronization
and 18 Hour release in the presence of E4orf4………………………………………..…56
Figure III-11: Cell Cycle fate of mCherry-H2B H1299 cells post G1/S Synchronization
with Hydroxyurea (HU) in the presence of E4orf4……………………………………...58
Figure III-12A: Analysis of E4orf4 induced cell death in 2n and 4n cell populations....61
Figure III-12B: Analysis of E4orf4 induced cell death in 2n and 4n cell populations....63
List of Tables
Table 1. Methods employed to synchronize H1299 cells in G0/G1……………………...47
Table 2. Comparison of E4orf4 expressing mCherry-H2B H1299 cell mitosis transit
compared to vector control AdrtTA…………………………………………………...…59
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List of Abbreviations
Abbreviation
aa
ACF
APC/C
ARM
BrdU
BSA
cAMP
CAR
CAV
Cdc
Cdk1
cDNA
CMV
Co-IP
CPE
CRE
DAPI
DMEM
DNA
ds/ssDNA
E1A
E1B
E2A
E3
E4
E4ARM
E4orf4
E4orf4 Point Mutants
E4orf6
eIF4E
FACS
FBS
FFU
FITC
g
G418
hr
H1299
HAdV
HIV-1
HTLV-1
Neera Sriskandarajah
Full Name
Amino Acid
ATP-utilizing Chromatin assembly and remodeling Factor
Anaphase Promoting Complex/Cyclosome
Arginine Rich Motif
Bromodeoxyuridine
Bovine Serum Albumin
cyclic-adenosine monophosphate
Coxsackie and Adenovirus receptor
Chicken Anemia Virus
Cell Division Cycle
Cyclin-Dependent Kinase 1
complementary DNA
Cytomegalovirus
Co-Immuno Precipitation
Cytopathic Effect
cAMP Responsive Element
4’,6-diamidino-2-phenylindole
Dulbecco's Modification Eagle's Medium
Deoxyribonucleic Acid
Double/Single-Stranded DNA
Adenovirus Early Region 1A
Adenovirus Early Region 1B
Adenovirus Early Region 2A
Adenovirus Early Region 3
Adenovirus Early Region 4
Early Region 4 Arginine-Rich Motif
Adenovirus Early Region 4, Open Reading Frame 4
E4orf4 point mutants are named as Original Amino Acid
+ Position Number + Mutant Amino Acid
Adenovirus Early Region 4, Open Reading Frame 6
Eukaryotic translation Initiation Factor 4E
Fluorescent-Activated Cell Sorting
Fetal Bovine Serum
Fluorescent Focus Unit (FFU)
Fluorescein isothiocyanate
grams
Geneticin
Hours
Human Lung Carcinoma cell line
Human Adenovirus
Human Immunodeficiency Virus 1
Human T-cell Leukemia Virus 1
10
HU
IP
kb
kDa
min
MLP
MLTU
MOI
MRN
mRNA
Mut
NES
NLS
NPC
Nup
Nup 205
OA
orf
p.i.
p53
PBS
PFU
PML
PP2A
PP2ABα
PP2ACdc55
pRb
RNA
SAC
S. cerevisiae
SH3
S phase
TPL
TPR
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Hydroxyurea
Immunoprecipitation
kilo bases
kilo Daltons
Minutes
Major Late Promoter
Major Late Transcription Unit
Multiplicity of Infection
Mre11/Rad50/Nbs1 protein complex
messenger RNA
Mutant/Mutation
Nuclear Export Signal
Nuclear Localization Signal
Nuclear Pore Complex
Nucleoporin
Nuclear pore complex protein Nup 205
(nucleoporin 205kDa)
Okadaic Acid
open reading frame
Post-Infection
tumor protein 53
Phosphate Buffered Saline
Plaque Forming Unit
Promyelocytic Leukemia
Protein Phosphatase 2A
Bα-containing form of PP2A
Cdc55-containing form of PP2A
retinoblastoma tumour suppressor protein
Ribonucleic Acid
Spindle Assembly Checkpoint
Saccharomyces cerevisiae
Src-homology 3
Synthesis Phase
Tri-Partite Leader Sequence
Tetratricopeptide Repeat
11
Original Contributions to Knowledge
0.01% foetal bovine serum (FBS) in media (DMEM) arrests p53-/- H1299 human lung
carcinoma cells in G0/G1.
H1299 cells expressing E4orf4 exhibit increased mobility, a major delay in mitosis, and a
significant proportion of cells demonstrate a failure in cytokinesis.
Confirmation by time-lapse imaging that cells expressing E4orf4 following release from
G0/G1 arrest largely fail to divide.
H1299 cells overexpressing E4orf4 undergo cell death at both G1 diploid and G1
tetraploid states.
Publications
Publications immediately relevant to the thesis
1. Cabon L, Sriskandarajah N., et al. Adenovirus E4orf4 Protein-Induced Death of p53-/H1299 Human Cancer Cells Follows a G1 Arrest of Both Tetraploid and Diploid Cells
Due to a Failure to Initiate DNA Synthesis. JVI, Dec 2013. 87(24): p. 13168-78.
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I. Introduction
1. Human Adenoviruses and their Classification
Adenoviruses are very common in the world’s human population, as 80% of
children under the age of 5 have already been exposed to the virus [1]. Wallace Rowe and
his colleagues first isolated Adenovirus from adenoid cell cultures in 1953 in the search
to identify the infectious agent causing the common cold [1]. Despite not being the
sought after common cold causing target, human adenoviruses (HAdV) were later
identified as being responsible for many acute infections affecting the respiratory, enteric,
and ocular systems [1-6]. In 1962, HAdV12, the first human oncogenic virus to be
discovered, was shown to transform hamster cells [7]; however, thus far adenoviruses
have not yet been causally implicated in any human malignancy [8].
Adenoviruses are part of the viral family called Adenoviridae which is divided
into two subclasses: Mastadenoviruses and Aviadenoviruses. Mastadenoviruses infect
mammals such as humans, monkeys, mice, horses and dogs while Aviadenoviruses infect
birds [9]. Thus far, there are more than 60 HAdv serotypes identified based on their
sensitivity to neutralizing antibodies [1, 9]. Each subtype is classified into different
species (A-F) distinguished by their ability to agglutinate red blood cells [10, 11].
Adenovirus subtypes are also classified according to their oncogenicity in rodents, DNA
sequence similarity/homology, virion protein electrophoretic mobility, as well as serum
profiles based on antibody binding to virion hexon and fiber proteins [3,12-14].
To date, serotypes belonging to species A and B and two serotypes belonging to
species D have been shown to cause tumors when subcutaneously injected into rodents
whereas the rest of the subgroups display lesser oncogenic potential [10]. Two HAdV
types have been most widely studied, Ad2 and Ad5 each of which belong to species C
[1,3].
Adenovirus research has advanced the field of molecular biology in many ways,
including through studies of messenger RNA splicing, adenoviral gene therapy, and a
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host of other topics. As such adenoviruses have served and will continue to serve as a
useful model system to explore fundamental cellular processes, as well as serve as
evolutionary models for other viruses [1-2, 4-6].
1.1. Adenovirus structure
Adenoviruses are large non-enveloped viruses of approximately 90nm in diameter
and a molecular weight of 150MDa. The adenovirus structure has been resolved by both
X-ray crystallography and cryo-electron microscopy (schematic shown in Figure I-1) [3].
Adenovirus virions are the infective form of the virus and consist of a protein shell called
the capsid that protects the viral core genome. Adenovirus virions display icosahedral
symmetry with 20 equilateral triangle faces and 12 vertices and are studded with knobbed
fibers [10]. The HAdV capsid, surrounds the viral core genome, and is comprised of 11
late viral structural proteins, composing the hexon and penton base shown in Figure I-1.
The two main functions of the capsid are to protect the core genome and arbitrate
virus entry into host cells. The capsid is composed of 252 units called capsomeres. The
iscosahedral symmetry of HAdV results from the arrangement of hexons (protein II) form
240 capsomeres, and the pentons (protein III) forming the remaining 12 units. There are
twelve vertices in the capsid containing a penton base, which serves as a base for the noncovalent association to a projecting fiber protein. Each of the rod and knob fiber (protein
IV) vertex proteins is crucial for adenovirus host cell attachment and entry during
infection [3,13]. Proteins II, III and IV are the main structural proteins.
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Figure I.1. Schematic diagram of the HAdV structure
The icosahedral capsid of adenovirus protects the core genome. The capsid is
composed of capsid proteins, hexons, penton bases, and fibers. Minor capsid proteins
include pIX, pIIIa, pVI and pVIII. The core genome consists of the viral DNA bound to
proteins V, VII and mu.
Adapted from [3].
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1.1.2. Adenovirus infection
Initiation of the HAdV infection begins with the adenoviral knob domain of the
fiber proteins making contact with high affinity cellular receptors, termed Coxsackie and
Adenovirus Receptor (CAR), as well as with integrin receptors. The binding of the
HAdV penton base to secondary cell surface integrin receptors, such as the αV family,
leads to migration of virus-receptor complexes to clathrin-coated pits [10]. This permits
the internalization of the partially disassembled virions by clathrin-mediated endocytosis.
Once in the cytoplasm the virion disintegrates in the acidic environment of the late
endosome releasing the vertex proteins (penton base, pIIIa, fiber, peripentonal hexons).
The uncoated virus particles are then released into the cytoplasm by liberating the
internal capsid protein pVI, which induces the endosomal membrane to break down. The
HAdV genome core is transported to the nucleus via interactions between hexons and the
microtubule network. After moving along microtubules, the virus particles are imported
by translocation through the host cell nuclear pore complex into the nucleus, where
finally the viral DNA is transcribed (Figure I-2) [3].
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Figure I.2. Schematic diagram of Adenovirus Infection
Adenovirus knob domain makes contact with cellular Coxsackie and Adenovirus
Receptor (CAR) and integrin receptors. HAdV penton base interaction with integrin
receptors results in the packaging of virus-receptor complexes into clathrin-coated pits.
The virion collapses under low pH of late endosome releasing the vertex proteins. Virus
particles in the cytoplasm are transported along the microtubule network and are
translocated through the host NPC into the nucleus.
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1.2. Adenovirus genome and gene expression
The adenovirus core genome is associated with four proteins bound to its linear,
condensed, double-stranded DNA, ranging in size from 30 to 38 kilo base pairs (Kbp)
[1]. Three of these core proteins are arginine-rich basic proteins V, mil (protein X), and
histone-like protein VII, which function together to protect and maintain folding of the
viral DNA. The fourth protein, pTP, binds the 5’ end of the viral dsDNA covalently and
acts as a primer for viral replication in the host’s nucleus (Figure 1-3) [15]. The viral
DNA contains inverted terminal repeat sequences approximately 100bp which serve as
origins of replication [3, 12].
The adenoviral genome is divided into a set of three transcription units: early,
delayed early (intermediate), and late phase each of which is defined by the onset of viral
DNA replication. During the early phase of the infection as soon as the viral DNA has
been translocated into the host-cell nucleus (about 2hpi), E1A, E1B, E2A, E2B, E3, E4
alternatively spliced mRNA’s are produced from six early transcription promoters
independently of viral DNA replication that encode regulatory proteins that prepare the
cell for optimal viral DNA replication. There are three preparatory steps that have
evolved to ensure effective viral replication. First, E1A expression promotes efficient
activation of all early viral transcription units. Second, E1A and E4 products induce host
cells to enter S phase. And third, E1B, E3 as well as adenovirus associated RNA (VARNA) inactivate interferon-based host-cell antiviral defense mechanisms [12]. Viral
DNA replication quickly follows at 8-10 hours post-infection (hpi) along with the
expression of delayed early (intermediate) genes such as IX, IVa2, E2 late. Finally, the
expression of five late mRNA’s, L1 to L5, by alternative splicing from one Major Late
Promoter (MLP) and five polyadenylation sites, induces progression to the late phase of
infection (10-24hpi). This expression also gives rise to structural proteins involved in
DNA packaging and assembly of viral particles. The host-cell then begins to break down
and virion progeny release occurs (24-36 hpi).
The assembly of transcription units is an evolutionary advantage first discovered
in adenovirus, that results in the production of multiple viral proteins through alternative
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splicing of full-length mRNA. In fact, products of early units often regulate the
expression of late products. For example, the Early Region 4 open reading frame 4
(E4orf4) protein promotes the alternative splicing of adenoviral late mRNA’s to enhance
late protein production by causing hypo-phosphorylaton and thus activation of cellular
SR splicing factors [15].
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Figure I-3. Transcriptional map and gene products of adenovirus type 5 (Ad5)
Families of early (E in purple), intermediate (in black), and late (L, in blue) mRNAs are
shown in relation to viral genome, divided into 100 map units (350nt). Names of proteins
encoded by mRNAs are labeled adjacent to the respective mRNA. The vertical bar on the
5’ end of mRNA’s correspond to transcriptional promoters while the 3’ end (arrowhead)
corresponds to polyadenylation sites.
Adapted from Acheson [10].
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1.2.1. The Adenovirus E1A gene
The Adenovirus Early Region 1A (E1A) is the first viral gene to be transcribed
following entry into the host cell nucleus [16]. E1A transcription is driven by a
transcriptional enhancer positioned at the E1A promoter and relies on transcription
factors provided by the host. E1A is essential for viral replication as viruses containing
mutations in E1A genes are highly deficient in early gene transcription, and thus yield
reduced amounts of progeny virions. [8]. Some adenovirus gene expression is regulated
by viral products as, for example, E4orf4 (discussed in section 2.0) autoregulates E4
transcription.
Through alternative splicing, the E1A gene produces five distinct mRNAs with
sedimentation coefficients (denoted for the slight different sedimentation rates in sucrose
gradients) of 9S, 10S, 11S, 12S, and 13S. However the latter two, 12S and 13S encode
proteins 243R and 289R, respectively [18-20]. They are the most abundant proteins
produced by the E1A transcription unit and differ by an alternatively spliced intron.
There are five conserved regions among adenovirus serotypes within E1A
proteins, the N-terminal region, CR1 (conserved region 1), CR2, CR3 and CR4 (Figure I4). E1A protein 13S/289R contains all of these domains, whereas 12S/243R lacks CR3
[12, 21]. It is through each of the three conserved regions of E1A that protein-protein
interactions occur. CR1 and CR2 are the main domains involved in adenovirus E1Ainduced oncogenic transformation as they contribute to the induction of host cell S phase
entry and further progression through the cell cycle [23-27]. CR1 is specifically
necessary to finish a round of the cell cycle [22-23] through interaction with chromatinmodifying complex proteins such as p300/CBP, p400, as well as with CDK inhibitors
such as p21 and p27, and proteasome components [23, 27].
E1A’s CR2 domain interacts with the Retinoblastoma (Rb) protein family of
pocket proteins via an LxCxE motif. Rb is known to bind and inhibit E2F cellular
transcription factors. This inhibition prevents E2F from targeting the genes required for
activating viral and cellular DNA synthesis and entry into S-phase. During infection, this
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function allows adenovirus to optimally target quiescent epithelial cells [22].
Adenovirus induces cell cycle entry and progression, through interaction between
the E1A-CR2 LxCxE motif and Rb family members Rb, p107 and p130 [28-31] thus
freeing E2F to activate expression of cell cycle-dependent genes [32-34]. Examples of
E2F activated target genes are cyclin-dependent kinase 2 (Cdk2) and cyclins A and E
which drive cells from the G0 phase to the S phase [12, 31-33]. Thus E1A can both
activate viral genes that are required for viral DNA replication, and E2F-dependent
cellular genes that are required for S phase entry [1].
CR3 is vital to activate early viral gene expression by associating with cellular
transcription factors present at these promoters rather than by binding DNA directly [3537]. Both the 12S/243R and 13S/289R forms of E1A proteins are important
transcriptional activators of several viral and cellular genes [36, 38]. For example, 243R
specifically activates the E2 region while 289R activates the transcription of all the
remaining early viral genes. E1A also moderates the expression pattern of host-cellular
factors required for transcriptional regulation, cell cycle progression, apoptosis, protein
degradation and even deoxynucleotide production for viral DNA replication [8, 36, 3839].
E1A is the major oncogene of hAdV. Through its CR1, CR2 domains and the Nterminal region, E1A is capable of promoting S phase entry, DNA synthesis and cell
transformation in otherwise non-dividing host cells. Such cells are capable of forming
tumours in nude mice, or in some other cases other rodents [8].
Neera Sriskandarajah
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1.2.2. The E4 transcription unit
There are seven different proteins encoded by Early Region 4 (E4) of human
adenovirus, E4orf1, E4orf2, E4orf3, E4orf3/4, E4orf4, E4orf6 and E4orf6/7 [40]. E4
proteins are involved in multiple biological processes that enhance viral replication [4145]. The rate of E4 transcription peaks during the early phase (4hpi) of adenovirus
infection and is maintained throughout infection, although the rate declines somewhat
later in infection [46]. Among all serotypes of HAdV, the E4 transcription units are
highly homologous. The adenovirus E4 gene expression is activated by E1A and
regulated at two levels: transcriptionally and post-transcriptionally [47]. In addition, the
E1A-dependent expression of the E4 transcriptional unit is subject to E4 negative
feedback regulation. In fact, E4orf4 has been shown to down-regulate the expression of
all E4 genes [40,48]. For example, E4orf4 inhibits the E1A-mediated transactivation of
E4 promoter through the cellular serine/threonine protein phosphatase 2A (PP2A) [49]
(discussed in section 2.1.2.1.1).
E4orf3, E4orf6, and E4orf4 (discussed in section 2) have been best studied in both
serotypes Ad2 and Ad5 [1]. Firstly, E4orf3 enhances viral replication by associating with
E1B55K and relocalizing it to structures within the nucleus such as the promyelocytic
leukemia (PML) nuclear bodies [50]. This relocalization to PML bodies forces the
reorganization of these nuclear bodies and of the MRN protein complex
(Mre11/Rad50/Nbs1), which is involved in DNA repair, to “track-like” structures [5153]. Sequestering of the MRN complex by E4orf3 expression lowers DNA damage
repair, promotes genomic instability, and contributes to uncontrolled cell proliferation.
Moreover, E4orf6 promotes p53 degradation by forming cellular Cullin-based E3
ubiquitin ligase complexes that allows interaction with E1B55K (section1.2.4) [54-57].
Along with p53, E4orf6/E1B55K ubiquitin ligase polyubiquitinates and targets substrates
such as Mre11, DNA ligase IV, Integrin α3, and Bloom helicase for degradation by the
26S proteasome [52, 58-61]. Both E4orf3 and E4orf6 increase viral DNA replication and
late viral protein synthesis as well as induce host cell shut-off [62-66].
Neera Sriskandarajah
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1.2.3. Late gene expression
The majority of structural proteins are produced during the late phse of gene
expression from the adenovirus genome [1]. Eighteen late adenovirus mRNAs are
produced under the Major Late Transcription Unit (MLTU). In fact, the MLTU is further
sub-divided into five sub-regions, L1 to L5. The major late promoter (MLP) is active at
low levels during the early phase of infection such that transcription progresses up to the
L3 region. Viral DNA replication triggers the full-level activation of MLP in order to
produce sufficient levels of late structural and scaffolding proteins as well as a protease
resulting in newly synthesized adenovirus genomes packaged into virions [12, 67).
Each of the multiple 18 late mRNAs produced by the MLTU, contains: one noncoding 200-nt, the tripartite leader (TPL) sequence (leaders 1, 2, and 3) at their 5’ ends
and 3’ splice and poly-adenylation sites. The TPL promotes efficient nuclear export,
translation and increases stability of late mRNAs, while the 3’ poly-adenylation sites
promote the essential process of alternative splicing [68]. Adenovirus has evolved
mechanisms to enhance this splicing activity, in particular, the action of E4orf4
mentioned earlier [15, 34, 49, 67-68].
Neera Sriskandarajah
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1.2.4. Host Cell Shut-off
Adenovirus employs several strategies to control host cell machinery to optimize
replication, including the preferential expression of viral products by reducing translation
of cellular proteins. This effect has been coined Host Cell Shut-off. Host Cell Shut-off is
exerted at two levels: post-transcriptionally and translationally. Post-transcriptionally for
example, the host cell selectively exports a higher proportion of newly synthesized late
viral mRNAs over cellular mRNA. In fact, host factors such as the nuclear pore complex
and early viral products such as E4orf6 and E1B-55K are vital and cooperate to
effectively block cellular mRNA transport to the cytoplasm by a mechanism that is not
yet understood [69-72].
Viral mRNAs are also preferentially translated over cellular mRNAs at late stages
of infection [73]. This effect is achieved by reducing, CAP-dependent translation of
cellular mRNA’s. HAdV late 100K protein of the L4 region (L4-100K) binds to scaffold
protein eIF4G causing the displacement of a kinase such that translation initiation factor
eIF4E (which normally binds to 5’-m7GpppN CAP structure on most cellular mRNAs) is
dephosphorylated. Thus the L4-100K/eIF4G interaction inhibits eIF4F complex
association with capped cellular mRNAs. The 5’ TPL in late viral mRNAs also
contributes to their preferential translation compared to cellular mRNAs. This is due to a
process called ribosome shunting, where 5’-UTR scanning is skipped and ribosomes are
immediately transferred to the initiation codon.
1.2.5. Cytopathic Effect (CPE)
The Cytopathic Effect (CPE) is the sum of the biochemical and morphological
changes following the replication of viral DNA and synthesis of all viral components for
virion release. Adenovirus specifically, alters causes host cellular morphology to show
characteristics of swelling, rounding, basophilic intra-nuclear inclusion bodies, [74]
nuclear shrinking (pyknosis), proliferation of nuclear membrane, vacuoles in cytoplasm,
syncytia (cell fusion), margination as well as breaking of chromosomes. Infected cells
eventually lift from the culture plate and lyse to release viral progeny.
Neera Sriskandarajah
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2. The Adenovirus E4orf4 protein
Adenovirus Early Region 4 Open Reading Frame 4 (E4orf4) protein is most well
known for its ability to induce p53-dependent-tumour cell specific killing when
expressed alone and at high levels [75-78]. E4orf4 is a small protein in the case of Ad2
and Ad5 (in species C), consisting of 114 amino acids and has a molecular mass of 14
kDa. Although E4orf4 does not share any sequence homology with any known
eukaryotic protein, it is conserved among all adenovirus serotypes with differences only
in the carboxy-terminus [78]. In the past, many studies were performed to determine the
role of E4 gene products of adenovirus. Using deletion and insertion mutants of the Ad5
E4 region, some studies found that E4orf4 is not essential for virus growth in human
tumor cells [62, 79], although recent studies in human primary cells suggested an
important role (P. Blanchette, unpublished). As stated above, E4orf4 has been shown to
be an important regulator of adenoviral gene expression by altering viral transcription and
splicing of late viral mRNAs [15, 34, 49, 79].
2.1. The structure of E4orf4
E4orf4 harbors two short motifs; a proline rich sequence at its amino-terminus
and an arginine-rich motif (ARM) in the second half of the protein [80]. It is believed that
the 11 amino acid proline-rich sequence (in Ad2, M1 VLPALPAPP; consensus
MxxPxLPxPP) could function as an SH3-binding site. One of E4orf4’s known interacting
partners is c-Src. c-Src normally binds proteins through SH3-motifs. However, mutating
the proline-rich motif does not disrupt E4orf4’s interaction with c-Src. The basic
arginine-rich motif (E4 ARM) between residues 66/9 and 75 within the E4orf4 protein
[81] resembles a sequence found in human immunodeficiency virus 1 (HIV-1)’s Tat and
Rev proteins, Chicken Anemia Virus Apoptin (CAV-Ap) [82, 83], as well as Rex from
human T-cell leukemia virus 1 (HTLV-1) [84-89]. The E4ARM functions as a strong
inducer of nucleolar targeting and is also necessary for E4orf4-mediated cell death [90]
The ARM differs from the normal Nuclear Localization Signal (NLS) in that arginines
are more prevalent than lysines. Interestingly the ARM is also required for E4orf4
binding to Src-kinase [90,91].
Neera Sriskandarajah
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In spite of E4orf4’s small size the crystal structure remains to be resolved, due to
high insolubility; however, software such as PredictProtein predict that E4orf4 would
have mostly a helical structure based on the primary protein sequence. E4orf4 is rendered
non functional and often unstable when portions of the protein are deleted or altered [76,
78]. Such a profile implies that E4orf4 may have tertiary and quaternary structure that
could be vital for function and stability [78].
……
2.1.2. The function of E4orf4
Although E4orf4 has been fairly intensely studied, little is known about the
function of E4orf4 within a viral context. Using adenovirus mutants lacking the
expression of E4orf4 (E4orf4- virus), it was found that E4orf4 plays a role in the
regulation of transcription, splicing, and signal transduction (reviewed in [92-93]). For
example E4orf4, with respect to adenovirus, down regulates early viral gene expression,
increases splicing of late viral mRNA’s, and up-regulates the switch to late viral gene
expression (section 2.1.2.3).
Most of the research carried out on E4orf4 has focused on its capacity to induce
dose-dependent and p53-independent cancer cell specific death, when expressed alone
outside of a viral context [77, 94-95]. As such, these studies often employed adenovirus
vectors lacking all viral products other than E4orf4.
Neera Sriskandarajah
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2.1.2.1. E4orf4 interacting partners
Given the abundance of molecular pathways that viruses hijack in order to
efficiently infect the host, viral proteins functionally interact with several host factors. As
mentioned earlier, the discovery of an E4orf4 interaction with the host Bα regulatory
subunit of PP2A was later been shown to be crucial for E4orf4-induced cell killing [15,
49, 96-97, 98-100]. In 2000, Lavoie and colleagues identified proto-oncogene tyrosine
kinase c-Src as yet another interacting partner of E4orf4 [101]. E4orf4-cSrc interaction
provided further advancements in the mechanism of cytoplasmic killing pathway
activated by E4orf4. Additional studies have revealed a few other interacting partners,
such as ASF/SF2 splicing factors, cellular transcription factor AP-1 [15, 34, 49], as well
as the ATP-dependent chromatin-remodeling factor ACF [96]. Although many E4orf4
interacting partners such as these have been found, the vast array of pathways that E4orf4
is implicated in during adenovirus replication, gene expression, and cell death still
remains to be elucidated.
2.1.2.1.1 Protein Phosphatase 2A (PP2A)
Protein phosphatase 2A (PP2A) is the dominant and most abundant cellular
serine/threonine phosphatase. PP2A has a wide range of substrate specificities and is
involved in the regulation of a multitude of cellular pathways [102-104]. It is a
heterotrimeric enzyme comprising a heterodimeric core holoenzyme and a variable
regulatory subunit. Subunits A and C represent the structural and catalytic subunits,
respectively forming the dimeric core enzyme of PP2A. In mammalian cells, there are 4
classes of regulatory B subunits, B, B’, B’’, B’’’ [105-106]. In addition, these four
diverse classes of B regulatory subunits are further divided into 16 members (4 members
for each B class). The B class for example has 4 different highly related members Bα, Bβ,
Bγ, and Bδ, each with a molecular mass of 55 KDa [107]. The diversity of regulatory B
subunits permits substrate specificity spatial and temporal functions of PP2A [105-106,
108].
Neera Sriskandarajah
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2.1.2.1.1.2. PP2A the Cell Cycle
Protein phosphatases and kinases play a significant role in cell cycle regulation
such that the cell cycle progresses unidirectionally in a timely and controlled manner. For
example, cell cycle entry is triggered by phosphorylation of cyclin D by cyclin dependent
kinase (CDK) 4/6. In order for the cell cycle to progress into S phase, cyclin D must be
dephosphorylated by a phosphatase and cyclin E must then be phosphorylated by CDK2
[109]. PP2A is amongst many phosphatases involved in multiple aspects of cell cycle
regulation. PP2A simultaneously inhibits all forms of Cdc25 phosphorylation and
activation and activates Wee1 to impede the formation of CDK1: cyclin B dimers [110,
111]. Rb and p107 pocket proteins are dephosphorylated also by PP2A (among other
phosphatases) in response to UV induced DNA damage as a defense mechanism to halt
the cell cycle and repair DNA damage [112]. In addition, PP2A negatively regulates
entry into mitosis, by dephosphorylating and inactivating Mitosis Promoting Factor
(MPF), such that the levels of activated Cdk1-cyclinB do not reach a high enough level to
promote G2 to M transition [113]. Moreover, regulation of phosphorylation status by
PP2A is also involved in other processes important in cell cycle progression such as
disassembly of the nuclear envelope [114].
2.1.2.1.1.3. E4orf4-PP2A Interaction
PP2A was the first protein discovered to bind E4orf4 in co-immunoprecipitation
(co-IP) experiments. The Bα isoform of PP2A, belonging to the B/B55 class of regulatory
subunits, was shown to be required for PP2A’s interaction with E4orf4 [76-78]. Our
group discovered that E4orf4 binds only to the B/B55 class of regulatory subunits.
Importantly, in the absence of interactions with the B subunit, E4orf4-induced cell death
was found to be greatly reduced, suggesting that this interaction is necessary for this cell
death pathway [115].
The association of Bα with E4orf4 allows the co-IP of the A and C subunits [49].
Moreover, E4orf4 complexed to PP2A is associated with phosphatase activity, which can
be inhibited by Okadaic Acid (OA), a potent inhibitor of both PP2A and PP1 [49, 116]. It
Neera Sriskandarajah
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was widely believed that E4orf4 utilizes PP2A activity to induce cell death; however,
studies by our group showed that inhibition of PP2A activity by E4orf4 binding was the
underlying cause of toxicity because: (1) inhibition of PP2A by low levels of OA or a
PP2A peptide inhibitor enhanced cell death; and (2) in vitro assays of PP2A activity
against protein substrates indicated that E4orf4 reduced phosphatase activity [117].
Recent extensive studies have elucidated the pattern of binding of E4orf4 to B55
subunits [97,118-119]. The crystal structure of the PP2A holoenzyme containing Bα
indicated that substrates interact exclusively with Bα [120]. Using a series of point
mutants in Bα and the closely related yeast CDC55 protein our group mapped the E4orf4
binding site to regions just “north” and “south” of the putative substrate binding groove
of Bα [119]. This interaction was predicted to block access of normal PP2A substrates
and in fact at high levels of E4orf4 this was found to be the case with the substrate p107.
Thus our group believes that E4orf4 tumour cell toxicity results from the inhibition of the
B55 class of PP2A enzymes and the failure to dephosphorylate key substrates necessary
for the survival of tumour cells. However, we believe that E4orf4 toxicity is an artifact of
overexpression and that E4orf4-PP2A interactions play a considerably different role at
lower levels of E4orf4 found during virus infection. In the case of the virus infectious
cycle we now believe, that E4orf4 recruits target phosphorylated proteins into the active
site of PP2A to be dephosphoylated [15, 49, 78, 96-97, 98-100]. One such target is ASF
(see below), section 2.1.2.3.
In terms of high level expression and cell killing, the relationship between
E4orf4’s function and PP2A interaction has been studied though mutational analysis, in
hopes of understanding the mechanism of E4orf4-induced cell death [78]. Marcellus and
colleagues were able to create E4orf4 point mutants along the full length of E4orf4, and
group these mutant into two classes, Class I or Class II. Class I mutants were identified to
have reduced interaction with PP2A-Bα, and therefore defective in inducing cell death by
E4orf4. On the other hand, Class II mutants were seen to bind PP2A-Bα similarly to wild
type E4orf4, however they exhibited a reduction in killing [78]. As such there may be
other players involved working in synergy with PP2A and E4orf4 to promote E4orf4Neera Sriskandarajah
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induced cytotoxicity [99].
2.1.2.3 E4orf4 and splicing
E4orf4 not only regulates transcription, but also post-transcriptional processes
such as splicing [17, 64-65]. One of E4orf4’s functions during virus infection is to
stimulate late mRNA production [121]. E4orf4 promotes the shift from early to late viral
gene expression by enhancing the alternative splicing and the production of late viral
mRNA L1-IIIa [41]. During the early phase of infection, SR (serine/arginine) splicing
factors) repress the alternative splicing of the L1 unit of the MLTU, such that only one
L1-IIIa pre-mRNA is made. SR factors bind to the 3RE intronic repressor element at the
IIIa 3’ splicing site, such that L1-52, 55K mRNA is produced during early phase. During
late phase, the SR factors are removed from the 3RE such that the IIIa form of L1 mRNA
can be produced. E4orf4 specifically associates with two hyperphosphorylated SR
proteins, SF2/ASF and SRp30c through their RNA recognition motif as well as with
PP2A [15]. E4orf4 targets hyperphosphorylated SF2/ASF and SRp30c to complex with
PP2A and become dephosphorylated such that the SR proteins are released from the 3RE,
thus alleviating the repression [15]. Once SR proteins are release, the splicing of IIIa-premRNA inducing the switch from early to late phase of viral gene expression [15, 121].
2.2. E4orf4 mediated cell killing and cell cycle
E4orf4 in cooperation with PP2A, induces p53-independent cell death in many
different cancer cells [76-78, 99, 122], as well as in the yeast S. cerevisiae [123]. In the
case of human tumour cells generation of a population of G1 tetraploids and evidence of
death by mitotic catastrophe was suggested [124, 125].
Earlier work by our group suggested that E4orf4 toxicity in yeast may involve
effects on the cellular Anaphase Promoting Complex/Cyclosome (APC/C). APC/C is an
E3-Ubiqtuin Ligase involved in cell cycle protein degradation that promotes the
progression of cells through mitosis and that is also very critical for a cell’s exit from
mitosis and into G1. During normal cell cycle progression there are two forms of APC
Neera Sriskandarajah
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that are sequentially activated, APCCdc20 and APCCdh1. APCCdc20 is active from early
mitosis to anaphase while APCCdh1 complex is active from anaphase and into G1 phase of
the cell cycle. Each form is activated sequentially to target its corresponding substrates by
polyubiquitination for degradation by the 26S proteasome such that cells can go through
mitosis and exit successfully into G1 and initiate a new round of DNA synthesis. Previous
studies in our lab using S. cerevisiae as a model system suggested that E4orf4 induces the
premature activation of APCCdc20 and the failure to activate APCCdh1, such that substrates
of APCCdc20 (securin) are degraded as early as S phase and substrates of APCCdh1 (Cdc20
and Plk1) are stable (Figure I-4) [111]. These results suggested that APC may also be
involved in E4orf4 killing of tumour cells and that premature activation of APCCdc20 or
inhibition of APCCdh1 could be involved.
Neera Sriskandarajah
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Figure I-4. APC and the cell cycle
This figure illustrates the steps involved in APC activation, degradation of APC
substrates, and the potential role of E4orf4 and PP2ABα in this process. E4orf4 induces
Cdk1 activity, and PP2A is believed to positively regulate the stability of Wee1 and may
negatively regulate formation of APCCdc20 via dephosphorylation of core proteins. Cdk1
is known to promote the activation of APCCdc20 and to inhibit activation of APCCdh1.
Adapted to the mammalian system from the yeast S.cerevisiae system depicted in Mui et
al. 2010 [111].
Neera Sriskandarajah
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In addition, our group performed two types of synchronization experiments
performed on H1299 cells [125]. The first case shown in figure I-5, depicts cells arrested
, in G1/S using hydroxyurea (HU). Cells were then infected with either the control vector
AdrtTA or vector over expressing E4orf4, AdE4orf4, and finally released. It was found
that from 2 to 12 hours, mock, control vector, and cells expressing E4orf4 all progressed
though S phase and G2M. Interestingly, from 14 hours onwards the controls continued
through mitosis and divided normally to yield 2 daughter cells with 2n DNA content,
whereas those expressing E4orf4 exhibited a population of cells that remained 4N and G1
tetraploids, with a clear defect in the reappearance of 2n cells. Thus, cells expressing
E4orf4 appeared able to continue DNA synthesis once initiated. In the second
synchronization experiment (Figure I-6), cells were arrested in G0G1 by nutrient depletion
and serum starvation, and then infected and released by the addition of fresh medium.
Once released the mock- and AdrtTA –infected cultures continued through the cell cycle;
however, cells infected with AdE4orf4 from around 6 hours exhibited very low levels of
4N S-phase cells. In fact most E4orf4 expressing cells remained 2N and therefore failed
to initiate DNA synthesis. Thus, depending on when E4orf4 is expressed within the cell
cycle, two populations appear to accumulate, one being G1 tetraploid and the other G1
diploid. Expression of E4orf4 at G1S resulted in 4n cells as cells continued to replicate
DNA and exhibited a defect in mitosis/cytokinesis. Expression of E4orf4 at G0G1 resulted
in more prominently 2n cells and cells that failed to initiate DNA synthesis. So cells
expressing E4orf4 can continue DNA synthesis but cannot initiate new rounds of
replication.
Given the strength of the evidence above pertaining to the many facets of
E4orf4’s mode of action, the precise mechanism of E4orf4 induced cell toxicity remains
to be identified, and as such further study was needed to reexamine E4orf4’s effect on the
cell cycle.
Neera Sriskandarajah
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Figure I-5. Cell cycle analysis by flow cytometry post-synchronization in S phase
with hydroxyurea (HU).
Mock-, AdrtTA-, or AdE4orf4-infected H1299 cells were examined by flow cytometry
following treatment with HU and released by the removal of drug and the addition of
fresh media. Cells were harvested at 2 hour intervals following release and analyzed by
flow cytometry.
Cabon, Sriskandarajah et al. 2013. [125].
Neera Sriskandarajah
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Figure I-6. Cell cycle analysis by flow cytometry post-synchronization in G0G1
growth arrest.
Mock-, AdrtTA-, or AdE4orf4-infected H1299 cells were studied by flow cytometry
following growth arrest in spent medium and addition of full medium and fresh serum.
Cells were harvested at 2 hour intervals following release and analyzed by flow
cytometry.
Cabon, Sriskandarajah et al. 2013. [125].
Neera Sriskandarajah
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3. Experimental Rationale
The adenovirus E4orf4 protein induces cell death in human cancer cells,
apparently leaving normal primary cells unharmed. For this reason, E4orf4 could be of
some therapeutic interest. Given the strength of the evidence supporting the role of
E4orf4 in affecting the cell cycle and initiation of DNA synthesis, a more in depth
investigation into the processes involved in E4orf4-induced cell killing was warranted.
Thus, the objective of this study is to reexamine the effect of E4orf4 on the cell cycle to
help delineate a mode of action of E4orf4 induced cell death.
The approach taken for the experiments described in this thesis involved first,
optimizing a protocol for arresting cells in G0G1 to allow further analysis of E4orf4
action. Upon identifying the proper conditions for synchronization, I further examined
the morphology of cells expressing high levels of E4orf4 as well as significant variations
in their cell cycle compared to controls. Specifically, I also studied the fate of the E4orf4
expressing G1-arrested diploid and tetraploid cells. Together with future experiments, this
research will perhaps reveal novel cellular factors and components of the cell cycle that
may synergistically work with E4orf4 to kill tumor cells.
Neera Sriskandarajah
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II. Materials and Methods
Cell culture
Two types of immortalized human cell lines were used in this research. First, the
human lung carcinoma cell line H1299 (ATCC no. CRL-5803) with a homologous
deletion of the p53 gene, as well as the fluorescence modified cell line mCherry-H2BH1299. Both cell lines under normal conditions were maintained in Dulbecco’s
Modification Eagle’s Medium (DMEM, Multicell, Catalogue Number 319-005-CL),
supplemented with 10% v/v Fetal Bovine Serum (HyClone), (without any antibiotics) at
37 °C under 5% CO2 (95% air). The mCherry-H2B-H1299 cell line specifically was
treated with 1ug/ul (Geneticin) G418 to maintain the selection of fluorescent mCherry –
H2B-H1299 over H1299.
G0/G1 and S phase cell synchronization
The final condition I developed to arrest H1299 cells in G0/G1 was to first plate
700 000 cells in 10mm dishes and serum starve cells in 0.01% Fetal Bovine Serum over 4
hours. To synchronize cells in S phase H1299 cells were incubated in (2mM)
hydroxyurea (HU) over 24 hours.
Release of Synchronized Cells
Medium was removed from each of the 6 wells along with any floating cells and
placed into sterile Eppendorf vials. Samples were centrifuged at 1200 rpm and the media
was aspirated. Any remaining pellet was resuspended in 4ml of 20% FBS media.
Adenovirus Vectors
Adenovirus vectors lacking the viral E1 region whilst expressing single gene
products such as E4orf4 or the rtTA inducer (control) under the cytomegalovirus
promoter previously described in [126] were used. The control vector used was AdrtTA,
expressing the rtTA inducer, while E4orf4 was overexpressed using AdE4orf4
(expressing hemagglutinin [HA]-tagged adenovirus type 2 [Ad2] E4orf4 protein). Initial
stocks of these viral vectors were amplified in 293 cells, and titered using both plaque
formation assays and fluorescence formation assays.
Neera Sriskandarajah
38
Infection
In all experiments, old media was removed and cells were infected for 1h at a
multiplicity of infection (MOI) of 50 PFU per cell in a low volume of the serum-free
culture medium. Under normal cell conditions, H1299 cells were infected at a stage of
80% confluence. For cell synchronization followed by infection, cells were infected at 24
and 48 hours, for G0/G1 and S phase respectively. After 1 hour of infection the serum free
media was replaced with either 10% serum complimented media, or 0.01% serum for
normal conditions and S-phase arrest as well as G0/G1 phase arrest respectively. S phase
arrested cells post infection were then retreated with 2mM HU to prolong arrest post
infection.
Cell Cycle Analysis by Flow Cytometry
Adherent and non-adherent H1299 cells grown in 6 well plates were harvested
using low volumes (200ul) of trypsin. Cells were washed twice with phosphate-buffered
saline (PBS) and pelleted by centrifugation at 1200 rpm for 5 minutes. Cells were then
resuspended in 500ul of PBS and under a vortex, the samples were fixed and
permeabilized by the slow addition of ice-cold 95% ethanol. Samples were either stored
at -20°C until cell cycle analysis was performed or immediately pelleted by
centrifugation at 1200rpm. Cells were then resuspended in 500ul of PBS and treated with
RNAse A (400 _g/ml, RNA 675.50; Bioshop Canada) for 40 minutes at 37°C. Lastly
cells were treated with Propidium Iodide (PI) 1ul (50mg/ml, PPI777; Bioshop Canada),
or 4’,6-diamidino-2-phenylindole (DAPI) kept on ice (4°C) in the dark until data
acquisition by either model: LSRII (Becton Dickinson Inc.), Guava technologies
(Millipore), or Cell Lab Quanta Sc Flow cytometer(Beckman Coulter). Results from the
first two cytometers were analyzed by FlowJo. For each sample, 20,000 events were
collected and measured for relative size (FSC) and granularity (SSC), and aggregated
cells were gated out. Acquisition of data from the Beckman coulter was analysed in
conjunction with Quanta collection and Quanta analysis software (Beckman Coulter).
For 2n/4n and live/dead sort experiments, sorted cells were further analyzed for
cell cycle DNA content as described in the previous section except that the cytoplasm
Neera Sriskandarajah
39
was first removed by incubation with Vindalov’s buffer consisting of 10 mM Tris base,
10 mM NaCl, and 0.1% Nonidet P-40 described in reference (41). This was performed in
order read the DNA content specifically of the nuclei passing through the flow cytometer
and to minimize debris post sorting.
BrdU assay
To evaluate the incorporation of BrdU and establish the proportion of G0/G1
synchronized cells that have been released into S-phase, cells were pulse-labeled with 10
uM BrdU (BRU222.250; Bioshop Canada) for 40 min. Cells were fixed for 24 hours, and
washed in PBS containing 0.5% BSA. DNA denaturation was performed with the
addition of 1.5M HCl for 20 min at room temperature, and neutralized by adding 0.1 M
sodium borate for 4 min. Cells were then centrifuged and washed, and fluorescein
isothiocyanate (FITC)-conjugated anti-BrdU antibody was added for 1 h at room
temperature. DNA was stained with propidium iodide (PI) before analysis by flow
cytometry.
Time Lapse Microscopy
To study the cellular morphology of H1299 cells overexpressing E4orf4,
mCherry-H2B H1299 cells were synchronized in either G0/G1 or S phase and infected
with AdE4orf4. Cells were then released as described earlier with the addition of 20% v/v
Fetal Bovine Serum (HyClone). Time lapse microscopy was performed using AxioVision
3 Microscope equipped with Axiocam HR (Zeiss, Thornwood, NY) Digital camera.
Pictures were taken at 15 minute intervals over 24 hours. Photos were taken using both
phase contrast (to observe whole cell morphology) and fluorescence microscopy
(looking at Histone 2B and hence the nuclei of the cells) at 10X magnification. S phase
arrested cells were released and photos were taken immediately after the addition of new
medium. G0/G1 arrested cells were released for 18 hours to provide sufficient time for cell
attachment prior to photo acquisition for another 24 hours from this time point. Analysis
of the time-lapse images was performed using Image J and Adobe Illustrator.
Neera Sriskandarajah
40
Cell Sorting
For the 2n/4n experiments, Vybrant DyeCycle violet stain (V35003; Invitrogen
Molecular Probes) was used to stain the DNA of live H1299 cells overexpressing control
vector AdrtTA and AdE4orf4. Samples were harvested using trypsin, rinsed with PBS
and pelleted by centrifugation at 1200rpm. Pellets were each resuspended in 100ul of
PBS and treated with the Vybrant DyeCycle violet stain at 10 uM for 30 minutes at 37°C.
Samples were then maintained at room temperature prior to sorting by flow cytometry.
Using a FACSAria sorter (Becton Dickinson), cells were sorted into two distinct
populations according to their DNA content (2n versus 4n).
For the live/dead sort experiments, cells were harvested as explained earlier, and
resuspended in PBS. And treated with PI without fixation. Using the same FACS Aria
sorter (Becton Dickinson), live cells and dying cells were sorted for, denoted by
PI+(dying cells) and PI- (living cells).
Neera Sriskandarajah
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III. Results
3.1 G0G1 Synchronization
A method for synchronizing human lung carcinoma H1299 cells in G0/G1 needed
to be developed in order to test E4orf4’s effect on the cell cycle linked to the induction of
cell death. Sugar, nucleotides and sufficient amino acids are required for cells to enter G1
and further progress into S phase. Thus, impeding the production or deceasing the supply
of these factors should cause the arrest of cells in G0/G1.
Low serum conditions are known to be able to arrest cells like mesenchymal stem
cells among others in G0/G1 [127]. However, I have attempted many different serum
conditions such as 1% 0.5% 0.1%, none of which appeared to work with H1299 cells;
however, 1% FBS treated H1299 cells were studied (see Figure III-1). The control cells
treated with 10% serum showed a normal cell cycle profile with approximately 64% of
the cells in G0/G1. However, H1299 cells treated with 1% FBS medium exhibited a much
lower percentage of cells in G0/G1 and little indication of arrest.
Figure III-1. G0/G1 Synchronization by 1% (low) serum conditions
Seven hundred thousand H1299 cells were seeded into 6cm plates in either 10% or 1%
FBS media for 48 hours. Cells were harvested and fixed/permeabilized with ethanol and
cell cycle analysis was performed as described in Materials and Methods.
Neera Sriskandarajah
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To achieve better cell cycle arrest several other approaches were tried
(summarized in Table 1). The next procedure was a method devised in our lab called
nutrient depleted serum starvation. This method consisted seeding 3 million H1299 cells,
plating them in 10 cm dishes, and growing them past full confluence for 5 days. The
medium was then harvested from each of the plates, and centrifuged at 1200 rpm to
remove any remaining cells. To arrest cells for experiments, cells were plated in the
overgrown media (Figure III-2). Unfortunately this method in two experiments gave
variable results, yielding 73% and 81% G0/G1 populations. This difference perhaps
occurred because of variations from experiment to experiment in the level of nutrient
depletion. Thus although this method was somewhat effective I sought a more consistent
protocol to achieve G0/G1 arrest.
Figure III-2. G0/G1 Synchronization by Nutrient depleted serum starvation
H1299 cells were treated with either 10% FBS media (control) or with two batches of
nutrient depleted serum media (protocol described earlier), over 72 hours to arrest cells in
G0/G1. The second and third panels show a variation in G0/G1 arrest.
Neera Sriskandarajah
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Next amino acid free (aa-) medium was tested to arrest the cells. However, with
this approach most cells died as shown by the significant loss in cell numbers compared
to the controls. In addition it was very difficult to obtain a clear cell cycle profile from the
H1299 cells treated with aa- media. In an attempt to improve the arrest, reduce cell death,
and achieve a better profile, 1% and 10% serum was added to the aa- free media and the
results are shown in Figure III-3. The results showed that treating H1299 cells with aamedia regardless of serum level did not induce sufficient arrest of the cells in G0/G1.
Figure III-3. G0/G1 Synchronization by Amino Acid (aa) free media+/- serum
H1299 cells treated with either 10% FBS serum (control), aa- media, aa- media
complimented with 1% FBS or aa- media complimented with 10% FBS. Cells treated
with aa- media show a decrease in the amount of cells in G0/G1 (43.3%), and an even
larger decrease with the addition of serum (33%).
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In a further attempt to arrest cells in G0/G1, I returned to the original idea of low
serum, however, I tried 0.01% serum. Figure III-4 shows the G0/G1 arrest obtained by
seeding 1 million H1299 cells in 6 cm plates in 0.01% FBS media. Using this method,
91.42 % of the cells were arrested in G0/G1 with the majority of the remaining cells
(7.21%) in SubG0.
Figure III-4: H1299 G0/G1 synchronization using 0.01% FBS media.
One million cells were seeded in 6cm dishes and incubated with 0.01% serum media over
48 hours. This resulted in a 91% G0/G1 synchronization.
Neera Sriskandarajah
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To optimize this condition further I varied the seeding amounts of H1299 for both
48 and 72 hours to find the best conditions for G0G1 synchronization.
Figure III-5: Varying seeding amounts with 0.01% serum
Five, seven and nine hundred thousand H1299 cells were seeded into 6cm dishes, and
incubated in 0.01% FBS media for either 48 hours or 72 hours. Cell cycle analysis was
then performed post harvesting, ethanol fixation/permeabilization, and treatment with
propidium iodide.
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It was found that seeding 700 000 cells and incubating them in 0.01% FBS
medium provided the best condition for the highest G0/G1 synchronization with minimal
cell death. For example, at 48 hours, cells had a 73.2 % G0/G1 arrest with 4.4% of the
cells in Sub G0, whereas at 72 hours 81% arrest with 6.47 % SubG0 cells. For future
experiments this seeding amount and either 48 hours or 72 hours were employed.
The following table summarizes some of the methods attempted to achieve a strong
G0/G1 H1299 cell synchronization.
Table 1: Methods employed to synchronize H1299 cells in G0/G1.
Method
1% Serum
Nutrient depleted
serum starvation
Amino acid free
media
Amino acid free
media +low serum
(1%)
0.01% Serum
Neera Sriskandarajah
%Sub G0
0.09
0
%G0/G1
47.46
80.8
%S
31.7
8.9
%G2M
18.1
9.6
0
43.3
9.5
6.3
0
33.6
15.7
12.5
7.21
91.42
1.22
0.15
47
3.2. G0/G1 Cell Synchronization and Release
Once the conditions for G0/G1.H1299 cell synchronization were obtained, I
determined if the arrested cells were able to be released as a synchronized population. In
the first panel (left) of Figure III-6, control (untreated) H1299 cells demonstrated a
regular cell cycle profile. In panel 2 (center), H1299 cells exhibited a good arrest in
0.01% medium, for 72 hours and in panel 3 (right) 15 hours following addition of 20%
serum a well synchronized population of cells in S phase indicating a good response to
serum addition.
Figure III-6: G0/G1 Cell Synchronization and Release
Control (untreated) H1299 cells were grown for 48 hours in 10% FBS media. Cells were
arrested with 0.01% FBS media for 72 hours. Following the addition of 20% FBS
medium, cells were grown for 15 additional hours. All samples were harvested, fixed,
permeabilized and treated with RNAse A as well as propidium iodide (PI), as described
in Materials and Methods for cell cycle analysis.
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To further confirm the release of cells from G1 into S phase a BrDU assay was
performed. BrDU is a thymidine analog that is incorporated into newly synthesized DNA
during S phase in replicating cells. Five cell samples were examined including: control
(untreated), G1S arrested (Hydroxyurea (HU) treated), G0/G1 arrested, as well as G0/G1
arrested and released for either 18 hours and 24 hours, and further incubated with BrdU
for 40 min. Cells then were subjected to acid DNA denaturation, treated with anti-BrdU
antibody tagged with fluorescein, as well as propidium iodide for flow cytometry
analysis. Figure III-9 shows that the BrDU incorporation as demonstrated by the 2dimentional horseshoe plot was very minimal (as expected) for both the G0/G1 arrest and
G1S arrest with 1.3 and 4.5 % of the cells in S phase respectively. Once G0/G1
synchronized cells were released (with 20% FBS media), there was a significant rise in
the population of cells in S phase, at approximately 18 hours, to 13.1%. Finally by 24
hours of release from G0/G1 synchronization, the cohort of cells in each phase of the cell
cycle looked similar to the control, with 29.1% of control cells in S phase and 28.1% in
cells that have been released 24 hours post G0/G1 arrest. Therefore the system I created to
synchronize H1229 in G0/G1 with 0.01% FBS media worked effectively both in terms of
arrest and cell synchronization. Thus this procedure can be employed for further studies
of the effects of E4orf4 on the cell cycle.
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Neera Sriskandarajah
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Figure III-7: Validation of G0/G1 release by a BrdU assay
Evaluation of the BrdU-incorporation into S-phase cells. Cells were arrested in
G1S (control), and G0/G1, and further released by the addition of 20% FBS media. Cells
were harvested and incubated with 10M BrdU for 40 min and further fixed in 90%
methanol. Partial DNA denaturation was performed with a low concentration of HCl. The
output of BrdU incorporated into the DNA, was observed by a Fluorescein-conjugated
anti-BrdU antibody. In addition, PI was added for the DNA content determination. Twodimensional flow cytometry results are presented with BrdU-positive population and the
respective cell cycle encircled boxes with their corresponding percentages beside them.
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3.3. Time-Lapse Microscopy of H1299 cells expressing E4orf4
In order to observe the morphological changes as well as alterations of the cell
cycle as a result of high level E4orf4 expression in H1299 cells, time-lapse microscopy
was performed. Specifically, mCherry-H2B H1299 cells, which are red-fluorescent for
histone H2B were employed to monitor the nuclear state of DNA during the cell cycle, by
fluorescence microscopy. At precisely the same time points as the fluorescent photos, the
cellular morphology was observed by phase contrast. To thoroughly study the effects of
E4orf4 on the cell cycle, six videos were made over the course of 18 hours post release
from three different conditions: asynchronously growing cells; G0/G1 arrested cells
following an 18 hour release in 20% v/v FBS medium; and G1/S arrested cells following
removal of HU (Supplemental video 1 and 2).
First, in the case of asynchronously growing cells, mCherry-H2B H1299 cells
were seeded onto 6 well plates with 10% FBS media. Cells were infected with either the
control vector AdrtTA or AdE4orf4 vector expressing E4orf4, for 18 hours. Fresh
medium (20% serum v/v) was then added to the cells at the zero time point, and a series
of photos were taken over the course of 18 hours. The results of the first two videos are
shown in Figure III-8, in which cells were blindly chosen, tracked, and assessed for M1:
timing to round up, M2: rounded 2 nuclei, M3: maintenance of cell rounding, cytokinesis,
failure of cytokinesis, time to re-adhere to the plate, as well as failure to enter mitosis.
Method of time lapse analysis adapted from Gascoigne, et al, 2008 [128].
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Figure III-8: Cell cycle fate of Asynchronous mCherry-H2B H1299 cells in the
presence of E4orf4
Graphical representation of time-lapse microscopy of asynchronous mCherryH2B H1299 cells +/- E4orf4. In each case 50 cells were selected at random and tracked
individually.
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H1299 cells expressing the control rtTA (left) demonstrated a quick transit
through mitosis. In contrast, H1299 cells expressing E4orf4 as shown by the right panel
exhibited increased cell rounding, and a higher percentage of cells exhibited a failure in
cytokinesis, rendering a rounded ‘two nuclei’ phenotype which may represent the G1
tetraploid population of cells described previously. In addition, E4orf4 expressing cells
that were able to complete cytokinesis successfully took a prolonged time to re-adhere to
the plate and enter a new round of the cell cycle. Although at an MOI of 50 most cells
should be infected with AdE4orf4 and express E4orf4, there were a few cells that
appeared to undergo regular transit through mitosis. However, it may be that these few
cells were uninfected or poorly expressed E4orf4.
Second, the effect of E4orf4 expression on the release of G0/G1 arrested cells by
addition of serum was studied. Cells were arrested in G0/G1 using 0.01% serum as
described earlier, infected for the last 18 hours of the arrest, and released with 20% FBS
serum. To assess this process further I first examined the morphology of these cells. It
was clear that from the moment of serum addition that the cells aggregated and looked
abnormal. However, over time the cells slowly started to adhere to the plate and began
growing as shown in Figure III-9. At time 0 when fresh medium was added, cells infected
with either control vector or vector expressing E4orf4 both exhibited some aggregation.
However, by 8 hours the cells started to adhere and by 18 hours most cells had settled
onto the plate. Thus an analysis of individual cells of these cultures by time-lapse
photography was performed as above (Figure III-10).
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Figure III-9: Aggregation of G0/G1 arrested mCherry-H2B H1299 cells
H1299 cells arrested in G0/G1 by 0.01% serum, and infected with AdrtTA or
AdE4orf4. Under phase contrast, photos were taken at 15 minute intervals for 24 hours.
The aggregated arrested cells begin slowly adhering to the plate.
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Figure III-10: Cell cycle fate of mCherry-H2B H1299 cells post G0/G1
Synchronization and 18 Hour release in the presence of E4orf4
Graphical representation of time-lapse microscopy of G0/G1 arrested mCherry-H2B
H1299 cells +/- E4orf4. In each case 50 cells were selected at random and tracked
individually.
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Cells expressing the control vector seemed to progress very quickly through
mitosis. A reasonable range of time in M1 or time to round up was evident, which we
define here as the beginning of mitosis. This variation may be perhaps because of
differences in the arrest in G0/G1 such that some cells were in G0 and some in G1. In any
case, G0/G1 arrested cells expressing E4orf4 exhibited a significant number of single cells
that were rounded up and unable to initiate DNA synthesis. There were a few cells that
underwent DNA replication but failed to undergo cytokinesis. This result obtained by
time-lapse, confirmed the flow cytometry studies that our group had recently published
(Cabon, Sriskandarajah et al, 2013) [125].
Finally, time-lapse microscopy was then performed on mCherry-H2B H1299 cells
following G1/S arrest with hydroxyurea (HU) and infection with AdrtTA control or
AdE4orf4. Figure III-10 shows that, cells expressing the control rtTA showed a rapid
transit through mitosis, with some variation in the time to round up (Red: begin mitosis).
Cells expressing E4orf4 at S phase however, contained a large proportion that either
remained 4N with two rounded up nuclei, delayed cytokinesis, or even 4N cells that
appeared to fail to complete cytokinesis and regressed into a single tetraploid cells that
re-adhered to the plate. Once again, these observations confirmed previous flow
cytometric results that suggested that G1/S phase arrested cells expressing E4orf4 could
complete DNA synthesis, but exhibited complications in completing cytokinesis, such
that there is a significant population of cells that remained tetraploid.
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Figure III-11: Cell cycle fate of mCherry-H2B H1299 cells post G1/S
Synchronization by Hydroxyurea (HU) in the presence of E4orf4
Graphical representation of time-lapse microscopy of G1/S arrested mCherry-H2B H1299
cells +/- E4orf4. In each case 55 cells were selected at random and tracked individually.
Thus all the video profiles demonstrated that cells expressing E4orf4 exhibited
Neera Sriskandarajah
58
increased mobility, and rounding up (Supplemental videos 2, 4, 6 ). The following table
summarizes some of the findings from the time-lapse videos (Supplemental videos 1 to
6). It is important to note that there is up to a 15 minute time variable for each point.
Table 2: Comparison of E4orf4 expressing mCherry-H2B H1299 cell mitosis transit
compared to vector control AdrtTA
Average Time in Mitosis
Condition:
Asynchronous
G0/G1 synchronization
G1/S synchronization
Range: Time in Mitosis [min, max]
Condition:
Asynchronous
G0/G1 synchronization
G1/S synchronization
% of E4orf4 expressing cells within range of
Control [min, max]
Condition:
Asynchronous
G0/G1 synchronization
G1/S synchronization
Ratio of Transit Through Mitosis
Condition:
Asynchronous
G0/G1 synchronization
G1/S synchronization
% Cells that Cytokinese
Condition:
Asynchronous
G0/G1 synchronization
G1/S synchronization
AdrtTA
AdE4orf4
87.61 min
114.6 min
186.3 min
843.9 min
1042.3 min
628.6 min
[30,120 ] min
[90,150] min
[105, 1050* ] min
N/A
N/A
N/A
N/A
N/A
N/A
2%
0%
100%**
1
1
1
9.63X Slower
9.09 X Slower
3.37 X Slower
88% (44/50)
100%(50/50)
94% (47/50)
10% (5/50)
4% (2/50)
16% (8/50)
*1050 is a maximum value, in this case because there were a few cells in the control
(rtTA) G1/S synchronization condition that remain rounded, mitotic, and lifted for the
whole course of the experiment. Otherwise the maximum would be 165 min and the % of
E4orf4 cells within the range of control for G1/S condition would then be 16% (8/50)**.
Cells expressing E4orf4 in any of the three conditions showed a significant
Neera Sriskandarajah
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increase in average time in mitosis. This effect is depicted as well by the ratio of transit
through mitosis, with E4orf4 expressing cells being between 3 and 10 times slower than
the control rtTA expressing cells. Only between 0 and 16% of E4orf4 expressing cells are
within the range of the control minimum and maximum time in mitosis. In addition,
E4orf4 expressing cells showed a clear failure in cytokinesis with respect to each
condition ranging from 4 to 16% of cytokinesing cells compared to controls, which range
from 88 to 100%. Therefore cells expressing E4orf4 exhibited an aberrant delay in
mitosis as well as a delay/failure in cytokinesis.
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3.4. E4orf4 induces cell death at both G1 diploid and G1 tetraploid states.
In our previous studies it was not clear if cells were killed by E4orf4 only when
they were induced to form G1 tetraploids or if in fact both 2N and 4N populations
arrested in G1 went on to die. To determine the fate of G1-arrested diploid and tetraploid
cells expressing E4orf4, two types of experiments were performed. In the first, H1299
cells were infected with either the control vector AdrtTA or vector expressing E4orf4,
AdE4orf4 for 3 days. At the 72hpi time point cells were harvested and treated with
propidium iodide without fixation and further separated by FACS. Propidium Iodide is a
DNA stain that is cell permeable only when cells are fixed or if the cells have started to
die. Thus this method was employed to sort specifically dying cells that were not yet at
the stage of full DNA fragmentation DNA. The top panels of Figure III-13A demonstrate
two cohort of cells classified as PI+ (dying) and PI- (living). The control AdrtTA infected
cells showed a 5.9% uptake of dye, whereas cells infected with AdE4orf4 had a
significantly increased percentage of 43.8% of cells that were PI+.
Nuclei from these dying cells were then analyzed by flow cytometry to determine
DNA content. The bottom two panels in Figure III-13A show that the dying nuclei from
AdrtTA-infected control cultures were largely 2n in DNA content (85.7%), with a small
amount of 4n nuclei (3.8%). The sorted PI + dying cells from the AdE4orf4 infected
H1229 cohort of cells, showed a larger proportion of 4n nuclei (10.8%) compared to the
control and a decreased percentage of 2n nuclei with 38.6% in keeping with the
accumulation of both G1 diploid and G1 tetraploid cells. These results suggested that both
2n and 4n cells probably die following expression of E4orf4.
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Figure III-12A: Analysis of E4orf4 induced cell death in 2n and 4n cell populations.
H1299 cells were infected with either AdrtTA or AdE4orf4 vectors for 72 h and sorted
for the uptake of PI (dead cells; top panels). The nuclei of these cells were then analyzed
by flow cytometry for DNA content (bottom panels).
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To confirm that both 2n and 4n cells die following expression of E4orf4 a second
approach was taken. Cells were again infected with either the control vector AdrtTA, or
vector expressing E4orf4 (AdE4orf4). At the 48hpi time point, unfixed live cells were
stained with Vybrant DyeCycle violet stain and subjected to separation by FACS to
isolate 2n and 4n cell populations. The sorted cells were then grown separately in culture
for an additional 48 hours, after which the unfixed cells were stained with propidium
iodide in order to determine the populations of dead/dying cells, given by high PI stain
shown in Figure III-13B. It was observed that AdrtTA-infected control cells, shared
similar levels of dead cells in both 2n and 4n recultured cells (14.2% and 11.9%,
respectively). Such was also the case with AdE4orf4- infected cells (18.4% and 19.7%,
respectively). Thus, these results also indicated that both G1 diploid and tetraploid cells
undergo E4orf4-induced cell death.
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Figure III-12B: Analysis of E4orf4 induced cell death in 2n and 4n cell populations.
H1299 cells were again infected with either AdrtTA or AdE4orf4 vectors for 48 h and
sorted for 2n and 4n live cell populations using the Vybrant DyeCycle violet stain. Cells
were then returned to culture for an additional 48 h and further analyzed for live/dead cell
populations.
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IV. Discussion
4.1. Development of a novel method of G0/G1 synchronization in p53-/- H1299
Human Lung Carcinoma cells.
The G1 phase restriction point of the mammalian cell cycle is the oldest,
recognized, and widely accepted control point regulating division cycle in mammalian
cells [129]. It is widely known that most cancer cells exhibit reduced sensitivity to G0
arrest. Cell lines such as HEK293 and U2OS cells may be G0/G1 arrested in DMEM
medium without FBS for 48 to 72 hours [130]. Interestingly, cells derived from the lung
appear to have a more difficult time arresting in G0/G1 using serum deprivation, as for
example, pulmonary microvascular endothelial cells (PMVEC’s) [131] and p53-/- human
lung carcinoma cells (H1299’s).
However, in this thesis I have developed a protocol for H1299 cells using 0.01%
FBS media over 48 to 72 hours, which can yield up to 90% G0/G1 synchronization. When
seeding the cells, the cells must be washed with PBS to remove any additional serum that
may increase the percentage of serum in the media. This low percentage is the right
critical balance between G0/G1 arrest and minimization of cell death often seen with full
serum deprivation. Although the phenotype of the cells appeared aggregated and
senescent, I have shown by flow cytometry, cell cycle analysis as well as time-lapse
microscopy that H1299 cells arrested in G0/G1 may be released in a synchronized manner
with the addition of 20% v/v FBS media.
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4.2. Cellular morphology of E4orf4 expressing cells
Although many studies with respect to cell death induced by E4orf4 have been
carried out throughout the years, the cellular morphological changes and cell cycle
patterns induced by E4orf4 had yet to be observed in real-time. Through synchronization
experiments and cell cycle analysis by flow cytometry, expression of E4orf4 at the G1/S
boundary resulted in G1 tetraploid (4n) cells as cells in G1/S had passed the restriction
point and continued to replicate DNA but exhibited a defect in mitosis/cytokinesis.
Expression of E4orf4 at G0/G1 resulted in G1 diploid (2n) cells that failed to initiate DNA
synthesis.
By comparison using time-lapse microscopy, mCherry-H2B H1299 expressing
E4orf4 at G0G1 and following release demonstrated a failure of the nuclei to replicate and
split into two daughter cells. When E4orf4 was introduced into cells at the G1/S boundary
and then released, cells continued DNA synthesis and remain G1 tetraploid as seen by the
replicated 4n cell forming a cleavage furrow, furrow retraction, and failure of cytokinesis
during the time-lapse (Supplementary Figure 6).
In addition to confirming the cell cycle effects of E4orf4 depicted in the flow
cytometry results (Cabon, Sriskandarajah et al. 2013) [125], E4orf4 expressing cells
showed an overall increased mobility, and a major delay in mitosis (from approximately
4 to 10X slower compared to controls). In addition, a significant proportion of cells that
failed in cytokinesis. The increased mobility may be linked to the effects E4orf4
previously reported on the Src-pathway [77,101]. There were also some aberrant cells
that showed blebbing, which is also characterisic of E4orf4 expression [77,101].
Further studies will be needed to identify the major E4orf4 targets in the mitotic
phase of the cell cycle that may be responsible for the G1 diploid and tetraploid states and
the eventual death of the E4orf4 expressing cells. One such target discovered in previous
studies by our group in yeast had suggested that APC may be a target for E4orf4 [111]. In
addition our group has shown in yeast that E4orf4 prematurely activates the early mitotic
form APCCdc20 as early as S phase, while inhibiting the activation of late mitotic/early G1
form APCCdh1 (111). APCCdh1 is known to be involved in the regulation of DNA synthesis
through the programmed degradation of its corresponding substrates such as Cdc6, Cdt1,
and Geminin. As such, it would be of great interest to pursue studies on the relationship
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66
between E4orf4 and APCCdh1, the link with E4orf4’s inhibition of DNA synthesis as well
as E4orf4 induced cell death [132-134].
4.3. G1 arrest is the critical event leading to E4orf4 induced cell toxicity
It has been known for over a decade that the human adenovirus E4orf4 protein,
when expressed alone at high levels, effectively kills human cancer cells. There continues
to exist considerable ambiguity about the events that induce cell death. Several studies
indicated that at least some cell types die by apoptosis [91, 93,134-139]; however, in
many cases clear apoptotic hallmarks are absent [93,123,135, 137, 140]. The mechanism
of these effects still remains uncertain. Earlier work indicated that a major target
responsible for E4orf4 toxicity in both human and yeast cells is the B55/Cdc55 regulatory
subunit of PP2A [78, 81,117, 134, 136, 139, 141, 143-146). Thus effects of E4orf4 on
PP2A or some as yet unidentified target may be responsible for the mitotic effects found
here and in earlier work.
Several reports suggested that E4orf4 induces a form of apparent mitotic arrest in
both human tumor cells and in yeast [78,81, 123, 142, 144-145). Our group has suggested
that effects of E4orf4 on mitotic progression might result in mitotic catastrophe, a
phenomenon known to lead to subsequent cell death by a variety of mechanisms,
depending on of the cell type [123]. From the synchronization experiments our group has
recently published (Cabon, Sriskandarajah et al, 2013) [125], it was suggested that two
populations of cells in the G1 phase of the cell cycle accumulate; G1 diploid as well as G1
tetraploid. These two states appear to be conditional upon where in the cell cycle E4orf4
levels become sufficient to induce effects.
As such, I have further studied fate of G1-arrested diploid and tetraploid cells
expressing E4orf4, to determine whether the cell death as a result of E4orf4
overexpression is due to the accumulation of cells in mitosis and the failure of
cytokinesis, or rather the dual G1 arrested diploid and tetraploid states. By sorting first for
living and dying cells by PI staining without fixation, cells expressing E4orf4 as expected
showed more than a seven fold increase in dying cells compared to the control. Cell cycle
Neera Sriskandarajah
67
analysis of these two cohorts determined that there was a large proportion of 4n nuclei
and a decreased percentage of 2n nuclei, yet maintaining two population G1 diploid and
G1 tetraploid cells (Fig III-12A). To confirm these results, I performed another
experiment where cells infected with either control vector or vector expressing E4orf4
were sorted for 2n and 4n populations using live cell Vybrant dye cycle stain. Cells were
recultured for 48 hours and stained with PI in their unfixed state, for live/dead analysis. It
was observed that both controls and cells expressing E4orf4 shared similar levels of dead
cells in both 2n and 4n recultured cells. Thus, these results indicated that both G1 diploid
(2n) and G1 tetraploid (4n) cells undergo E4orf4-induced cell death. This suggests that it
is the arrest itself and not the formation of tetraploids that is the critical event leading to
death.
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4.4. Concluding Remarks
The work presented in this thesis provides new insights into the ongoing
investigation into the mechanism of action of the adenoviral protein E4orf4. A new useful
model for G0/G1 synchronization of p53-/- H1299 human lung carcinoma cells was
developed and employed in time-lapse microscopy. The results of this project also
demonstrated that E4orf4 expressing cells exhibit a major delay in mitosis and failure in
cytokinesis and that the induction of tumor cell death at both G1 diploid and G1 tetraploid
states. As such, E4orf4 induced cell toxicity, which if exploited, makes it an attractive
target for future novel anti-tumor therapy.
V. Supplementary Videos
5.1. Asynchronous mCherry-H2B H1299 cells infected with AdrtTA over 18 hours
5.2. Asynchronous mCherry-H2B H1299 cells infected with AdE4orf4 over 18 hours
5.3. mCherry-H2B H1299 cells post G0/G1 Synchronization infected with AdrtTA +18
Hour release over 18 hours
5.4. mCherry-H2B H1299 cells post G0/G1 Synchronization infected with AdE4orf4 +18
Hour release over 18 hours
5.5. mCherry-H2B H1299 cells post G1/S Synchronization by Hydroxyurea (HU)
infected with AdrtTA
5.6 mCherry-H2B H1299 cells post G1/S Synchronization by Hydroxyurea (HU) infected
with AdE4orf4
All videos are available at:
http://teodorolab.mcgill.ca/Movies.html
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69
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