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GSBS Dissertations and Theses Graduate School of Biomedical Sciences
2004-04-29
Pathways Linking Deregulated Proliferation to Apoptosis: a Dissertation
Harry A. Rogoff University of Massachusetts Medical School
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Repository Citation Rogoff HA. (2004). Pathways Linking Deregulated Proliferation to Apoptosis: a Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/18tf-2t22. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/63
This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. A Dissertation Presented
Harr Andre Rogoff
Submitted to the Faculty of the
University of Massachusetts Graduate School of Biomedical Sciences, Worcester
In partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
April 29, 2004
Program in Immunology and Virology COPYRIGHT NOTICE
Pars of this dissertation have appeared in the following publications:
Rogoff, H.A., Pickering, M. , Debatis, M. , Jones, S. , and T.F. Kowalik (2002). E2F1
Induces Phosphorylation of p53 that is Coincident with p53 Accumulation and Apoptosis.
Mol. Cell. BioI. 22: 5308-5318.
Rogoff, H.A. , Pickering, M. , Frame, F. , Debatis, M. , Sanchez, Y. , Jones , S. , and
F. Kowalik (2004). Apoptosis Associated with Deregulated E2F Activity is Dependent on E2F1 and AtmlbsllChk. Mol. Cel. BioI. 24: 2968-2977. Michelle Kelliher, Ph.D., Chair of Committee
Michael Brodsky, Ph.D., Member of Committee
Maria Zapp, Ph.D., Member of Committee
Stephen Doxsey, Ph.D., Member of Committee ACKNOWLEDGMENTS
I would like to thank my mentor Tim Kowalik for his guidence, support, and
friendship during my graduate career. Joining Tim s lab was possibly the best decision I
made during my graduate career and provided me the best possible place to grow as a
scientist. I would also like to thank the members of my committee, Michelle Kelliher
Stephen Doxsey, Maria Zapp, Michael Brodsky, and Karl Munger for their thoughtful
advice on my research projects. I am also grateful to the members of the Kowalik lab for
ther contributions. Fiona Frame has been a scientific colleague and, most of all, a good
friend. Fiona contributed Chapter V Figure 1B and Chapter V Figure 8. Michelle Debatis
contributed Chapter III Figure 5. Mary Pickering contributed Chapter III Figure 4 and
Chapter III Figure 12A. I would also like to thank Brad Stadler and former lab member
Jon Castilo for their helpful scientific advice, for making the Kowalik lab an enjoyable place to work, and for their friendships outside of science.
I would like to thank my parents, Martin and Linda Rogoff, for their support
during my years in graduate school. I would also like to thank the four legged members
of the Rogoff household; Andy, Bada, Marley, Teddy Bear, and Chloe for bringing love
and relaxation to my homelife. Most of all, thank you to my wife Tina, who has provided daily love and encouragment throughout my graduate career. ABSTRACT
Proper regulation of cellular proliferation is critical for normal development and
cancer prevention. Most, if not all, cancers contain mutations in the Rb/E2F pathway,
which controls cellular proliferation. Inactivation of the retinoblastoma protein (Rb) can
occur through Rb loss, mutation, or inactivation by cellular or viral oncoproteins leading
to unrestrained proliferation. This occurs primarily by de-repression and activation of the
E2F transcription factors, which promote the transition of cells from the G to S phase of
the cell cycle. In order to protect against loss of growth control, the p53 tumor suppressor
is able to induce programmed cell death, or apoptosis, in response to loss of proper Rb cell cycle regulation.
E2F! serves as the primary link between the Rb growth control pathway and the p53 apoptosis pathway. While the pathway(s) linking E2F! to p53 activation and apoptosis are unclear, it has been proposed that E2F! activates p53-dependent apoptosis
RF by transactivation ofpI leading to inhibition of Mdm2-promoted degradation ofp53.
We tested this hypothesis RF , and found that pI is not required for E2F!-induced
apoptosis. Instead , we find that expression of E2F I leads to covalent modifications of p53 that correlate with p53 activation and are required for apoptosis.
The observation that E2F! induces covalent modification ofp53 is consistent with the p53 modifications observed following DNA damage. We therefore hypothesized that
E2F! may be activating components of the DNA damage response to activate p53 and kill cells. Consistent with the DNA damage response, we find that E2FI-induced apoptosis is compromised in cells from patients with the related disorders ataxia
telangiectasia and Nijmegen breakage syndrome, lacking functional Atm and Nbsl gene
products, respectively. E2FI-induced apoptosis and p53 modification also requires the
human checkpoint kinase Chk2, another component of the DNA damage response. We
find that the commitment step in E2F I- induced apoptosis is the induction of Chk2.
Having found that E2FI requires DNA damage kinases to induce apoptosis, we
next examined events upstream of kinase activation. To this end, we observe relocalization of the DNA damage repair MRN complex (composed of Mrel I , NbsI , and
Rad50) to nuclear foci specifically following expression of E2F!. Expression of E2FI also induces relocalization of the DNA damage recognition proteins yH2AX and 53BPI
to nuclear foci, consistent with the location of these complexes observed following DNA
double strand breaks. As a consequence of activating some or all of these DNA damage
signaling proteins, expression of E2F I blocks cell cycle progression in diploid human
fibroblasts. The observed block in cell cycle progression is found to be , in part, due to activation of a p2I-dependent cell cycle checkpoint.
The E7 protein from the oncogenic human papilomavirus (HPV) is able to bind to and inactivate members of the Rb family. HPV infects quiescent, non-cycling cells that lack expression of DNA replication machinery that is essential for replication of the viral genome. By expression of the E7 protein, HPV is able to bypass normal Rb-mediated growth control and induce quiescent cells to enter S phase where the host cell DNA replication enzymes are present for viral replication. We find that expression of E7 can also result in apoptosis that is dependent specifically on E2F!. Additionally, E7-induced V11
apoptosis, like E2FI-induced apoptosis, requires Atm, NbsI , and Chk2. Expression of
, like that of E2FI , induces E2Fl-dependent covalent modification of p53 that correlates with apoptosis induction.
These findings demonstrate that deregulation of the Rb/E2F growth control pathway leads to activation of an apoptosis program with some similarity to the pathways activated by DNA damage. Our observations suggest that E2FI not only functions as sensor for deregulation of Rb, but may also play an important role in regulating cellular growth control in response to other oncogenic stimuli......
V11
T ABLE OF CONTENTS
COPYRIGHT NOTICE......
ACKNOWLEDGEMENTS......
ABSTRACT...... ,...... v
TABLE OF CONTENTS...... viii
LIST OF FIGURES...... ix
CHAPTER I. Introduction...... I
CHAPTER II. Materials and Methods......
CHAPTER III. E2FI induces phosphorylation of p53 that is coincident with
p53 accumulation and apoptosis...... 44
CHAPTER IV. Apoptosis associated with deregulated E2F activity is
dependent on E2F I and Atmlbs I /Chk ...... 71
CHAPTER V. E2F I induces Mre II foci formation and blocks cell cycle
pro gression in human fi bro blasts...... II 0
CHAPTER VI. Discussion ...... 138
CHAPTER VII. Appendix A...... 156
CHAPTER VIII. References...... 161 ......
LIST OF FIGURES
Figure 1. 1. Cyclin/Cdk regulation of the cell cycle ...... 5
Figure 1.2. Rb/E2F regulation of the cell cycle...... 7
Figure 1.3. Schematic representation of the E2F family...... 10
Figure 1.4. Models of Rb/E2F regulation ofE2F regulated promoters ...... 15
Figure 1.5 Model of p5 3 activation following cellular stress...... 26
Figure 1.6. Model of DNA double strand break signaling ...... 31 AR protein accumulation...... Figure 3. 1. E2FI induces p I9
Figure 3.2. E2FI induces apoptosis in the absence of pI 9ARF AR ...... Figure 3.3. E2FI induces p53-dependent apoptosis in the absence of pI 9
Figure 3.4. E2FI reqires pI9ARF to induce p53 accumulation...... AR and Mdm2 to increase p53 protein levels...... 53 Figure 3.5. E2FI requires pI9
Figure 3.6. Expression ofE2FI or E2F2 results in an increase in p53 protein
levels and levels ofthe phospho-serine 18 form of p53 in
wildtype MEFs......
Figure 3.7. E2FI specifically induces accumulation ofp53 and the phospho- AR ...... 57 serine 18 form ofp53 in cells lacking pI9
Figure 3.8. E2F2 does not induce apoptosis in wildtype MEFs ...... AR protein accumulation...... 60 Figure 3.9. E2F I and E2F2 induce p I9 AR to induce p53 protein and alter levels of the Figure 3. 10. E2F2 requires pI9
phospho-serine 15 form of p53 ...... "'''''' ......
Figure 3. 11. E2FI induces p53 protein and levels of the phospho-serine 15
and phospho-serine 20 forms of p5 3 in cells lacking p I9AR ...... 63 Figure 3.12. p53 phosphorylation correlates with E2FI- induced apoptosis ......
Figure 3. 13. Caffeine inhibits E2FI-induced apoptosis ...... 66 Figure 3.14. Model ofE2FI-induced apoptosis and p53 accumulation...... 69
Figure 4. 1. E2F I-induced apoptosis is reduced in human fibroblasts lacking
functional Atm or Nbsl ...... 74
Figure 4.2. E2FI-induced apoptosis is reduced in mouse embryo fibroblasts
lacking atm......
Figure 4.3. E2FI and E2F2 require Atm and Nbsl to efficiently induce
phosphorylation of p53...... 76
Figure 4.4. AdE2FI and AdE2F2 are expressed to similar levels in normal
, and NBS fibroblasts...... """'" ""'"'' ...... 78 Figure 4.5. Increased doses of AdE2F2 does not lead to apoptosis...... 79 Figure 4.6. Increased doses of AdE2F2 leads to increased levels of phospho-
serine 15 p53 but not phospho-serine 20 p53
Figure 4.7. Dominant negative Chk2, but not dominant negative ChkI reduces
E2F I-induced apoptosis ...... 82
Figure 4. 8. siRNA targeting ofChk inhibits E2FI-induced apoptosis ...... 83
Figure 4. 9. DN-Chk, but not DN-ChkI , inhibits phosphorylation ofp53 at
serine 20...... 85 Figure 4. 10. siRNA targeting ofChk inhibits E2FI-induced phosphorylation
of p53 at serine 20......
Figure 4.11. Expression ofE2FI , not E2F2, leads to increased Chk2 protein
levels in normal, AT, and NBS cells...... 87
Figure 4. 12. Expression of E2F Ie13 fails to induce apoptosis, increase Chk
protein levels or induce phosphorylation of p53 ...... """'"'''''' 88 Figure 4. 13. Expression ofE2FI or E2F21eads to an increase in the phospho-
serine 1981 form of Atm while leaving total Atm protein levels
unchanged...... 90
Figure 4. 14. E2FI , leads to an increase in , not E2F2 Chk2 mRNA levels but
not Atm mRNA levels...... 91
Figure 4. 15. E2FI and E2F2 cooperate with Chk in apoptosis induction...... 93
Figure 4. 16. Chk2 cooperates with E2F2 to induce phosphorylation of p53 at
serine 20...... 94
Figure 4. 17. HPV- I6 E7 induces apoptosis in human fibroblasts ...... 96
Figure 4. 18. HPV- I6 E7-induced apoptosis is reduced in cells lacking
functional Atm or Nbsl ......
Figure 4. 19. siRNA targeting ofE2FI or Chk inhibits E7-induced apoptosis ...... 98 Figure 4.20. siRNA knockdown of E2F2 and E2F3 ...... 100
Figure 4.21. HPV- I6 E7 requires E2FI but not E2F2 or E2F3 to induce
apoptosis ...... 101 X11
Figure 4.22. HPV- I6 E7 expression results in p53 accumulation and
modification, similar to those observed following expression of
E2F I ...... 102
Figure 4.23. HPV- I6 E7 expression results in an increase in Chk2 protein
levels and levels of the phospho-serine 1981 form of Atm
while leaving total Atm protein levels unchanged...... 104
Figure 4.24. HPV- I6 E7 requires E2FI and Chk to increase the levels of the
phospho-serine 20 form ofp53 ...... 105
Figure 4.25. HPV- I6 E7 requires E2FI to induce Chk2...... 106
Figure 4.26. Model ofp53 activation following deregulation ofE2FI ...... 108
Figure 5. 1. E2F I specifically induces Mre II foci formation...... 113
Figure 5.2. Expression ofE2FI , but not E2F3a, results in an increase in the
phospho-serine 20 form ofp53 ...... 114
Figure 5.3. E2FI , but not E2F3a, induces apoptosis in human fibroblasts ...... 115
Figure 5.4. E2FI fails to induce MreII foci in NBS cells ...... 116
Figure 5. 5. E2F I induces Mre II foci in AT cells...... 118
Figure 5.6. E2FI induces relocalization of DNA damage response proteins...... 119
Figure 5.7. Expression ofE2FI induces MreII foci that do not correlate with
BrdU incorporation...... 120
Figure 5. 8. E2FIl-283 induces MreII foci formation similar to full length E2FI ...... 121
Figure 5.9. E2FIe13 induces MreII foci formation similar to E2FI ...... 122
Figure 5. 10. E2FI , but not E2F3a, blocks cell cycle progression...... 124 ......
X11
Figure 5. 11. Expression of E2F I and E2F3a results in increased levels of the
phospho-serine 1981 form of Atm 125
Figure 5. 12. Roles for Nbsl and 53BPI in blocking cell cycle progression...... 127
Figure 5. 13. Expression of E2F I leads to accumulation ofp2I protein ...... 128
Figure 5. 14. Role for p2I in blocking cell cycle progression...... 129
Figure 5. 15. Role for p53 in blocking cell cycle progression and p2I
accumulation...... 130
Figure 5. 16. Reduced levels of 53BPI ofp2Ileads to enhanced E2FI-induced
apoptosis ...... 133
Figure 5. 17. Model of E2F 1- induced apoptosis and growth arrest...... 13 5
Figure 6. 1. Model ofE2F signaling network...... 149
Figure AI. E2F I induces apoptosis...... 157
Figure A2. siChk2 inhibits E2F I-induced apoptosis ...... 158
Figure A3. DNChkI and DNChk do not effect E2FI expression...... 159
Figure A4. Specific requirement for E2FI in HPV- I6 E7 induced apoptosis...... 160 CHAPTER I
INTRODUCTION A. Cell Cycle
The cell cycle is a tightly controlled sequence of events in which genetic material
is precisely copied so that each daughter cell will receive an identical copy of the genetic
material following cell division. The eukaryotic cell cycle is divided into mitosis (M
phase), the phase in which chromosomes are segregated and cell division occurs, and an interphase period that occurs between subsequent mitosis. Interphase consists of a gap
phase that occurs following cell division and prior to DNA replication known as the G
phase. DNA is then replicated during S phase, and is followed by another gap phase, G
prior to cells re-entering mitosis. Many cells in the body reach a state of terminal differentiation where they will exit from the cell cycle following mitosis and enter a
resting, or G , state (Figure 1. 1). Malfunctions during the cell cycle, including errors in
DNA replication, spindle formation, cytokinesis, or DNA damage can result in activation
of a cell cycle checkpont to allow for repair to take place so that mutations are not passed to daughter cells (365).
B. Cell eycle regulation
1. Cyetins and eyclin dependent kinases
Precise control of the cell cycle is necessary for cells to grow and divide at the proper time and to ensure passage of properly replicated DNA to daughter cells. Multiple levels of regulation are present to ensure that the cell cycle will not advance to the next phase if the cellular or environmental conditions are not conducive for cell cycle progression. Progression from one phase of the cell cycle to the next is achieved mainly by the action of cellular cyclins and cyclin-dependent kinases (Cdk) (Figure 1.2). Cdk are a family of serine-threonine protein kinases that control progression of the cell cycle by phosphorylating target proteins at specific times (272, 318). Cdk protein levels remain constant during the cell cycle, so their activity is regulated by expression of the cyclin family of proteins (281 , 318, 319). Timed expression and rapid ubiquitin mediated proteolysis limits cyclin expression to specific intervals of the cell cycle, limiting the activity of the Cdk to certain phases of the cell cycle (102, 126 , 281 , 282, 330).
Beginning in G , the cyclin D family (consisting of cyclin DI , D2, and D3) forms a complex with Cdk4 or Cdk6. The cyclin D-Cdk4/6 complex begins the cells entry into S phase primarily by phosphorylation of the retinoblastoma (Rb) family of proteins (364).
This is followed by cyclin E-Cdk2 activity and additional Rb phosphorylation pushing the cell from G into S phase (298). The complex of cyclin A and Cdk2 is required for cells to progress through S phase (121 , 422). In the G and M phases of the cell cycle cyclin A forms a complex with CdkI , and this complex is required for transition from to M (446). Mitosis is then further regulated by activity of the cyclin B-Cdki complex
(11 , 200) (Figure 1. 1).
2. Cdk inhibitors
Additional layers of Cdk regulation take the form of cyclin dependent kinase inhibitors and protein localization. Cyclin dependent kinase inhibitors play important roles in coordinating proliferation during normal development and differentiation, as well as during cellular stress (63, 223). Cyclin dependent kinase inhibitors can be divided into INK4a the INK4 family and the Cip/Kip family (367). The INK4 family, including pI6 target Cdk4 and Cdk6 and prevent their association with cyclin D (45). Without cyclin D association, Cdk4/6 remain inactive and the cell cycle is unable to progress through G
(325, 360). The Cip/Kip family of cyclin dependent kinase inhibitors, including p2I and p27, are able to inactivate multiple cyclin-Cdk complexes and are able to block cell cycle progression in G and, to a lesser extent, Sand M phases of the cell cycle (139, 150 221
320). Unlike pI6, p2I and p27 promote a stable association between cyclin D and their
Cdk partners (215). Following cellular growth signals, increased levels of cyclin D-Cdk4 complexes will titrate p2I and p27 from cyclin E-Cdk2 complexes allowing for progression through G I (32). The localization of both Cdk activators and inhibitors also contributes to proper cell cycle progression. For example, to prevent inappropriate mitosis the cyclin B-Cdki inhibitor Wee I is kept in the nucleus, while the cyclin B-Cdki activator Cdc25 is sequestered in the cytoplasm prior to M phase (144, 237, 312, 451).
3. The retinoblastoma protein family
In the late G phase of the cell cycle, cells must pass through the G restriction point in order to enter into S phase and begin DNA replication (307). Passing the G restriction point means that the cell can continue through the remainder of the cell cycle
and back into G without additional growth factor requirements (307, 363). The G restriction point is controlled by the retinoblastoma (Rb) family of proteins. The Rb
family consists ofthree related genes;pRb (known as Rb), pI07, andp130 (387). Rb was
originally identified due to homozygous loss of the Rb allele that predisposes children to Figure 1.1. Cyetin/Cdk regulation of the eell eycle. Eukaryotic cells replicate DNA during S phase and divide during mitosis (M phase).
Separating the S and M phases of the cell cycle are two gap phases, G and G2. Activity of cellular cyclins and cyclin dependent kinases (Cdk) regulate progression from one phase of the cell cycle to the next. Sites of activity of Cyclin-Cdk complexes during the cell cycle are depicted. development of retinoblastoma, a malignant tumor of the retina (116 , 118 , 203 , 224). pI07 and p130 have been identified more recently, and have structural and functional similarities to Rb (59, 105, 120, 135 355 414 456 461). Together, Rb, pI07, and p130 are known as the pocket proteins due to a large, conserved protein-protein interaction domain (58, 220). Studies of Rb family knockout mouse models and cells from those mice reveal some functional overlap between family members in regards to development
(62, 222) and cell cycle regulation (67, 345). Rb family members also play important roles in terminal differentiation (67), however, the role of individual Rb family members in differentiation is unclear and may depend on the experimental system analyzed (44
293 334 352 453).
The Rb family fuction mainly as negative regulators of cell cycle control. During
, the Rb family are in an active, unphosphorylated state (429). In this state, the pocket proteins are able to interact with transcription factors, such as the E2F family, that control
the entrance of the cell into S phase (4, 429). While bound to Rb, the E2F family are
unable to transactivate the expression of genes required for S phase (4, 429). A major
function of the Rb family of proteins is to repress transcription from E2F regulated
promoters (5 , 89 , 109 , 114). At the restriction point, the Rb family becomes
sequentially phosphorylated by the cellular cyclin-Cdk complexes (225). D type cyclins
complexed to either Cdk4 or Cdk6 phosphorylate Rb during Gl (104, 193). This is
followed by cyclin E-Cdk2 phosphorylation leaving Rb in a hyperphosphorylated state
(154, 206, 230). In this state, the Rb family is unable to bind to and inhibit activity of the
S phase promoting E2F family members (55) (Figure 1.2). ..
Eukaryotic G Cell Cycle R 1
Figure 1.2. Rb/E2F regulation of the cell cycle.
Rb/E2F complexes regulate entry of cells from the G to S phase of the cell cycle. Prior
to the restriction point (R), Rb/E2F complexes repress E2F regulated promoters
preventing entry into S phase. Phosphorylation of Rb at the restriction point by
cyclin/Cdk complexes promotes dissociation of Rb and E2F, allowing expression of E2F regulated genes. 4. The E2F family c-- The Rb family controls cell cycle progression mainly through their ability to
interact with the E2F family of transcription factors. E2F was first identified as a cellular
transcription factor capable of binding to two consensus E2F "TTTCGCGC" sites within
the adenovirus E2 promoter (208, 209, 286). Human E2FI was later cloned by screening
cDNA libraries for proteins that interact with the pocket domain of Rb (148, 186, 362).
Many cellular genes regulated by the E2F family have been identified including genes
involved in controlling cell cycle regulation, DNA synthesis, and DNA replication. These
genes include cyclin A, cyclin E, cyclin DI , dihydrofolate reductase (DHFR), thmidine
kinase (TK), DNA polymerase a, and ORCI (382). E2F homo logs have been identified
in mouse (232), Drosophila melanogaster (297), yeast (249, 418), Xenopus laevis (315
391), and plants (326, 359).
To date, seven members of the human E2F family have been described (69, 81
404) (Figure 1.3). The E2F family can be subdivided into categories based on their ability
to interact with the Rb family and on their transcriptional activity. E2FI , E2F2, and
E2F3a are potent activators of transcription and interact mainly with Rb (3 , 148 , 149
174, 183 , 226, 227, 324). E2F4, and E2F5 are transcriptional repressors, and interact
primarily with the Rb family members pI07 and p130 (26, 153, 171 270 347 414). The
functions of the remaining members of the E2F family remain somewhat unclear. E2F3b
encoded by an alternate transcript from the E2F3 locus, interacts with Rb and may
function as a transcriptional repressor in quiescent cells (227). E2F6 shares little
homology with the other E2F family members outside of the DNA binding domain, does not interact with any of the Rb family members, lacks a transactivation domain, and is reported to repress transcription by interaction with polycomb proteins (275 , 403).
Likewise, E2F7 is unable to interact with Rb proteins, lacks a transactivation domain, and appears to act as a transcriptional repressor (69 81).
In cells , E2F proteins act in conjunction with their heterodimeric binding partners the DP family of proteins. The DP family consists of DPI and DP2. DPI was first identified as a ubiquitously expressed binding partner for E2FI that enhanced the transactivation ability of E2FI (18 , 125 , 149, 213). DP2 shares high homology to DPI and is also acts as an E2F binding partner that can enhance the transactivation ability of
E2F family members (299, 435 , 458). Unlike DPI , DP2 is expressed as tissue specific splice variants (72, 299, 339 458). In vitro, all combinations ofE2F-DP heterodimers can form and recognize similar E2F consensus binding sites (38, 226, 458). The large number of E2F-DP combinations possible in cells suggests that different heterodimers may differentially regulate genes. While some evidence of this exists (398), the role of the different E2F-DP heterodimers in regulating gene expression remains unclear.
5. Importanee of the E2F family in growth control
The important and unique roles for the E2F proteins have become partially realized through genetic studies in mice. Cells from mice lacking E2F I display limited cell cycle defects except for a prolonged Go phase, suggesting that E2FI may have a role in timing S phase entry from quiescence (113 , 425 , 449), while cells lacking E2F2 show little defect in proliferation (228). Cells from mice lacking E2F3 exhibit a delayed cell j .""""" '"...': , ...,... ' ,. ~~~- .'''' ' _.. .. "
Cyclin A Binding Dimerization Pocket Protein Binding
DNA Binding Marked Box Transactivation
''''''M'' .W"W E2F1 I"yJM"%'mm
E2F2 :''r- ?:''r r*-*-" M"' E2F3a F1l7k;;;IV..I'
"c""F'?- E2F3b r"' O=I I..
E2F4 r"7;57 -0=
f1r' E2F5 ISHZ
E2F6 I'YmHA, ?!,,"'D
E2F7
Figure 1.3. Sehematie representation of the E2F family.
The E2F family consists of seven members, distinguised by their transactivational ability and preference for association with Rb family members. E2FI , E2F2, and E2F3a are potent activators of transcription, and interact mainly with Rb. E2F4 and E2F5 are transcriptional repressors, and interact preferentially with pI 07 and p 130. E2F6 and E2F7 lack Rb family binding and transactivation function, and may function as transcriptional repressors. Shaded regions represent conserved domains. cycle , failure to upregulate many E2F responSIve genes II response to mitogen
stimulation, centriole duplication, and premature centriole separation (167 , 343).
However, cells from mice lacking all three activator E2Fs show severe proliferative
defects and fail to induce E2F target genes that are critical for the transition from G to S
(438). Cells lacking E2F4 or E2F5, or both, do not display the cell cycle defects or loss of
gene expression associated with loss ofE2F3 (166 331). However, loss ofE2F4 or E2F5
does result in a failure to properly exit from the cell cycle in response to growth
INK4a (37 inhibitory signals such as expression ofpI6 , 166 331).
E2F family members also playa role in the regulation of differentiation, or exit
from the cell cycle. Repression of E2F 1 regulated promoters is essential to promote and
maintain exit from the cell cycle in various cell types (82, 424). Mutation of Rb in some
cell types can lead to re-entry into the cell cycle and uncontrolled proliferation and an
increase in apoptosis (60 , 179 , 219). Repression of E2FI in some cell types is accomplished by the activity of the C/EBPa transcription factor (322). Active repression of E2F regulated genes by a complex composed of p130 and E2F4 or E2F5 is also essential for maintaining the differentiated state of some cell types (28, 274, 322). Taken together, E2F family members can have different roles depending on the cells state of differentiation.
6. Rb/E2F regulation of cell cycle progression
Cellular growth control by the retinoblastoma protein family occurs primarily through Rb-mediated repression of E2F regulated promoters and active transcriptional . .
repression by Rb/E2F complexes. While it is unclear which Rb/E2F function is most
important in vivo both mechanisms have been shown to occur in vitro. Rb inhibition of
E2F-mediated transactivation occurs by virtue of Rb binding to the E2F transactivation
domain and inhibiting transcriptional activation of E2F responsive promoters (Figure 1.4
A) (114, 137, 147 341). The importance ofE2F repression by Rb is demonstrated by the
observations that ectopic expression of E2FI , E2F2, or E2F3 is sufficient to drive
quiescent cells into S phase (76, 183 211).
Rb can also actively repress E2F-mediated transcriptional activation by
recruitment of chromatin remodeling enzymes, including HDACs, SWI/SNF complexes
and methyl transferases (136, 137 290 417 443). HDACs, or histone deacetylases, are a
family of enzymes involved with the removal of acetyl groups from histone tails that
promotes chromatin condensation (201 207) and have been shown to be associated with
transcriptional repressor complexes (8, 130, 140, 141 , 145). HDACI and HDAC2 contain
LxCxE motifs, allowing them to bind to Rb at the LxCxE binding site, while HDAC3
lacks the LxCxE motif and binds to Rb at another location (34, 243 , 247). E2F family
members bind to Rb at a site distinct from the LxCxE motif and HDAC3 binding site
allowing complexes containing HDACI , HDAC2, or HDAC3 , Rb, and E2F to form.
Interaction between Rb and HDACs is at least partially responsible for Rb-mediated
repression of E2F responsive promoters (34, 216 , 243 , 247 , 457). Additionally,
recruitment of HDACs to Rb/E2F repressor complexes serves to inhibit the function of
the histone acetyl transferases (HAT) p300/CBP that is recruited to promoters by
interaction with E2FI (405). HATs function by adding acetyl groups to histone tails relaxing chromatin structure, and allowing transcriptional machinery access to promoters
(201). By recruitment ofHDACs to corepressor complexes, Rb is able to actively repress
E2F -mediated transcription by altering chromatin structure (Figure 1.4 B). Repressor complexes containing pI 07 and p 13 0 have not been characterized as thoroughly as Rb repressor complexes, but studies have shown association of pI 07 and p 130 with HDAC proteins (112, 169, 194 386).
Active repression by Rb also occurs by recruitment of A TP-dependent SWI/SNF complexes. Rb recruits SWI/SNF complexes by interaction with the ATPase components of the complexes, Brgi and Brm (87, 375, 389). Similar to HDAC complexes, repressor complexes containing Brm, Rb, and E2F can form at promoters containing E2F binding sites (406). Dominant-negative ATPase mutant Brgi or Brm can block Rb-mediated growth suppression (87, 388) and expression of Brgi in cells lacking BrgI and Brm results in a growth arrest that is dependent on interaction with Rb (87). Additionally, expression of p I61NK4a induces a Rb-dependent growth arrest that requires functional
Brgi (434 457).
The formation of multiple Rb/E2F repressor complexes during the G phase of the cell cycle prevents expression of S phase promoting genes and thus prevents entry into S phase. However, different Rb/E2F complexes also form at promoters during the different phases of the cell cycle. Most cell cycle regulated promoters bind both E2FI 3/Rb complexes and E2F4 5/pI07 or p130 complexes. The use of chromatin immunoprecipitation (ChIP) allows analysis of promoter occupancy during distinct phases of the cell cycle and has provided a picture of the dynamic regulation by Rb/E2F complexes (329, 395 , 431). Regulation ofthe b-myb gene is an example of expression by
derepression. The b-myb promoter is occupied by an E2F4/pI07 repression complex during Gl. During S phase, the E2F4/pI07 complex dissociates and E2F4 no longer binds
, 431). to the b-myb promoter allowing expression of b-myb during S phase (395
Regulation of the dihydrofolate reductase (DHFR) promoter is an example of
transcriptional activation. The DHFR promoter is occupied in Go and by an
E2F4/pI07 repression complex. As cells progress from G into S phase, the complex is replaced by an E2FI/Rb complex, and following hyperphosphorylation ofRb, free E2FI
allowing expression of DHFR in early S phase (Figure 1.4 C). This complex is quickly replaced in late S phase with an E2F4/pI07 repression complex (431). Consistent with their proposed roles as activator E2Fs, E2FI , 2, and 3 occupy many E2F responsive promoters during S phase, while E2F 4 is the predominant E2F occupying these same promoters during Go and G . During S phase, some of the promoters tested also showed preference to binding by E2FI , E2F2, or E2F3 , such as DHFR , TK (thymidine kinase),
and Cdc6 while other promoters such aspI07 and Cdc2 showed no preference for E2F binding (329, 395 431).
7. Rb family independent regulation of E2F
In addition to regulation by the Rb family of proteins, the E2F family is also subject to Rb independent regulation. The majority of information regarding E2F regulation has come from studies of the E2FI family member. The association of E2FI with Rb during Gl serves to protect E2FI from degradation. When Rb becomes Transcriptional Repression
Active Transcriptional Repression
Transcriptional Activation
Figure 1.4. Models of Rb/E2F regulation of E2F regulated promoters.
(A) Binding of Rb/E2F complexes to E2F regulated promoters represses E2Fs transactivation function. (B) By recruiting chromatin remodeling enzymes such as
HDACs or SWI/SNF complexes to promoter regions, Rb is able to actively repress transcription. (C) Dissociation of Rb from E2F allows expression from E2F regulated promoters. phosphorylated as cells enter into S phase, E2FI becomes acetylated by p300/CBP enhancing E2FI stability and DNA binding activity (253). As cells proceed into mid S phase, the cyclin A/Cdk2 complex physically associates with E2FI at a conserved N- terminal domain (212 310 444). Subsequent phosphorylation of both E2FI and the DP heterodimeric binding partner by the cyclin A/Cdk2 complex at the S to G phase transition reduces E2FI DNA binding ability and promotes E2FI degradation by the proteasome (131 , 252).
Because of the ability of E2FI to drive cells into the cell cycle, E2FI protein levels must be very tightly controlled. When Rb dissociates with E2F I at the G to S transition, E2FI protein begins to be degraded by the ubiquitin-proteasome pathway (42). AR (mouse pI9 The degradation process is initiated by the human pI4 ) protein that
SKP2 binds to the C-terminus of E2FI and recruits the p45 ubiquitin protein ligase (25
251). This leads to poly-ubiqutination of E2FI and degradation by the proteasome.
Additionally, in response to some stimuli, E2FI can be phosphorylated by the ataxia telangiectasia mutated (Atm) kinase and the human checkpoint kinase, Chk2 (235 , 383).
These phosphorylation events stabilize E2FI protein, possibly by interfering with p45 SKP2 binding, and can also alter E2FI DNA binding specificity (383).
8. E2F regulated genes
The role for E2F in regulating genes involved in the G to S phase transition, S phase progression, and DNA replication has been confirmed by numerous micro array studies. Genes induced by E2F expression include genes involved in S phase promotion such as cyclin E genes required for the synthesis of DNA such as ribonucleotide reductase, thymidine kinase and DNA polymerase , and genes involved in assembly of pre-recognition complexes at origins of replication including those encoding Orc proteins, Cdc6, and Mcm proteins (173 187 244 279 332 380). Microarray analysis has also identified additional classes of genes that are regulated by E2F proteins. These include genes involved in DNA damage recognition, DNA repair, and apoptosis (279
332). The findings that E2F proteins regulate genes encoding DNA damage recognition and repair enzymes is not surprising given that DNA replication is an error prone process.
Additionally, in response to DNA damaging agents, E2FI is upregulated, providing a mechanism for increased expression of DNA damage recognition and repair enzymes
(279). E2FI can also downregulate expression of genes encoding anti-apoptotic factors
(314), and upregulate expression or pro-apoptotic factors (279). The ability of E2F proteins to regulate such a diverse set of genes suggests that E2F function is crucial to many biological pathways, although it remains unclear as to how E2F regulation may
segregate by biological pathway.
C. Deregulation of the cell cycle
1. Deregulation of the eell cycle by small DNA tumor viruses
Much of what is known regarding cell cycle regulation originally came about
through studies of the small DNA tumor viruses. Small DNA tumor viruses, including
adenoviruses, human papillomaviruses (HPV), and polyomaviruses such as SV 40 are
dependent on host cell DNA replication machinery for replication of viral genomes. However, the small DNA tumor viruses replicate in terminally differentiated, non- dividing cells that lack the required DNA replication machinery. In order to replicate, a virus must push cells into S phase where host cell DNA replication machinery is available for replication of viral genomes (39 , 78).
The induction of S phase by the small DNA tumor viruses is accomplished by virally encoded proteins that inactivate members of the Rb family of proteins. Each of the small DNA tumor viruses accomplishes this through a conserved LxCxE motif that binds and inactivates Rb family members. The adenovirus EIA protein contains an LxCxE motif and binds with high affnity to all three members of the Rb family of proteins (95
13 8 , 231 , 260). By binding to Rb family members, E I A disrupts the association of
Rb/E2F complexes allowing expression of E2F responsive promoters, resulting in induction of S phase (12, 16, 19, 95 , 138 , 163 , 231 , 260, 328, 455). HPV encodes the
LxCxE motif containing E7 protein that also binds to and inactivates Rb family members resulting in increased E2F activity and deregulation of the cell cycle (54, 80, 302, 436).
Expression of E7 also results in degradation of Rb family members by the proteasome providing another mechanism to bypass cellular growth control (27 , 33 , 128). The polyomavirus SV 40 encodes the large T antigen that is also capable of binding to Rb family members and blocking Rb-mediated G growth arest (74, 90, 103 432, 454).
2. Cell eycle deregulation and caneer
Long before the mechanisms of cell cycle deregulation by the small DNA tumor viruses were known, these viruses were found to be associated with tumor formation. Adenovirus was the first human virus identified to induce tumors in an animal model
(402). HPV is capable of inducing tumors in humans, and may be the causative agents in most cervical cancers (31 , 88, 464, 465). SV 40 was first isolated from monkey kidney cells (392) and is capable of transforming some rodent cell types (92, 122- 124, 399 400).
There is also some evidence that SV 40 may be associated with some human cancers
(202). In all cases, the small DNA tumor viruses require the function of the oncogenes
EIA, E7 or large T for efficient transformation (79, 93 , 94, 180, 313 , 344).
In addition to virus-associated transformation, cancer often results from cellular mutations that result in loss of normal growth control and lead to unrestrained proliferation. The high frequency of cell cycle regulators mutated in human cancers demonstrates the importance of proper cellular growth control for the prevention of cancer (363). The cell cycle mutations most often observed in human cancers include overexpression of cellular cyclins, loss of cyclin dependent kinase inhibitors, and loss of
Rb either by mutation, deletion, or inactivation (262).
Loss or mutation of cellular Cdks although rare, has been associated with some
tumors. Cdk4 amplification and Cdk4 and Cdk6 mutations have been identified in
melanomas, sarcomas, and gliomas (91 , 433). CdkI and Cdk2 overexpression has also been reported in a subset of colon cancers (198 , 447). Occuring more frequently in human tumors is the overexpression or deregulation of cellular cyclins. Overexpression
of cyclin DI often occurs as a result of a gene translocation that puts the cyclin DI gene under the control of the gene encoding the immunoglobulin heavy chain (133, 278, 430).
Cyclin D2, cyclin D3, cyclin A and cyclin E have all been reported to be amplified or overexpressed in some tumors , and elevated levels of cyclin E expression may correlate with shorter survival (85 170 197 218 357).
In addition to alterations in expression of Cdks and cyclins, mutations in both Cdk activators and inhibitors occur in human cancer. Deregulation or overexpression of the
Cdk activating enzme Cdc25 results in deregulated activation of cyclin-Cdk complexes entry into S phase, and is associated with tumor formation. Cdc25A and Cdc25B are activated by c-myc, an oncogene that is frequently mutated in tumors, and are often overexpressed in breast cancer (119, 291). Loss or mutation of the cyclin dependent
kinase inhibitorspI6 p27 andp2Ileads to unscheduled inactivation ofRb. Mutations in cyclin dependent kinase inhibitors often correlates with tumor aggressiveness and serves as a marker for poor patient prognosis (100, 188, 369).
The Rb family of tumor suppressors is one of the most frequently studied cell cycle regulators due to its involvement in viral and mutation-induced tumors. As discussed earlier, the Rb protein is the target of many viral oncoproteins that deregulate the cell cycle in order to provide a conducive environment for viral replication. In human tumors, the Rb protein is mutated in retinoblastoma, lung cancer, and acute lymphoblastic leukemia (162, 410). Moreover, it is estimated that greater than 90% of human tumors contain mutations in the Rb growth control pathway (133). While mutations in the Rb growth control pathway are common in human tumors, mutations in E2F family members
are rarely found in human tumors. E2F expression has been reported to be either reduced or amplified in different tumor types (99, 106 , 110 , 157, 177). The lack of direct E2F family mutations in human tumors suggests that cells must maintain a certain level of r::, E2F proteins in order to properly grow and avoid apoptosis (discussed below).
D. Apoptosis
1. Overview of apoptosis
Cell death has long been observed as a function of normal development (248).
The observation that cells appeared to be destined to die at specific times during
development was proposed in 1965 by Lockshin and Wiliams and termed programed cell
death (239). The term apoptosis, Greek for falling of leaves, was coined in 1972 to
describe the morphological changes associated with programmed cell death (195 , 196).
Apoptosis is characterized morphologically by cellular shrinking, condensation of the
chromatin, nucleosomal DNA fragmentation, convolution of the plasma membrane
known as blebbing, and formation of the dying cells into apoptotic bodies that are
surounded by an intact plasma membrane (415 , 442). Since the initial characterization of
the phenomenon of apoptosis in the I970s, there has been extensive work done to
determine its biological importance and underlying molecular mechanisms. The apoptosis
program has been found to be vital for normal development, for maintaining tissue
homeostasis, and for proper function of the immune system. Deregulation of the
apoptosis program can lead to disastrous consequences such as neurodegeneration
autoimmunity, and in its absence, cancer.
2. The p53 tumor suppressor The p53 protein plays an essential role in mediating both cellular growth arrest
and apoptosis in response to diverse environmental stimuli. The importance of p53 in
maintaining genomic stability is suggested by findings that greater than 50% of human
tumors contain mutations in the p53 gene (159). Activation of p53 was first observed in
cells exposed to ultraviolet radiation (250). It was then demonstrated that p53 was
essential for mediating a cellular growth arrest in response to irradiation or DNA-
damaging agents (192). The role of p53 in apoptosis induction was discovered by the
introduction of p53 into a p53 deficient cell line (452). The ability of p53 to induce apoptosis was later shown to correlate with the ability of p53 to act as a tumor suppressor
(393). Depending on the cellular context or duration or extent of DNA damage, p53 can
induce a cellular growth arrest, primarily through transactivation of the p2I promoter, to
allow for repair of damaged DNA or induce apoptosis (168 , 192, 229). Consequently, p53 has become known as a sensor of genomic stability due to its ability to respond to
damaged DNA, hypoxia, nucleotide depletion, viral oncogene expression, and other
genotoxic stresses (323).
3. Apoptotic targets of p53
The apoptosis program has been divided into two major pathways, the intrinsic pathway and the extrinsic pathway (2). Much of the data involving p53-mediated apoptosis have linked p53 to activation of the intrinsic, or cellular stress pathway. In the apoptosis pathway, the p53 protein functions as a sequence specific transcription factor
(204). Activation of the apoptosis program by p53 has been linked to its transactivation function (15 , 57 , 181 , 349). Perhaps the most important proapoptotic transcriptional targets of p53 in the intrinsic pathway are members of the Bcl-2 family. Transactivation by p53 of the Bc1- 2 family members Bax, Bid, Puma and Noxa ultimately leads to permeablization of the outer mitochondrial membrane, release of proapoptotic factors from the damaged mitochondria, activation of cysteine proteases known as caspases, and apoptosis (2, 214, 269, 284, 295, 350, 368). p53 also transactivates components of the
apoptotic machinery, including ApafI and the effector caspase, caspase-6. Apafl helps to initiate the caspase cascade by activating caspase-9 (190, 276, 336), while an increase in caspase-6 protein levels may sensitize cells to release of cytochrome-c from mitochondria
(245).
The extrinsic apoptosis pathway involves activation of cell surface death receptors by their appropriate ligands, resulting in caspase activation and apoptosis (14). The contribution of p53 to apoptosis resulting from activation of the extrinsic pathway remains unclear. p53 may elevate sensitivity to death receptor signaling by transactivation of genes encoding cell surface receptors or ligands. Direct transcriptional
targets of p53 include Fas and Fas ligand and studies using mutant p53 constructs have proposed a role for p53 in upregulation of these receptors as a means of promoting apoptosis in tumor cells (129, 280 301).
In addition to the two major apoptotic pathways, p53 is able to induce many apoptotic genes that are not directly associated with these pathways. These genes include p53AipI which is able to disrupt mitochondrial function and induce apoptosis in some tumor cell lines (257 , 296). PTEN is another transcriptional target of p53 that plays an /-
important role in p53-mediated apoptosis (378). PTEN does not act as a direct inducer of
apoptosis, but instead acts to inhibit the antiapoptotic protein Akt. Akt is a kinase that
inhibits cell death by phosphorylating numerous targets. PTEN functions as
phosphatase, preventing activation of Akt and the subsequent cellular survival signals
(420).
4. p53 aetivation
In response to cellular stressors , p53 is covalently modified, including
phosphorylation at numerous N- and C- terminal serine residues and acetylation on C-
terminal lysine residues (10). While it remains unclear as to which posttranslational
modifications are required for p53 activation under specific cellular stressors, these
modifications play an important role in mediating p53 function. p53 modifications have
been shown to increase transactivation function, increase protein stability, alter protein-
protein interactions, and even change promoter specificity (10) (Figure 1.5).
Several cellular kinases have been identified that play critical roles in the
activation of p53 following DNA damage, the best studied of the stress responses that
activates p53. These kinases include Atm, the kinase mutated in ataxia telangiectasia
(AT), Atr, the Atm and Rad3 related kinase, and their downstream kinase substrates
checkpoint kinase I (ChkI) and checkpoint kinase 2 (Chk) (1, 372). The role of Atr in regulating the p53 response to DNA damage is not well understood due in part to the
early embryonic lethality of atr mouse embryos (36, 71). However, it has been proposed that Atr is activated in response to certain types of DNA damage and can phosphorylate p53 on serine 15 (43 134 199 217). Atr is also able to phosphorylate and activate ChkI (132, 146 289), which can subsequently phosphorylate the N-terminus of p53 (370).
The role of Atm in activating p53 following DNA damage is better understood. In response to y-irradiation or genotoxic drugs that induce DNA double strand breaks, Atm is activated and can directly phosphorylate p53 at serine 15 (191). In cells from AT patients, there is a delay in activation of p53 following y-irradiation (374). In addition to directly phosphorylating p53 , Atm can phosphorylate and activate the human checkpoint kinase Chk2 (35 , 52 , 258, 259, 264). Chk2 is able to further phosphorylate p53 at additional N-terminal serine residues including serine 15 and serine 20, causing increased p53 stability and transcriptional activation (53 , 155 370). The importance ofChk in this pathway is demonstrated in dominant negative Chk2 expressing cells (53) and Chk2 deficient mice, which exhibit a defect in apoptosis and a decrease in p53 stabilization in response to y-irradiation (396).
5. Deregulated protiferation and p53
Inactivation of Rb by cellular or viral oncoproteins or loss of Rb wil stimulate cells to bypass GO/Gl and enter S phase (96 , 101 , 273 , 286). Ectopic expression of viral oncogenes, such as the high risk HPV - 16 E7 protein, can also lead to the induction of apoptosis (164, 304). The ability of E7 to induce apoptosis is dependent on its ability to interact with Rb (304). E7-induced apoptosis can be inhibited by expression of the viral 48-
Figure 1.5. Model of p53 activation following cellular stress.
The Atm and Atr kinases are activated by certain forms of DNA damage or replicative
stress. Atm and Atr can directly phosphorylate p53 at serine 15 as well as phosphorylate
and activate ChkI and Chk. ChkI and Chk can subsequently phosphorylate serine 20.
These modifications may stabilize p53 by promoting interaction with p300/CBP and inhibiting interaction with Mdm2. Additional kinases and acetyl transferases (shown in green) are activated in response to certain types of stress and may be important increasing p53 stabilzation and transactivation fuction. protein E6 (304). The HPV E6 protein is able to bind to and promote the degradation of
p53 (351). The studies with HPV and other small DNA tumor viruses suggest a link
between Rb/E2F cell cycle control and p53-mediated apoptosis. There is evidence that
ectopic expression of various E2F family members can induce both S phase progression
and apoptosis depending on the levels of expressed cDNA or cell type context (56 , 152
211 324 419 440 462). However, in fibroblasts, exogenous expression ofE2FI , but not
E2F2 or E2F3 , causes apoptosis (76, 210).
The link between E2FI and p53 dependent apoptosis has been demonstrated by genetic studies in mice. Apoptosis observed in transgenic mouse models expressing a
fragment of the SV -40 large T antigen and in the central nervous system of Rb- mouse
embryos is p53 dependent and associated with E2FI (246, 305, 408). Expression of an
E2FI transgene in the skin of K5E2FI transgenic mice results in increased proliferation
and p53 dependent apoptosis (316). Additionally, mice deficient for E2F I have an excess of matue T cells due to a defect in thymocyte apoptosis (113).
Expression of E2FI leads to an accumulation of p53 protein (152, 211). A potential mechanism for the stabilization of p53 observed following expression of E2F is activation of the pI9AR/Mdm2 pathway. Expression of E2FI has been shown to
transactivate the expression of the mouse ARF p I ~RF and human p I4 promoters (25 , 172
335). pI~RF encodes a protein that modulates the activity of Mdm2 (321 , 459). Mdm2 is an E3-1ike ubiquitin ligase that regulates the stability of p53 by promoting its degradation by the proteasome (115 , 117, 142, 160). By inhibiting Mdm2 activity, p19ARF allows for stabilization, activation, and accumulation of p53 protein (321 , 459). Thus, it has been ARF hypothesized that pI9 is a key protein linking the Rb/E2F and p53 pathways (25
189). One hypothesis for how aberrations in the Rb/E2F pathway are recognized by p53
is that de-regulated E2F expression activates p53 by inducing pI ~RF
D. DNA damage and E2F
1. DNA double strand break reeognition
Activation of p53 often occurs as a result of damaged DNA. The p53 activation
pathways begin when specialized proteins recognize a DNA break. The DNA damage
sensors are sensitive enough to recognize a single double strand break, an important task
for maintaining genomic integrity and preventing inactivation of critical genes or
chromosomal abnormalities (333 , 416). The double strand break repair pathway is
divided into two major pathways, non-homologous end joining (NHEJ) and homologous
recombination (HR). The NHEJ pathway is the predominant form of repair activated by
double strand breaks.
Within thirty minutes of the induction of DNA double strand breaks caused by ionizing radiation or genotoxic drugs, a complex containing MreII , NbsI , and Rad50
(MRN complex) relocalizes to sites of the DNA breaks (285). The Mre II component contains both exonuclease and endonuclease activities, and is thought to process broken
DNA ends prior to repair (308, 407). Nbsl is required for proper localization of the complex to DNA breaks following ionizing radiation (47). The role ofRad50 in the MRN complex is to hold the broken DNA ends in close proximity allowing for MreII process the broken DNA ends, and repair by other DNA repair factors (70, 161). Three distinct MRN complexes have been observed in cells. Type I foci are thought be involved
in telomere length regulation and can be observed in untreated cells (241 , 437). Type II and Type III foci form early and late, respectively, following exposure of cells to ionizing radiation, and have been termed ionizing radiation induced foci (IRIF) (255). Type IV
MRN foci are associated with normal cellular DNA replication, and may form in order to repair DNA breaks that arise during S phase (64). Type IV foci are thought to relocalize to sites near origins of DNA replication by virtue of an interaction between E2F I and the
Nbsl component of the complex (254).
2. Checkpoint signating
Following relocalization of the MRN complex to sites of DNA double strand breaks, a cell cycle checkpoint is activated resulting in a pause in cell cycle progression or apoptosis. Cells from patients with the rare human disorders Nijniegen breakage syndrome (NBS) and ataxia-telangiectasia-like disease (A TLD), lacking functional Nbsl and Mre II , respectively, fail to undergo growth arrest in response to ionizing radiation
(371 , 384, 394). A functional MRN complex is required for activation of Atm (412) that leads to activation of Chk2 and subsequent phosphorylation and activation of p53 as discussed.
In addition to MRN relocalization, other DNA damage signaling proteins localize to sites of double strand breaks, including yH2AX and 53BP1. Following DNA damage the histone protein H2AX becomes phosphorylated by Atm, and yH2AX rapidly relocalizes to sites of DNA breaks at or near the formation of MRN foci (205 , 338). yH2AX relocalization is though to be important for the recruitment of DNA repair factors
to sites of DNA breaks (309). 53BPI is a DNA damage response protein originally
identified due to its binding to p53 and enhancement ofp53 transactivation function (175
176). 53BPI is recruited to nuclear foci following DNA damage by binding to yH2AX
(426). The importance of 53BPI in cell cycle checkpoint activation has been
demonstrated by mouse models and siRNA gene knockdown experiments in human cells.
Loss of 53BPI results in a defect in Sand G /M checkpoint activation, hypersensitivity to
ionizing radiation, and the development of thymic lymphomas (84, III , 423 , 427).
53BPI , like H2AX, is phosphorylated by Atm (327) and is required for the Atm-
dependent activation of Chk (427) (Figure 1.6).
3. E2F! and DNA damage signating
Following treatment of cells with DNA damaging agents, E2FI protein
accumulates (30, 158, 165, 235, 266, 294), and is phosphorylated at an N-terminal Atm recognition sequence that is unique to E2FI among the E2F family members (235). This phosphorylation of E2FI is largely dependent on Atm and is required for efficient E2FI
stabilization following DNA damage (235). Chk has also been shown to phosphorylate
and stabilize E2FI following DNA damage, and this modification has been shown to be required for E2FI dependent apoptosis following DNA damage by altering its promoter
specifcity (383). Additionally, DNA damage induced apoptosis is compromised in thymocytes from E2Fr mice (235), suggesting that E2FI has multiple roles in DNA damage signaling. 53BP1
Growth Apoptosis Arrest
Figure 1.6. Model of DNA double strand break signating.
DNA double strand breaks are initially detected by the MRN complex, composed of
MreII , NbsI , and RadSO. Relocalization of 53BPI and yH2AX to sites at or near DNA double strand breaks and phosphorylation of these components by Atm acts to transduce through Chk2, the detection of a DNA break. Phosphorylation and activation of pS3 by
Atm, Chk, and possibly other kinses, can lead to activation of a cell cycle checkpoint resulting in growth arrest or apoptosis. E. Thesis aims
The objective of this dissertation was to determine the pathway(s) by which deregulation of the Rb growth control pathway results in p53-dependent apoptosis. Given AR can modulate p53 stability through inhibition of Mdm2 activity, it had been that pI9 proposed that this was the pathway by which E2FI induces apoptosis. We tested this
p I ~RF is not required hypothesis , and the data presented in Chapter III demonstrate that for E2FI-induced apoptosis. Instead, apoptosis correlated with the ability of E2FI to induce covalent modification of p53. The observation that E2FI could induce p53 modifications similar to those observed following DNA damage, prompted us to examine the role of DNA damage kinases in mediating signaling to p53 and apoptosis. The experiments in Chapter IV demonstrate a role for the DNA damage kinases Atm and induced Chk2, as well as the DNA damage recognition and repair protein Nbsl in E2FI-
apoptosis. Comparing the ability of E2F family members E2FI and E2F2, we found that correlated with p53 the induction of Chk2 expression was specific for E2FI and
activation and apoptosis.
To determine if ectopic E2F expression properly modeled Rb family inactivation
we confrmed our results obtained by E2F overexpression by expression of HPV - 16 E7.
In this biologically relevant context, E7 specifically required E2FI and activation of Atm
Nbs I , and Chk2 to induce apoptosis. The experiments in Chapter V address the role of
Nbs I in this apoptosis pathway. Expression of E2F specifically induced the
relocalization of the Nbs I containing MRN complex to nuclear foci that correlated with
the formation of 53BPI and yH2AX containing foci. Due to the role of the MRN complex in cell cycle checkpoint activation following DNA damage, we hypothesized that a similar cell cycle checkpoint may be activated following expression of E2FI. We found that expression of E2FI in normal human fibroblasts blocked cell cycle progression that is mediated, in part, by activating a p2I-dependent checkpoint. These findings provide further insight into the pathways linking deregulated proliferation control to p53 and apoptosis. CHAPTER II
MA TERIALS AND METHODS A. Cell culture
1- Early passage wildtype and genetically matched p53- and MdmT /p5T MEFs were isolated from mouse embryos as described (184). MEFs derived from pI~RF- mouse embryos were a generous gift from Charles Sherr (St. Jude Children s Research
I- Hospital, Memphis , TN). MEFs derived from pI~RF- /p5T mouse embryos were a
generous gift from Gerard Zambetti (University of Tennessee, Memphis , TN). Ink4a
MEFs were kindly provided by Ronald DePinho (Harvard Medical School, Boston, MA).
atm I- and genetically matched wildtype mice were purchased from The Jackson
Laboratory, Bar Harbor, ME. 293 cells were generously provided by Joseph Nevins
(Duke University Medical Center, Durham, NC). MEFs and 293s were cultured in
Dulbecco s modified Eagle medium (DMEM) supplemented with 10% fetal bovine
serum and I % penicilin-streptomycin. Primary human dermal fibroblasts GM003I6B
and GM02270A (Normal), primary human embryonic lung fibroblasts GMOI604 (HEL
fibroblasts), GM03395C and GM05823C (AT), and GM07I66A (NBS) were obtained
from Coriell Cell Repositories, Camden, NJ. Human cells were cultured as recommended
by Coriell.
B. Adenoviral veetors
Recombinant adenoviruses encoding p53, E2FI , E2F2, and E2F3a have been
described previously (76, 211 , 354). An adenovirus encoding an E2FI DNA binding
mutant, E2FI 132 (183), was created by homologous recombination in 293 cells (211
288). An adenovirus encoding the E2FIl-283 mutant was a generous gift from W. Douglas Cress (H. Lee Moffitt Comprehensive Cancer Center, Tampa, FL). The p53N/C virus was
generated from the pCB6+p53N/C construct generously provided by Karen Vousden
(National Cancer Institute, Frederick, MD). The ChkI , DN-ChkI , Chk2, DN-Chk2, and
HPV - 16 E7 recombinant adenoviruses were created by homologous recombination in
Escherichia coli (143). DN-ChkI contains an aspartic acid to alanine substitution at
position 330. Plasmids encoding Chk2 and DN-Chk2 constructs were generously
provided by David Johnson (M.D. Anderson Cancer Center, Smithvile, Texas). DN-
Chk2 contains a serine to alanine substitution at position 347. A plasmid encoding HPV-
16 E7 was generously provided by Karl Munger (Harvard Medical School, Boston
Massachusetts). Control viruses encode either an empty expression cassette or 13-
galactosidase (f3-gal). Infection with control virus had no effect on parameters tested
relative to mock infection (data not shown). Viruses were propagated in 293 cells and
purified by centrifugation through cesium-chloride gradients. Virus titers were
determined by immunohistochemical staining for the adenovirus hexon protein with an
anti-adenovirus antibody (Biodesign International) and visualized using a 3
diaminobenzidine (DAB) substrate kit from Vector Laboratories (50).
C. Virus infection
Cells were washed once with PBS and serum free DMEM containing virus was added to the cells. Infections were carried out at 37 C in 5% carbon dioxide (C0 ) for I hour (211). The viral innoculum was then removed and replaced with DMEM containing the appropriate seru concentrations and cultured under the conditions described above. D. Western blot analysis
Whole cell extracts were harvested from recombinant adenovirus infected cells at
24 hours post infection unless otherwise noted. Cells were washed twice with cold PBS
and lysed in whole-cell extract buffer (SO mM HEPES, 2 mM magnesium chloride 2S0 mM sodium chloride , 0. 1 mM EDTA, I mM EGTA , 0. 1% Nonidet P- , I dithiothreitol, Ix mammalian protease inhibitor cocktail (Sigma), Ix phosphatase inhibitor cocktails I and II (Sigma)) by incubation for 30 min on ice followed by sonication. Soluble proteins were separated by centrifugation at 13 000 x g in a micro centrifuge and supernatants stored at - C. Proteins were separated by SDS-P AGE analysis and transferred to PVDF membrane (Perkin Elmer) by electro blotting. pI9
ARF was detected using anti-pI9 polyclonal antisera at I :SOO (Novus), E2FI was detected using monoclonal antibody KH-95 at I :500 (Santa Cruz Biotechnology), E2F2 was detected using polyclonal antisera C- 20 at I :SOO (Santa Cruz Biotechnology). p53 protein was detected using monoclonal antibody DO- I at I :500 or polyclonal antisera Ab-7 at
I :2000 (Oncogene Research Products), phospho-serine 15 and -serine 20 forms of p53 the phospho-threonine 68 form of Chk, and the phospho-serine 345 form of ChkI were detected using polyclonal antisera specific for each modification at I: I 000 (Cell
Signaling Technologies). ChkI was detected using polyclonal antisera FL-476 at 1:500
(Santa Cruz Biotechnology). Chk was detected using poly clonal antisera H-300 at 1:500
(Santa Cruz Biotechnology) or monoclonal antibody clone #7 at I :500 (Lab Vision
Corporation). Atm was detected using polyclonal antisera Ab-3 at I: ISO (Oncogene Research Products) and the phospho-serine 1981 form of Atm was detected using polyclonal antisera at I :600 (Rockland). p2I was detected using monoclonal antibody F- at I :400 (Santa Cruz Biotechnology). Actin was detected using polyc1onal antisera I-I9 at
I: I 000 (Santa Cruz Biotechnology). Immunoreactive proteins were detected with a chemiluminescence kit (Perkin Elmer) according to the manufacturers recommendations.
Actin blots are shown as protein loading controls. Relative changes in the levels of p53 were estimated from scanned images of western blots using Multianalyst software
(BioRad).
E. Apoptosis analysis by flow eytometry
At the indicated times post infection, cells were trypsinized, combined with any floating cells, pelleted, washed with PBS, repelleted, and resuspended in 400 l of PBS.
All centrifugations were at SOO x g for 5 min at 4 C. Subsequently, cells were fixed in cold ethanol (final concentration, 70%) and stored at 4 C. Cells were processed for propidium iodide (PI) staining as described (211). Flow cytometric analysis was performed by the University of Massachusetts Medical School Flow Cytometry Core
Facility. Cells undergoing apoptosis were defined as the population that contains less than a 2N DNA content.
F. Apoptosis analysis by ELISA
Cells were plated in 10 cm dishes at 6000 cells per cm , or 24 well plates at I x
cells per well. Virus infections were performed 24 hours after plating. At 96 hours post infection, cells were centrifuged at 500 x g for 10 min at 4 , lysed, and Cell Death
Detection ELISAplus assay performed as described by the manufacturer (Roche). The ordinate axis represents DNA fragmentation relative to control which is defined as
Error bars represent standard deviations calculated from experiments performed in triplicate.
G. Immunohistochemieal staining for p53 protein aeeumulation
Cells were infected with the appropriate adenovirus constructs and
immunohistochemically stained for p53 protein as described (210). AdCon, AdE2FI
AdE2FI 13 were infected at an MOI of 100. At the time of harvest (48 hpi), cells were
washed three times with PBS and then fixed for 15 min each in 3.7% formaldehyde
followed by methanol. The cells were then washed in PBS- 5% Tween 20. Cells were
then incubated with anti-p53 monoclonal antibody pAb42I (Oncogene Research
Products) in the presence of 1% BSA in PBS- 5% Tween-20 for 45 min at room
temperature. The cells were washed three times with PBS- 5% Tween 20, and bound
antibody was detected using Vectastain DAB substrate kit as described by the
manufacturer.
H. p53 immunofluorescenee
Cells were infected with AdCon, AdE2FI , or AdE2F2 at an MOI of 100 and at 24
hpi the cells were fixed in 3.7% formaldehyde followed by methanol. Fixed cells were
incubated with anti-p53 monoclonal antibody pAb42I at I :200 (Oncogene Research Products) and polyclonal antisera specific for phospho-Serine 15 p53 at I :200 (Cell
Signaling Technologies). Bound antibodies were detected using fluorescein isothiocyanate (FITC) or Rhodamine Red-X conjugated secondary antibodies (Jackson
Immunoresearch Laboratories, Inc. and Southern Biotechnology Associates, Inc.
I. Cell fraetionation immunofluorescence
Glass coverslips were pre-treated with 40% hydrochloric acid for 2 min followed by a 5 min wash with 70% ethanol. Cells were plated on treated coverslips at 3000 cells per cm . Cells were infected with recombinant adenoviral vectors and at 24 hpi the cells were washed three times with PBS and incubated for 5 min in cytoskeleton buffer (10 mM PIPES pH 6. 100 mM NaCl, 300 mM sucrose, 3 mM MgCh, I mM EGTA, 0.
Triton X- IOO) and 5 min in cytoskeleton stripping buffer (10 mM Tris-Hcl pH 7.4, 10 mM NaCl, 3 mM MgCh, 1% Tween 40 , 0. 5% sodium deoxycholate). Cells were then washed 3 times with PBS and fixed in fixing buffer (200 mM sucrose and 4% paraformaldehyde in PBS, pH 7.4) for 30 min at room temperature and permeablized
(0.5% Triton X- IOO, 20 mM HEPES pH 7.4 50 mM NaCl, 3 mM MgCh, 300 mM sucrose) for 15 min at room temperature (267). Fixed cells were then blocked in 10%
FBS for I hour at room temperature. Cells were then incubated with MreII monoclonal antibody I2D7 at I: 1500 (GeneTex), y H2AX polyclonal antisera at I :600 (Upstate
Biotechnology), 53BPI polyclonal antisera at 1:400 (Novus Biologicals), or Nbsl polyclonal antisera at 1:400 (Novus Biologicals). Bound antibodies were detected using FITC or Rhodamine Red-X conjugated secondary antibodues (Jackson Immunoresearch
Laboratories, Inc. and Southern Biotechnology Associates, Inc.
J. S phase analysis
Cells were plated at 3000 cells per cm . Cells were infected with recombinant
adenoviral vectors and pulsed with 10 mM 5-Bromo- deoxyuridine (BrdU) (Roche)
immediately following infection. At 24 hpi, cells were fixed and permeablized as
described above. Following permeablization, cells were incubated in 50 mM glycine for
10 min at room temperature. DNA was denatured with 4 N HCl with 0.1 % Triton X- IOO
for 10 min at room temperature, and the samples washed extensively with PBS followed
by a wash in 50 mM glycine (254). Cells were then blocked in 10% FBS and incubated
for I hour with polyclonal antisera specific for BrdU at 1:200 (Immunologicals Direct).
Bound antibody was detected using a Rhodamine Red-X conjugated secondary antibody
(Jackson Immunoresearch Laboratories, Inc).
K. Northern blot analysis
Poly-(A) RNA was isolated using the Micro-FastTrack mRNA isolation kit from
cells as described by the manufacturer (Invitrogen). Total cellular RNA was isolated'
using Trizol as described by the manufacturer (Invitrogen). Biotinylated Chk, Atm, and
GAPDH probes were generated by PCR using primers as described by the manufacturer
(KPL). Hybridized probes were visualized with a chemiluminescence kit (KPL) as described by the manufacturer. Blots were sequentially probed and stripped. PCR primers used to make biotinylated probes:
ChkF (5' ATGTCTCGGGAGTCGGATGTTG-
ChkR (5' GCACCACTTCCAAGAGTTTTTGAC-
AtmAF (5' ACGATGCCTTACGGAAGTTGC-
AtmAR (5' GGACAGAGAAGCCAATACTGGACTG-
GAPDHF (5' CAAGGTCATCCATGACAAC-
GAPDHR (5' TGGTCGTTGAGGGCAATG-
L. RNA interferenee
siRNAs used in this study were generated by Qiagen, Valencia, CA. siRNA
oligonucleotides were transfected into cells at a concentration of 100 nM using
Lipofectamine 2000 (Invitrogen) as described by the manufacturer. Control siRNAs
(siCon) recognize either GFP or retrovirus L TR, and had no effect on parameters tested
relative to mock transfection. siRNAs used in this study:
siGFP (5' CGUAAACGGCCACAAGUUC-
siLTR (5' GAUCCAGCAUAUAAGCAGC-
siE2FIa (5' GGCCCGAUCGAUGUUUUCC-
siE2FI b (5' -CUGACCAUCAGU ACCUGGC-
siE2FIc (5' - GUCACGCUAUGAGACCUCA-
siChka (5' -CUCCAGCCAGUCCUCUCAC- Chkb (5' -GAACCUGAGGACCAAGAAC- siE2F2a (5' -GUGCAUCAGAGUGGAUGGC- siE2F2b (5' CAAGAGGCUGGCCUAUGTG- siE2F3a (5' -AGCGGUCAUCAGUACCUCU- siE2F3b (5' -CUGUUAACCGAGGAUUCAG- sip2Ia (5' AGGCCCGCUCUACAUCUUC- sip2Ib (5' CUAGGCGGUUGAAUGAGAG- siAtr (5' GACGGUGUGCUCAUGCGGC- siAtm (5' -GCGCCUGAUUCGAGAUCCU- siNbsia (5' -AUCAUGCUGUGUUAACUGC- siNbsib (5' AUGUUGAUCUGUCAGGACG- sipI4 a (5' ATCCTAAAGGCCGCGGAGT- sipI4ARFb (5' AATAACACCTTCGCTGTC- sip53 (5' GCAUGAACCGGAGGCCCAU- CHAPTER III
E2Fl INDUCES PHOSPHORYLATION OF p53 THAT IS COINCIDENT WITH p53 ACCUMULATION AND APOPTOSIS A. Introduction
During normal cell proliferation, E2F family members modulate the expression of (287). many genes involved in the transition from Gl to S phase and DNA replication E2F transcriptional activity is regulated by interaction with members of the retinoblastoma (Rb) family of proteins (137). Inactivation of Rb by cellular or viral oncoproteins or loss of Rb wil stimulate cells to bypass GO/Gl and enter S phase (96 , or E2F3 is exogenously 101 273 286). S phase induction also occurs when E2FI , E2F2 , but not expressed in quiescent cells (76 , 183 211). In fibroblasts, expression of E2FI
E2F2 or E2F3 , can result in both S phase progression and apoptosis (76, 210).
Apoptosis induction by E2FI occurs primarily by activation of the p53 apoptotic signaling pathway. The link between E2FI and p53-dependent apoptosis has been , 408 demonstrated in cell culture and by genetic studies in mice (211 , 246, 305, 324
440). It has been proposed that the E2F I transcription factor serves as a link between the
Rb/E2F proliferation pathway and the p53 apoptosis pathway by inducing the expression
of p I ~RF a protein that regulates p53 stability. We tested this hypothesis to determine
the requirement for p I ~RF in the E2F I apoptosis pathway and to better understand the
mechanism ofE2FI-induced apoptosis. /-
B. p19ARF is not required for E2Fl-mediated apoptosis
ARF HumanpI4 and mouse pI~RF promoters contain multiple E2F binding sites that are responsive to ectopic expression of E2FI (25, 172 , 335). Because pI ~RF mRNA levels increase upon E2FI expression (76), we wanted to determine whether the elevated AR protein levels. Indeed mRNA levels corresponded to an increase in p I9 , we found that infecting MEFs with an adenovirus encoding E2FI (AdE2FI) led to increased levels of AR protein (Figure 3. AR expression leads to p53 accumulation via P I9 1). Given that p I9 inactivation of Mdm2 functions and that E2FI expression leads to p53-dependent AR was required for E2FI-mediated apoptosis, we determined whether pI9 , p53- dependent apoptosis. We find that E2FI expression induced apoptosis regardless of 2N DNA content (Figure pI~RF status as measured by the percentage of cells with sub-
2). Apoptosis is observed at all multiplicities of infection (MOI) in the wildtype MEFs
ARF but only at the highest dose ofE2FI in the pI~RF- MEFs, suggesting that while pI9 is not required, it may contribute to E2FI-mediated apoptosis. MEFs infected with an adenovirus encoding an E2FI DNA binding mutant, E2FI 13, showed little or no increase in apoptosis over controls. The percentage of apoptotic cells was also measured
I- I- apoptosis in pI ~RF- and p I ~RF- /p5T MEFs to test the requirement for p53 in the
I- observed inpI~RF- MEFs. The pI~RF- /p53- MEFs were less susceptible to apoptosis
as compared to MEFs lacking only pI ~RF (Figure 3.3). In addition, expression of E2FI
l- l- also induced apoptosis in ink4a MEFs (unpublished observations). ink4a mice do not
INK4A express p I9AR because of the deletion of a shared exon with p I6 which encodes a ...... ,-, *:.
wt MEFs p19ARF-I- MEFs
p19ARF
ns- !I.. !c.nc:.
actin -
Figure 3.1. E2Fl induees pl9ARF protein aeeumulation.
Wildtype andpIgARF- MEFs were infected wth AdCon, AdE2FI , or AdE2FI 13 at MOIs of25 (E2Fbow) or 100 (E2Fhi). Cells were harvested and lysates generated at 24 hours AR protein levels. post infection (hpi). Western blot analysis was performed to detect p I9
Actin blot is shown as protein loading control. /- , --.--. . . .. - .".------
WT MEFs :: l Con
t4 t4
E2F1 e13 B::% ::J bcJ
E2F1 tGJ br.: J k :' p19ARF-I- MEFs
con :ll ID 11J.J E2Fl e132 Ewll
E2F1 :QT ll :
() DNA content
Figure 3.2. E2Fl induces apoptosis in the absence of pl9ARF
Wildtype and pI ~RF- MEFs were infected with AdCon, AdE2FI , or AdE2FI 132 at
MOIs of 100 300 , or 500. Cells were harvested for PI staining and flow cytometry at 72 hpi. Percentages represent cells with sub-2N DNA content. /- /- ,, ...... "'.... ""
19ARF-I- MEFs
Con :
E2Fl e13z
E2Fl fJ%
p19ARF /p53 1- MEFs
Con l:UJ t;t_
E2Fl e13z
() DNA content
ARF Figure 3.3. E2Fl induees p53-dependent apoptosis in the absenee of pl9
pI ~RF- and pI ~RF- /p53- MEFs were infected with AdCon, AdE2FI , or AdE2FI 13 at
MOIs of 100, 300 , or 500. Cells were harvested for PI staining and flow cytometry at 72 hpi. Percentages represent cells with sub-2N DNA content. /- /-
cyclin dependent kinase inhibitor (361). Thus, E2FI can induce apoptosis in pI~RF
deficient MEFs that are generated from independent genetic lesions in mice.
C. p19ARF and Mdm2 eontribute to p53 aecumulation AR is necessary for Given that apoptosis does not require pI~RF we asked ifpI9
E2FI-mediated changes in pS3 protein levels. To address this question we expressed
E2FI in pI~RF- MEFs and assessed pS3 protein levels by immunohistochemistry. As
shown in Figure 3.4, E2FI expression resulted in increased levels of endogenous p53
protein in wildtype MEFs. When E2FI was expressed in pI ~RF- MEFs, fewer cells
stained positive for pS3 protein and more modest changes in p53 protein levels were
observed. The changes in endogenous pS3 protein levels observed by
immunohistochemical staining following E2F I expression correlate with the changes
observed by immunoblotting for endogenous p53 (Figure 3. 10). Likewise , Nip et.a!.
observed an increase in the half-life of pS3 following E2FI expression in 32D.3 cells, a AR expression (292). In both MEF genotypes tumor cell line that lacks pI4 , expressing
the E2FIe13 DNA binding mutant had little or no effect on pS3 protein levels. These
results are consistent with a model where p I9AR can contribute to E2F I-mediated
changes in pS3 protein levels.
pI9ARF affects pS3 protein levels by binding to Mdm2 and preventing it from promoting the degradation of pS3 (321 , 459). Therefore, we determined if Mdm2 is necessary for E2F I-mediated accumulation of p53. It is not possible to duplicate the experiments described above for Mdm2 because there are no cell lines or MEFs available /-
9ARF- Con Con
E2Fl
Figure 3.4. E2Fl reqires pl9ARF to induce p53 accumulation.
Wildtype andpI~RF- MEFs were infected with AdCon, AdE2FI , or AdE2FI e13 at an MOI of 100. Cells were fixed and immunohistochemically stained for p53 protein levels at 48 hpi. that lack Mdm2 expression but retain endogenous, wildtype p53. However, MEFs can be
1- on p53 derived from Mdm2- /p53- embryos (184, 263). To analyze the effects ofE2FI protein levels in these cells , we introduced p53 in trans by infecting cells with an Adp53 recombinant adenovirus, essentially creating a "pseudo MdmX MEF. To control for the
I- addition of exogenous p53 transcript, wildtype pI ~RF- and p5 3- MEFs were also infected with Adp53 at the same low MOL As observed in the immunohistochemical and immunoblot analysis for endogenous p53 , E2FI expression resulted in a substantial (?6 fold) increase in exogenous p53 levels in wildtype MEFs in comparison to control infected cells when measured by immunoblot analysis (Figure 3. 5 and Figure 3.10).
I- Others have made similar observations in pI ~RF- MEFs following retroviral gene transfer of E2FI (463). p53 also accumulated in response to E2FI in p5T MEFs that ectopically expressed p53 (Figure 3.5). Therefore, exogenously expressed p53 protein is affected by E2FI in a manner similar to the endogenous protein. Infection with AdE2FI resulted in a modest ( 2.4 fold) increase in total p53 protein levels in Adp53-infected pI~RF-I- MEFs as compared to AdCon infection of the same cell type (Figure 3.5). The
I- basal levels of ectopically expressed p53 were higher in p I ~RF- MEFs relative to
wildtype cells. This observation is consistent with the data obtained by immunostaining
and immunoblotting for endogenous p53 inpI~RF- MEFs (Figure 3.4 and Figure 3.10),
although the difference is exaggerated when cells are infected with Adp53 (Figure 3.5).
I- Others have also reported higher basal levels of p53 in pI~RF- MEFs (73). E2FI
expression minimally affected p53 protein levels in Adp53- infected MdmX /p53- MEFs.
We suspect that the Mdm2 deficiency elevates baseline levels ofp53 in these cells due to ..-,. , ..,, /-.-
!: LL LL (+ p53 J Uo WN NW NW p53
llIlt(l - actin WT MEFs p53
iiJI: .ITI -actin p19ARF - p53 -actin p53 -
,,17 p53
TTr.'\ '-' -actin Mdm2 - /p53 -
Figure 3.5 E2Fl requires pl9ARF and Mdm2 to inerease p53 protein levels.
Wildtype p53- pI~RF- or MdmT /p5;- MEFs were infected with AdCon, AdE2FI or AdE2FI 13 at MOIs of25 (E2Fbow) or 100 (E2Fhi). Cells were harvested and lysates generated at 24 hpi. Western blot analysis was performed to determine levels of p53 protein. Actin blots are shown as protein loading controls. /-
an absence or greatly reduced level of p53 ubiquitin ligase activity. E2F Ie13 had little or no effect on p53 protein levels in each cell type , consistent with a transcriptional mechanism being at least parly responsible for E2FI-mediated accumulation ofp53. The
reduced ability of E2F I to alter p53 levels above controls in p I ~RF- MEFs, or in MEFs lacking Mdm2 but expressing p53, is consistent with a model where E2FI modulates p53 protein levels, in large part, through the pI9 /Mdm2/p53 pathway.
D. E2F expression leads to the phosphorylation of p53
In response to DNA damage, p53 is activated by covalent modifications
including the phosphorylation at certain serines and acetylation of lysine residues. Of
these modifications, phosphorylation of serine 15 on human p53, or serine 18 on mouse
p53 , is commonly observed in response to DNA damage (20, 43). Given that both DNA AR to activate p53 damage (189) and E2FI do not necessarily require pI9 , we determined
whether E2FI-mediated activation of p53 function is coincident with p53
phosphorylation. To detect phospho-serine 18 , we used an antibody specific for this
modification. We find that E2F I , and to a lesser extent E2F2, expression in wildtype
MEFs results in an increase in endogenous p53 protein levels and the phospho-serine 18
form ofp53 as detected by immunoflourescence (Figure 3.6). When merged it is apparent
that only a subset of the cells with an increase in total p53 has increased levels of the
phospho-serine 18 form ofp53. Total p53 protein and the phospho-serine 18 form ofp53
were present at low levels in cells infected with control virus (Figure 3.6). This data suggest that E2FI may signal p53 accumulation and p53 phosphorylation by separate pathways.
E. Differential abilties of E2Fl and E2F2 to phosphorylate p53 and induee
apoptosis
To discriminate between E2FI-specific induction of p53 and increased E2F
activity in general, we measured the levels of p53 in extracts of cells infected with an
adenovirus encoding E2F2 in comparison to E2F I. E2F2 is fuctionally similar to E2F I;
E2F2 is a potent inducer of S phase when expressed in quiescent cells, is normally
induced at the G to S transition, specifically interacts with Rb, and induces a similar
aray of genes when overexpressed (76 , 174 , 226, 279, 358). However, like other E2F
family members, E2F2 is unable to induce apoptosis in REF52 cells (76, 210). We find
that E2F2 expression does not lead to apoptosis in MEFs, even though it is capable of
inducing the expression of pI9AR protein (Figure 3. 8 and Figure 3.9). Ectopic E2F2
expression did result in increased levels of endogenous p53 protein in wildtype MEFs
although not as effectively as E2FI (Figure 3.6 and Figure 3.10). However, expression of
I- E2F I , but not E2F2 , alters p53 protein levels in p I ~RF- MEFs (Figure 3. 7 and Figure
10).
We next compared the ability ofE2FI and E2F2 expression to induce a change in
p53 phosphorylation. We find that ectopic expression of either E2FI or E2F2 leads to an
increase in the levels of both endogenous p53 and the phospho-serine 18 form of p53 in
wildtype MEFs, although the levels of phospho-serine 18 p53 is less in E2F2 expressing DAPI total p53
Con
E2Fl
E2F2
Figure 3.6. Expression of E2Fl or E2F2 results in an increase in p53 protein levels
wild type MEFs. and levels of the phospho-serine 18 form of p53 in
Wildtype MEFs were infected with AdCon, AdE2FI , or AdE2F2 at an MOI of 100. Cells
were fixed and stained for total p53 (rhodamine Red-X) and the phospho-serine 18 form
ofp53 (FITC) at 24 hpi. DAPI staining is shown in blue. Merged images are also shown. /-
DAPI
Con
E2Fl
E2F2
Figure 3.7. E2Fl specifcally induces accumulation ofp53 and the phospho-serine form of p53 in cells lacking pI9ARF pI~RF- MEFs were infected with AdCon, AdE2FI , or AdE2F2 at an MOI of 100. Cells were fixed and stained for total p53 (rhodamine Red-X) and the phospho-serine 18 form ofp53 (FITC) at 24 hpi. DAPI staining is shown in blue. Merged images are also shown. sample (Figure 3. 10). Similar results were observed by immunoflourescence staining for
I- p53 and phospho-serine 18 p53 inpI~RF- MEFs (Figure 3.7). Expression ofE2FI , but not E2F2, leads to increased levels of the endogenous phospho-serine 18 form of p53 in pI~RF-I- MEFs (Figure 3.7 and Figure 3. 10). In addition to phosphorylation at serine 15
(the human equivalent to serine 18 in mouse p53), phosphorylation of serine 20 (serine
23 in mouse) on human p53 is commonly observed following DNA damage (53 , 155
370). Since we have had difficulties detecting mouse phospho-serine 23 p53 by immunoblot analysis, cells were coinfected at a low MOI with an adenovirus encoding human p53 , which when phosphorylated can be detected by the anti-phospho-serine 20 antibody. Using this approach, we find that E2FI expression results in an increase in the phospho-serine 20 as well as the phospho-serine 15 forms of p53 in both wildtype and pI~RF-I- MEFs (Figure 3. 11 A and Figure 3.11 B).
E2F2 expression results in a slight increase in both the phospho-serine 15 and
I- phospho-serine 20 forms ofp53 in wildtype but not inpI~RF- MEFs (Figure 3.11 A and
Figure 3. 11 B). This inability ofE2F2 to affect the phosphorylation ofp53 in the absence ofpI~RF was also observed for the endogenous mouse p53 (Figure 3.7 and Figure 3.10).
The ability of E2F2 to affect p53 protein levels was, unlike E2FI , solely dependent on pI ~RF since both endogenous and exogenous p53 protein levels were unchanged when
I- E2F2 was expressed inpI~RF- MEFs while E2FI expression leads to an increase in p53
I- in both wildtype and pI~RF- MEFs (Figure 3. 10 and 3. 11 C). We note that the differential ability of E2FI and E2F2 to alter the ectopically expressed p53 in pI ~RF- " - - ......
Con.
br:1. :1 .. - .. lJ:Ili Li:iII .. ..
E2F1 . l::-o ...... k:J .DE2F
o DNA content
Figure 3.8. E2F2 does not induee apoptosis in wildtype MEFs.
Wildtype MEFs were infected with AdCon, AdE2FI , or AdE2F2 at MOIs of 100 300, or
500. Cells were harvested for PI staining and flow cytometry at 72 hpi. Percentages represent cells with sub-2N DNA content. /- .., .. ~~~..
wt MEFs P 1 9ARF- MEFs
p19ARF ns- II fJIIT .r' iillllJ. L .
actin
ARF Figure 3.9. E2Fl and E2F2 induee pl9 protein aeeumulation.
, AdE2F2, or AdE2FI Wildtype or pI~RF- MEFs were infected with AdCon, AdE2FI at an MOI of 100. Cells were harvested and lysates generated at 24 hpi. Western blot AR protein. Actin blot is shown as analysis was performed to determine levels of p I9 protein loading control. /- .-
wt MEFs p' 9ARF- - MEFs
L..- NL. NL. Or: L..- NL. UJ UJ UJ UJ UJ p53 -
Ser'8 p53
actin
Figure 3.10. E2F2 requires p19ARF to induce p53 protein and alter levels of the phospho-serine 15 form of p53.
Wildtype or pI ~RF- MEFs were infected with AdCon, AdE2FI , AdE2F2, or AdE2FI e13 at an MOI of 100. Cells were harvested and lysates generated at 24 hpi. Western blot analysis was performed to determine levels of total p53 protein and levels of the phospho-serine 18 form of p53. Actin blot is shown as protein loading control. MEFs appears to be exaggerated relative to the endogenous protein in these experiments.
These results suggest that E2F2 utilizes the pI9 /Mdm2 pathway to affect p53 levels.
F. p53 phosphorylation contributes to E2Fl-mediated apoptosis
Having found a correlation between E2FI-induced phosphorylation of p53 and
apoptosis, we determined if these covalent modifications are required or contribute to
E2FI-mediated apoptosis. Studies of the DNA damage response have suggested that it is
unlikely that a single p53 modification is responsible for activating p53 dependent
apoptosis (48), so we initially addressed this question by using . a p53 mutant in which
most of the N- and C- terminal amino acid residues known to be phosphorylated upon
DNA damage have been changed to alanines. This mutant, p53N/C, has been shown to
activate expression of p2I , Mdm2 and Bax reporter constructs as effectively as wildtype
p53 and has been reported to induce apoptosis in response to certain stimuli (13). Mdm2
has also been shown to regulate the levels of p53N/C (13). Indeed, we find that p53N/C
protein accumulates to levels similar to wildtype p53 when expressed by recombinant
adenovirus transduction (Figure 3. 12 A). To measure apoptosis p53- MEFs were
transduced with AdE2FI or AdE2F2 and either wildtype Adp53 or Adp53N/C (Figure
12 B). Without the addition of p53 , levels of apoptosis in E2FI or E2F2 expressing
samples were similar to AdCon infected cells. Cells expressing both E2F I and wildtype
p53 underwent apoptosis. In contrast, apoptosis was greatly reduced in cells transduced
with E2FI and p53N/C encoding adenoviruses suggesting a contribution by these
,oJ" (+p53)
Ser15 pS3 wt MEFs
Ser15 pS3 - pl9ARF-
(+p53)
Ser20 pS 3 MEFs
Ser20 pS3 - pl9ARF-
(+p53)
wt MEFs actin -
p 1 9ARF- actin -
Figure 3.11. E2Fl induees p53 protein and levels of the phospho-serine 15 and phospho-serine 20 forms ofp53 in cells laekingpl
(A) Western blot analysis of the phospho-serine 15 form ofp53 in wildtype andpI~RF-
MEFs infected with AdCon, AdE2FI , or AdE2F2 and an MOI of 100. (B) Western blot
I- analysis of the phospho-serine 20 form of p53 in wildtype and p I ~RF- MEFs infected with AdCon, AdE2FI , or AdE2F2 and an MOI of 100. (C) Western blot analysis of total
I- p53 protein levels in wildtype and pI ~RF- MEFs infected with AdCon, AdE2FI , or
AdE2F2 and an MOI of 100. Cells were harvested and lysates generated at 24 hpi. Blots were sequentially probed and stripped. Actin blot is shown as protein loading control. potential phosphorylation sites to apoptosis. As expected, E2F2 did not induce apoptosis when co-expressed with either p53 or p53N/C.
We next examined the role of p53 kinases on apoptosis signaling in response to
E2FI expression by using caffeine, a potent inhibitor of several of these kinases. We find that there is a dose dependent decrease in apoptosis in E2FI expressing MEFs following treatment with increasing doses of caffeine (Figure 3. 13). Treatment of cultured cells with caffeine prior to ionizing radiation results in a delayed and attenuated accumulation of p53 protein (192). This defect in p53 accumulation is due to the inhibition ofthe ataxia telangiectasia mutated (Atm) and the Atm-Rad3-related (Atr) kinases that phosphorylate p53 on serine 15. Inhibition of Atm and Atr wil also block subsequent signals that
activate the checkpoint kinases ChkI and Chk2 and perhaps other kinases that phosphorylate p53 on serine 20 (29 , 348). The observations that blocking p53
phosphorylation either by mutating potential phosphorylation sites on p53 or treatment
with the p53 kinase inhibitor, caffeine , can significantly compromise apoptosis
demonstrates that p53 phosphorylation is a critical step in E2FI mediated apoptosis. '" " .. ..",. ,. ..."
p53-1- MEFS Adp53 Adp53N/C
p53 -%SN4 actin \81:.. lIn' UII.AiII,
+ wt p53 + p53N/C
8% .. 0% .00 Con '"
E2F1
-- :2
E2F2 ""
Figure 3.12. p53 phosphorylation correlates with E2Fl-indueed apoptosis.
(A) p5T MEFs were infected with AdpS3 or Adp53N/C at MOIs of 10, 20 , or SO. Cells were harvested and lysates generated at 24 hpi. Western blot analysis was performed to detect total p53 protein levels. Actin blot is shown as protein loading control. (B)
Analysis of apoptosis inp5T MEFs infected with AdCon, AdE2FI , or AdE2F2 at an
MOI of 500 and coinfected with AdpS3 or AdpS3N/C at an MOI of 20. Cells were harvested for PI staining and flow cytometry at 72 hpi. Percentages represent cells with sub-2N DNA content. Con E2F1
Caffeine
Figure 3.13. Caffeine inhibits E2Fl-induced apoptosis.
Analysis of apoptosis in wildtype MEFs infected with AdCon or AdE2FI at an MOI
SOO. Caffeine was added at the indicated doses to the culture medium immediately following viral infection. Cells were harvested and apoptosis detected at 72 hpi. Error bars represent standard deviation of experiment performed in triplicate. /- /-
G. Diseussion
The signaling mechanism by which the RB/E2F pathway in general, and E2FI AR specifically, communicates with pS 3 has been thought to involve the p I9 /Mdm2 AR and Mdm2 are required for the majority of p53 pathway. Indeed, we find that pI9
protein accumulation observed following E2FI expression. However, contrar to the role
of the pI9AR pathway in p53 accumulation, we find that E2FI induces apoptosis in
14). It is unlikely that E2FI induction ofp73 MEFs lackingpI~RF (see model, Figure 3.
p I ~RF- is directly responsible for the apoptosis observed in MEFs since we show that
bothpI~RF andp53. These results are E2FI failed to induce apoptosis in MEFs lacking
p I ~RF in contrast to earlier studies which concluded that is important for E2F I-mediated
(409) and Tolbert et.al. apoptosis (73 , 463). However , reports published by Tsai et.al.
Rb (401) show thatpI~RF was not required for apoptosis in deficient mouse embryos or
following Rb inactivation by transgenic expression of a fragment of the SV -40 large- T
antigen, respectively. Likewise , a report published by Russell et. al. showed that crossing
an E2FI transgenic mouse into apI~RF- background does not reduce apoptosis caused
by expression of the E2F I trans gene (342). Thus, studies using several different models
arrive at the conclusion that the apoptosis associated with deregulated E2F activity does
not require pI
It is conceivable that forcing cells into S phase by ectopic E2F expression can
induce the apoptotic response. However, it is not likely to simply be ectopic S phase
induction following E2F expression that triggers apoptosis, since both E2F I and E2F2
can induce S phase at similar efficiencies (76), and we find that E2F2 does not induce apoptosis in MEFs. Moreover, there does not appear to be a specific phase of the cell cycle in which E2FI induces apoptosis (75, 211). Our findings that ectopic E2FI , but not
E2F2 , expression results in increased p53 phosphorylation in the absence of p I ~RF and that this covalent modification ofp53 contributes to E2FI-mediated apoptosis, suggests that E2FI may activate a cellular response similar to DNA damage.
The observation that caffeine inhibits E2F I-mediated apoptosis suggests that the
action of one or more p53 kinases is likely to be important for E2FI signaling. Signaling
cascades that activate protein kinases responsible for phosphorylating N-terminal
residues on p53 upon DNA damage are well documented (10). Candidate kinases include
ataxia telangiectasia mutated (Atm), the Atm-Rad3-related protein (Atr), ChkI and Chk.
Atm and Atr phosphorylate p53 at serine 15 as well as activate the Chk kinases by
phosphorylation (20 217 238 259). Active Chk kinases can then phosphorylate p53
at serine 15 and serine 20 (370).
Recently, Atm/Atr has been shown to phosphorylate the N-terminus ofE2FI , but
not E2F2 or E2F3 (235). This observation, together with the data presented here leads us
to speculate that ectopic E2FI expression or activation of endogenous E2FI upon
phosphorylation by Atm, leads to increases in the activity and, perhaps, levels of one or
more of the p53 kinases, which then phosphorylate p53 and promote apoptosis. Given
that the E2FI DNA binding mutant, E2FI 13, did not induce p53 phosphorylation or
apoptosis, transcriptional activation of one or more p53 kinases might provide a
mechanism for E2FI activation ofthis pathway. However, overexpression of Atm/Atr .. -
Rb / E2F1 E2F1 Kinase(s) P53 Apoptosis
p19ARF Mdm2 p53 p21 E2F1
E2F2 E2F3 S Phase genes Phase
Figure 3.14. Model ofE2Fl-indueed apoptosis and p53 aeeumulation.
E2FI , E2F2 , and to a lesser extent, E2F3 can signal p53 accumulation through the pI9AR/Mdm2 pathway. p53 phosphorylation contributes to E2FI-mediated apoptosis and AR may function to attenuate E2F proliferation- can occur in the absence of p I ~RF P I9 promoting activity as shown by the dashed lines. Activation of the p I9AR /Mdm2 pathway may increase levels of p53 , contributing substrate to the p53 kinases activated by E2FI , as shown by the dashed arrow. ChkI , or Chk2 kinases has been found to be insufficient for their activation (238, 259).
Therefore, an additional signal(s) may be necessar to stimulate their activity.
It is conceivable that E2FI , E2F2 , or E2F3 could signal throughpI~RF/Mdm2 increase p53 protein levels and that the increased pools of p53 would provide more substrate for the p53 kinases activated by E2FI. Thus , the pI ~RF/Mdm2 pathway may act as both an attenuator of proliferation by targeting E2F family members for degradation and as an amplifier of a DNA damage signal by increasing pools of p53 available for phosphory lation. The decision to undergo growth arrest or apoptosis would then depend on the cellular context or extent of DNA damage.
Our data implicates p53 phosphorylation as a key step in E2FI-mediated p53- dependent apoptosis. These observations raise the possibility that E2FI signaling and
DNA damage response pathways may converge and involve the same or related kinases to activate p53. Alternatively, E2FI may contribute to or be a component of the DNA damage pathway. Given these possibilities, it is possible that a role of E2FI may be to amplify DNA damage signals resulting in p53-mediated apoptosis. CHAPTER IV
APOPTOSIS ASSOCIATED WITH DEREGULA TED E2F ACTIVITY IS DEPENDENT ON E2Fl AND Atm/Nbsl/Chk2 A. Introduetion
Having found that E2FI can induce p53-dependent apoptosis in the absence of pI~RF we wanted to determine the pathway(s) by which E2FI activates p53 to induce cell death. The findings presented in chapter III demonstrate a requirement for covalent modification of p53 in the E2FI apoptosis pathway, and that the p53kinase(s) activated by E2FI are sensitive to caffeine. The apoptosis program initiated by DNA damage also correlates with p53 modification, with a subset of DNA damage responsive p53 kinases being sensitive to caffeine (10). Therefore, we hypothesized that E2FI may activate one or more DNA damage response kinase cascades resulting in p53 activation and apoptosis.
Kinases that have been shown to phosphorylate the N-terminus ofp53 in response to DNA damage include ataxia telangiectasia mutated (Atm), the Atm-Rad3-related protein (Atr), and the human checkpoint kinases ChkI and Chk2. Atm and Atr
phosphorylate p53 at serine 15 as well as activate the Chk kinases by phosphorylation
(20 217 238 259). Active Chk kinases can then furher phosphorylate p53 at serine
15 and serine 20 (370). Activation of downstream kinase activation by Atm can also
require Nbsl (40, 234, 460). To address which kinase(s) may be involved in E2FI-
mediated p53 activation and apoptosis, we used primary human fibroblasts from patients
with ataxia telangiectasia and Nijmegen breakage syndrome, lacking functional Atm and
Nbsl gene products, respectively. Using cells from these patients, we tested the
hypothesis that E2FI and deregulation ofRb family members by expression of the HPV-
16 E7 protein induces apoptosis through activation of a pathway that shares some
similarities to the apoptosis program induced by DNA damage. B. Roles for Atm and Nbsl in apoptosis induction
Atm kinase activity is often induced in response to cellular stress leading to phosphorylation of many substrates, including serine 15 on p53. Given that ectopic E2FI expression also results in phosphorylation of serine 15 on p53 (340), we determined whether Atm was required for E2FI-mediated apoptosis and p53 phosphorylation. We found that E2Fl-mediated apoptosis was compromised in fibroblasts isolated from an AT patient ectopically expressing E2FI (Figure 4. 1). Similar results were obtained from AT fibroblasts isolated from a different donor (data not shown). In addition, apoptosis was
I- also reduced following expression of E2FI in atm MEFs (Figure 4.2). The apoptosis observed in fibroblasts ectopically expressing E2FI appears to be specific to E2FI because the related E2F family member, E2F2, was unable to induce apoptosis (Figure
1).
We next examined the ability ofE2FI and E2F2 to induce the phosphorylation of serine 15 and serine 20 residues on p53 in the absence of Atm. We found that in cells lacking Atm, both E2FI and E2F2 were still able to induce total p53 protein levels AR (340). However (Figure 4.3) likely due to induction of p I4 , phosphorylation of p53 at serine 15 was reduced and phosphorylation at serine 20 was absent in AT cells following
E2FI expression (Figure 4.3). An increase in the phospho-serine 15 form ofp53 was also observed following E2F2 expression in normal, but not AT, cells (Figure 4.3). Because
E2F2 expression led to lower levels of the phospho-serine 15 form of p53 than E2F I , it was possible that E2F2 was not inducing apoptosis because it is not as efficient at c: 8
Con E2F1 E2F2 Con E2F1 E2F2 Con E2F1 E2F2 Normal NBS
Figure 4.1. E2Fl-indueed apoptosis is redueed in human fibroblasts laeking funetional Atm or Nbs1. , and NBS Apoptosis analysis in normal human dermal fibroblasts, AT fibroblasts (AdE2Fl), fibroblasts. Cells were infected with recombinant adenovirus encoding E2FI
E2F2 (AdE2F2), or a control virus (AdCon) at an MOI of 1000. Cells were harvested and
represent standard apoptosis detected at 96 hours post infection (hpi). Error bars
deviation of experiment performed in triplicate. Con E2F1 Con E2F1 afm
Figure 4.2. E2Fl-indueed apoptosis is redueed in mouse embryo fibroblasts laeking atm.
I- Apoptosis induction in wildtype and atm MEFs. Cells were infected with AdE2FI or
AdCon at an MOI of 500. Cells were harvested and apoptosis detected at 72 hpi. Error bars represent standard deviation of experiment performed in triplicate. Normal NBS
Con E2F1 E2F2 Con E2F1 E2F2 Con E2F1 E2F2
p53
actin
Normal NBS
Con E2F1 E2F2 Con E2F1 E2F2 Con E2F1 E2F2
Ser15
actin
Ser20
actin
Figure 4.3. E2Fl and E2F2 require Atm and Nbsl to effieiently induee phosphorylation of p53.
Western blot analysis of p53 protein levels following infection with AdCon, AdE2FI
AdE2F2 at an MOI of 1000. Cells were harvested and lysates generated at 24 hpi. The phospho-serine 15 and phospho-serine 20 forms of p53 were detected with polyc1onal antisera specific for each modification. Actin blots are shown as protein loading controls. inducing Atm activity as E2F1. To control for the differences in activation by E2FI and
E2F2 of the kinase(s) that phosphorylate p53 on serine 15, we expressed E2F2 at doses
that resulted in levels of serine 15 phosphorylation and p53 accumulation that were
similar to levels observed following E2FI expression (Figure 4.6). Even at these elevated
doses, E2F2 still did not induce apoptosis (Figure 4.5) or phosphorylation of p53 at serine
20 (Figure 4.6). Because E2F2 can also activate Atm resulting in p53 phosphorylation at
serine 15 in the absence of apoptosis, these results suggest that while E2FI requires Atm
. to signal apoptosis, Atm activation is not the commitment step for apoptosis induction.
In addition to directly phosphorylating p53 on serine 15 (20 , 43), Atm also
activates other kinases that lead to p53 phosphorylation on serine 20 (53 , 155 259 370
443). Since downstream kinase activation by Atm can require Nbsl (40 234 460), we
asked whether functional Nbsl protein was necessary for E2FI-induced apoptosis. We
found that E2FI-induced apoptosis was compromised in fibroblasts from NBS patients
similar to the reduction observed in AT cells (Figure 4. 1). Although expression of E2FI
was found to be slightly lower in AT cells than in normal cells (Figure 4.4), increased
amounts of E2F I stil did not induce apoptosis in AT cells (data not shown). Multiple
forms of both endogenous and exogenously expressed E2FI are routinely observed by
western blot, likely due to posttranslational modifications associated with E2F
regulation (212 252 253 310). It remains unclear as to what patterns ofposttranslational
modifications contribute to the observed altered mobility of E2FI. Although E2FI was
able to induce total p53 protein levels in NBS cells (Figure 4.3), we observed a modest Con E2F1
E2F1
actin
Con E2F2
E2F2
actin
Figure 4.4. AdE2Fl and AdE2F2 are expressed to similar levels in normal, AT, and
NBS fibroblasts.
Normal, AT, and NBS fibroblasts were mock infected or infected with a low dose (MOI of 100) of either E2FI or E2F2. Cells were harvested and lysates generated at 24 hpi and levels ofE2FI or E2F2 were compared by western blot analysis. Actin blots are shown as protein loading controls. Con E2F1 E2F2
Figure 4.5. Inereased doses of AdE2F2 does not lead to apoptosis.
Normal human fibroblasts were infected with AdCon, AdE2FI at an MOI of 1000, or increasing doses of AdE2F2 (MOI of 1000, 1500, 2000, or 2500). Cells were harvested and apoptosis detected at 96 hpi. Error bars represent standard deviation of experiment performed in triplicate. p53
actin
Ser15
actin
Ser20
Figure 4.6. Increased doses of AdE2F2 lead to inereased levels of phospho-serine 15 p53 but not phospho-serine 20 p53.
Normal human fibroblasts were infected with AdCon, AdE2FI at an MOI of 1000, or increasing doses of AdE2F2 (MOI of 1000, 1500, 2000, or 2500). Cells were harvested and lysates generated at 24 hpi. Cellular lysates were analyzed for levels of p53 and the phospho-serine 15 and phospho-serine 20 forms ofp53. Actin blots are shown as protein loading controls. decrease in the levels of the phospho-serine 15 form and a large decrease in the levels of the phospho-serine 20 form ofp53 in NBS cells following E2FI expression (Figure 4. 3), demonstrating that functional Nbsl protein is required for E2FI-mediated apoptosis and for signaling p53 phosphorylation at the serine 20 residue. Ectopic E2F2 expression found to be similar in all three cell types (Figure 4.4), failed to induce apoptosis in NBS cells (Figure 4. 1) but did cause an increase in both total p53 levels and the phospho- serine 15 form of p53 (Figure 4.3). These results are consistent with a mechanism whereby E2F2 alters the phospho-serine 15 form of p53 in NBS cells through its ability to activate Atm.
c. Chk2 is required for E2Fl-mediated apoptosis
Given that phosphorylation of p53 on serine 20 correlates with apoptosis, we proceeded to use this as a marker to identify any additional kinase(s) that may contribute to E2FI-mediated apoptosis. Among the Atm-induced kinases that require functional
Nbsl protein for activation and that directly phosphorylate p53 on serine 20, is the human
checkpoint kinase Chk2 (40, 234, 370). To examine the role of Chk2 in E2FI-induced
apoptosis, we coexpressed E2FI with a kinase defective form of Chk2 (DN-Chk2) to
inhibit Chk2 kinase activity in fibroblasts. We observed a reduction in apoptosis when
E2FI was coexpressed with DN-Chk. Apoptosis levels did not appreciably change with
a dominant negative form of ChkI (DN-ChkI) (Figure 4.7), another DNA damage
responsive kinase that is capable of phosphorylating p53 on serine 20 (370). Expression c 50
U: 30
CD 20
DN- DN- DN- DN- Con E2F1
Figure 4.7. Dominant negative Chk2, but not dominant negative Chkl reduees
E2Fl-induced apoptosis.
Normal human fibroblasts were infected with AdCon or AdE2FI at an MOI of 1000 and
coinfected with a recombinant adenovirus encoding dominant negative ChkI (DN-ChkI)
or dominant negative Chk2 (DN-Chk2) at an MOI of 1000. Cells were harvested and
apoptosis detected at 96 hpi. Error bars represent standard deviation of experiment
performed in triplicate. siCon siChk2a siChk2b Time: 24 48 24 48 24 48 Chk2
5 60
Con E2F1 E2F2 Con E2F1 E2F2 siCon siChk2b
Figure 4.8. siRNA targeting of Chk2 inhibits E2Fl-indueed apoptosis.
(A) HEL fibroblasts were transfected with siChka, siChkb, or a control siRNA (siCon).
Cells were harvested and lysates generated at 48 hours post transfection. Actin blot is shown as protein loading control. (B) HEL fibroblasts were transfected with siChkb or siCon 24 hours prior to infection with AdCon, AdE2FI , or AdE2F2 at an MOI of 250.
Cells were harvested and apoptosis detected at 96 hpi. Error bars represent standard deviation of experiment performed in triplicate. . '- -
ofDN-ChkI or DN-Chk alone did not alter levels ofE2FI protein (data not shown). To
confirm the involvement ofChk in E2FI-mediated apoptosis, we used siRNAs to reduce
the levels of Chk2 in cells (Figure 4. 8 A). We observed a reduction in apoptosis
following E2FI expression in cells transfected with siChkb (Figure 4.8 B), and no effect
of this siRNA following expression ofE2F2 (Figure 4. 8 B). Similar results were obtained
using siChka (data not shown).
We next determined the involvement of Chk in E2F 1- induced pS 3 accumulation
and modification. Since Chk directly phosphorylates p53 on serine 20, we asked ifDN-
Chk2 expression or Chk2 siRNA transfection could block E2FI-induced p53
phosphorylation. We found that DN-Chk2 expression and the Chk2 siRNA reduced the
levels of the phospho-serine 20 form ofp53 following E2FI expression but had no effect
on either total p53 levels or the levels of the phospho-serine 15 form of p53 (Figure 4.
and 4. 10). Coexpression of DN-ChkI was unable to inhibit E2FI-induced p53
accumulation and had only a modest effect on p53 phosphorylation (Figure 4.9).
D. E2Fl specifically induees Chk2 expression
We have shown that E2F I requires Atm, Nbs I , and Chk2 to efficiently induce
apoptosis. However, E2F2 is also able to activate Atm resulting in phosphorylation of
p53 at serine 15 but does so without inducing apoptosis. We confirmed the activation
state of Atm using an antibody that recognizes a modified form of Atm observed
following DNA damage that correlates with Atm activation (17). Expression of either
E2F I or E2F2 led to an increase in the levels of the phospho-serine 1981 form of Atm . . "
Con E2F1
p53
actin III:
Ser15
actin
Ser20
actin
Figure 4.9. DN-Chk2, but not DN-Chkl, inhibits phosphorylation of p53 at serine
20.
Normal human fibroblasts were infected with AdCon or AdE2FI at an MOI of 1000 and coinfected with DN-ChkI or DN-Chk2 at an MOI of 1000. Cells were harvested and lysates generated at 24 hpi. Western blot analysis was performed to determine levels of p53 protein and levels of the phospho-serine 15 and phospho-serine 20 forms of p53.
Actin blots are shown as protein loading controls. Con E2F1
en . p53
actin
Ser15
actin
Ser20
actin
Chk2
actin
Figure 4.10. siRNA targeting ofChk2 inhibits E2Fl-induced phosphorylation ofp53 at serine 20.
HEL fibroblasts were transfected with siCon or siChk2. Twenty four hours post transfection, cells were infected with AdCon or AdE2FI at an MOI of 250. Cells were harvested and lysates generated at 24 hpi. Western blot analysis was performed to determine levels of p53 protein, levels of the phospho-serine 15 and phospho-serine 20 forms of p53 , and levels of Chk2 protein. Actin blots are shown as protein loading controls. Normal NBS
Con E2F1 E2F2 Con E2F1 E2F2 Con E2F1 E2F2
Chk2
actin
Chk1
actin
Figure 4.11. Expression of E2Fl, not E2F2, leads to inereased Chk2 protein levels
normal AT, and NBS eells.
Normal human fibroblasts, AT fibroblasts, and NBS fibroblasts were infected with
AdCon, AdE2Fl , or AdE2F2 at an MOI of 1000. Cells were harvested and lysates generated at 24 hpi. Western blot analysis was done to determine levels of ChkI and
Chk2 protein. Actin blots are shown as protein loading controls. chk2
actin
p53
actin
Ser15
acUn
Ser20 Con E2F1 e132 actin
Figure 4. 12. Expression of E2Fl 132 fails to induee apoptosis, inerease Chk2 protein levels or induce phosphorylation of p53.
(A) Normal human fibroblasts were infected with AdCon, AdE2FI , or AdE2FI 13 at an
MOI of 1000. Cells were harvested and apoptosis detected at 96 hpi. Error bars represent standard deviation of experiment performed in triplicate. (B) Normal human fibroblasts were infected with AdCon, AdE2FI , or AdE2FI 13 at an MOI of 1000. Cells were harvested and lysates generated at 24 hpi. Western blot analysis was done to determine levels of Chk2 protein or levels of (C) p53 protein and the phospho-serine 15 and phospho-serine 20 forms of p53 . Actin blots are shown as protein loading controls. while leaving the total Atm protein levels unchanged (Figure 4. 12). The difference between E2F I and E2F2 signaling appears to be the ability of E2F I to stimulate Chk2 activity, which results in an increase in the phospho-serine 20 form of p53 and correlates with E2FI-induced apoptosis. Because Atm activation is upstream of Chk2 in signaling to p53 , E2FI expression must have an additional effect(s) downstream or independent of
Atm that is specific to E2FI for apoptosis induction. We found that expression ofE2FI but not E2F2, led to an increase in the levels of Chk2 protein, and this increase occurred in the absence of Atm or functional Nbsl (Figure 4. 11). E2FI expression also results in accumulation of the phospho-threonine 68 form ofChk (data not shown), a modification observed following DNA damage that may be associated with Chk2 activation (259
264). The phospho-threonine 68 modification of Chk may not be a reliable marker of
Chk2 activation (6, 356, 439). Instead, we examined the Chk substrate p53 serine 20 residue as a marker for Chk activation. Whle expression of E2F I resulted in an increase in Chk protein levels in the absence of functional Atm or Nbs I (Figure 4. 11), we did not observe an increase in the phospho-serine 20 form of p53 in these cells (Figure 4. 3).
Induction and activation of Chk2 by E2FI appeared to be specific because E2FI expression did not result in an increase in ChkI protein (Figure 4. 11) or in an increase in the phospho-serine 345 form of ChkI (data not shown). Chk2 protein accumulation
appeared to result from an increase in Chk2 mRNA levels following E2FI expression
(Figure 4. 13 A). E2F2 expression did not lead to an increase in Chk2 protein levels
(Figure 4. 11) or Chk2 mRNA (Figure 4. 13 A). Expression of an E2FI DNA binding Atm Ser1981 Atm
Figure 4.13. Expression of E2F! or E2F2 leads to an increase in the phospho-serine
1981 form of Atm while leaving total Atm protein levels unehanged.
Normal human fibroblasts were infected with AdCon, AdE2FI , or AdE2F2 at an MOI of
1000. Cells were harvested and lysates generated at 24 hpi. Western blot analysis was performed to determine levels of Atm and levels of the phospho-serine 1981 form of
Atm. ..
L. () WC\
Chk2
GAPDH
Atm
GAPDH
Figure 4. 14. E2Fl, not E2F2, leads to an inerease in Chk2 mRNA levels but not Atm mRNA levels.
(A) Northern blot analysis for Chk2 mRNA isolated from normal human fibroblasts infected with AdCon, AdE2FI , or AdE2F2 at an MOI of 1000. GAPDH shown as
loading control. (B) Northern blot analysis for Atm mRNA isolated from normal human fibroblasts and infected as in (A). GAPDH shown as loading control. mutant, E2FI 13, did not lead to an increase in Chk2 protein levels over control cells
(Figure 4. 12) suggesting that Chk may be induced by a transcriptional mechanism. We also did not detect apoptosis , p53 accumulation, or p53 phosphorylation following expression of E2FIe13 (Figure 4. 12). Since neither E2FI nor E2F2 induced the
expression of Atm mRNA (Figure 4. 13 B), induction of Chk2 expression appears to be specific to E2F I and correlates with the induction of apoptosis.
E. Chk2 and E2F eooperate in apoptosis induction
Given that the induction of Chk2 mRNA and protein levels following expression of E2FI is associated with E2FI-specific apoptosis, we next asked whether Chk2 cooperates with E2FI to signal apoptosis. We found that coexpression of Chk2 with a reduced amount of E2F I encoding virus resulted in enhanced levels of apoptosis whereas expression of Chk alone had a nominal effect on apoptosis levels (Figure 4. 14).
Thus, it appears that Chk is limiting for E2FI-induced apoptosis. This observation raises the possibility that the different abilities of E2FI and E2F2 to activate the apoptosis
program lies in the capacity of E2F I to induce Chk2 expression. Given this possibility,
we determined if Chk2 could cooperate with E2F2 to induce apoptosis if provided
trans . We found that co expression of Chk2 with E2F2 permitted E2F2 to induce apoptosis (Figure 4. 14) and led to an increase in the phospho-serine 20 form of p53
(Figure 4. 15). Coexpression ofE2FI or E2F2 with ChkI had no effect on apoptosis (data not shown). The sudden onset of serine 20 phosphorylation at the point where cells are E2F1 E2F2 Con 500
Figure 4.15. E2Fl and E2F2 cooperate with Chk2 in apoptosis induction.
Normal human fibroblasts were infected with AdCon, AdE2Fl , or AdE2F2 at an MOI of
SOO and coinfected with the indicated doses of AdChk2. Cells were harvested and apoptosis detected at 96 hpi. Error bars represent standard deviation of experiment performed in triplicate. g g
Con E2F1 E2F2 000 000 000 a a a 00000 Chk2 a .. L! .. C' L! a .. L! .. C' L! p53
actin
Ser20
actin
Figure 4.16. Chk2 eooperates with E2F2 to induce phosphorylation of p53 at serine
20.
Normal human fibroblasts were infected with AdCon, AdE2FI , or AdE2F2 at an MOI of
500 and co infected with the indicated dose of AdChk2. Cells were harvested and lysates generated at 24 hpi. Western blot analysis was performed to determine levels of total p53 protein and levels of the phospho-serine 20 form ofp53. Actin blots are shown as protein loading controls. undergoing apoptosis in response to coexpression of E2F2 and Chk2 may mean that
additional events are required to lead to phosphorylation ofp53 through Chk2. These data
suggest that an E2FI-specific increase in Chk2 expression is essential for p53 activation
and apoptosis induction.
F. Apoptosis induetion by HPV-l6 E7 is dependent on E2Fl and Atm/Nbsl/Chk2
We next examined the role of Chk2 in apoptosis resulting from deregulation of endogenous E2F activity. The human papiloma virus (HPV) type 16 E7 protein binds to and inactivates Rb family members resulting in the release of Rb-associated factors including E2F proteins (277). We found that expression of the HPV- I6 E7 protein resulted in apoptosis induction in human fibroblasts plated at low density (Figure 4. 16).
Similar to the apoptosis observed following ectopic E2F I expression, apoptosis resulting from Rb family inactivation by HPV- I6 E7 required functional Atm and Nbsl proteins
(Figure 4. 17). Since Rb inactivation by E7 results in release of five E2F family members from Rb proteins, we used siRNAs targeted to E2FI to address the requirement for E2FI in E7 induced apoptosis. We screened three siRNAs targeted to E2FI for their ability to inhibit E2FI expression (Figure 4. 18 A). siE2FIc was used for the remainder of the experiments shown, but similar results were obtained with the other E2FI siRNAs (data not shown). We found that pre-treatment of cells with siE2F I blocked the ability of E7 to induce apoptosis (Figure 4. 18 B and data not shown). Having observed that E7 induces apoptosis through E2F! , we next determined ifE7-induced apoptosis requires Chk. We found that reducing Chk1evels with an siRNA decreased the ability ofE7 to induce g, 12
Q) 4
Con E2F1
Figure 4.17. HPV-16 E7 induees apoptosis in human fibroblasts.
HEL fibroblasts were infected with AdE2FI at an MOI of250, or AdCon and AdE7 at an
MOI of 1000. Cells were harvested and apoptosis detected at 96 hpi. Error bars represent
standard deviation of experiment performed in triplicate. Con Con Con Normal NBS
Figure 4. 18. HPV-16 E7-induced apoptosis is reduced in eells lacking funetional
Atm or Nbs1.
Normal, AT, and NBS fibroblasts were infected with AdCon or AdE7 at an MOI of 1000.
Cells were harvested and apoptosis detected at 96 hpi. Error bars represent standard deviation of experiment performed in triplicate. E2F1
actin
Con Con Con siCon siE2F1 c siChk2b
Figure 4.19. siRNA targeting ofE2F1 or Chk2 inhibits E7-indueed apoptosis.
(A) Western blot analysis ofE2FI protein levels in cells transfected with siCon, siE2FIa siE2FI b, or siE2FIc. Cells were harvested and lysates generated at 48 hours post transfection. Actin blot is shown as protein loading control. (B) Analysis of apoptosis in cells transfected with the marked siRNA 24 hours prior to infection with AdCon or AdE7 at an MOI of 1000. Cells were harvested and apoptosis detected at 96 hpi. Error bars represent standard deviation of experiment performed in triplicate. 1;'
apoptosis (Figure 4. 18 B). To confirm the requirement of and determine the specificity
for E2FI in E7-mediated apoptosis, we used siRNAs targeted to E2FI , E2F2 or E2F3
(Figure 4. 19). siE2F2a and siE2F3a were used for the remainder of the experiments
shown, but similar results were obtained with the other E2F2 and E2F3 siRNAs (data not
shown). We found that neither siE2F2 nor siE2F3 were able to block apoptosis induced by E7 expression (Figure 4.20). These data suggest that apoptosis resulting from
deregulation of endogenous E2F activity occurs specifically through E2FI and the
Atmlbs/Chk pathway.
G. HPV- 16 E7 induees Chk2 expression and p53 Modifieation
We next asked ifE7 expression induces modifications to p53 similar to E2F1. We
found that E7 expression resulted in an increase in p53 levels, and the levels of the
phospho-serine 15 and phospho-serine 20 forms ofp53 (Figure 4.21). Additionally, Chk
protein levels were elevated following either E2FI or E7 expression (Figure 4.22 A).
phospho-serine 1981 Similar to E2F I , expression of E7 resulted in an increase in the
form of Atm while leaving total Atm protein levels unchanged (Figure 4.22 B).
Expression of E7 also resulted in an increase in the phospho-threonine 68 form of Chk2
(data not shown). These observations suggest that E7 is able to activate Atm kinase
activity resulting in an increase in active Chk kinase.
We next determined whether E2FI was required for E7 to increase Chk and p53
levels, and modifications to p53. We found that E2FI is not required for much of the
observed increase in p53 or the phospho-serine 15 form ofp53 following E7 expression 100
E2F2
actin
ro
E2F3
actin
Figure 4.20. siRNA knockdown of E2F2 and E2F3. siRNAs targeted to E2F2 or E2F3 were transfected into HEL fibroblasts. Cells were harvested and lysates generated at 48 hours post transfection. Western blot analysis was performed to determine effciency of siRNA targeting of E2F2 or E2F3. Actin blots are shown as protein loading controls. " ' . ,- ;.
101
Con Con E7 Con E7 Con E7 siCon siE2F1c siE2F2a siE2F3a
Figure 4.21. HPV-l6 E7 requires E2Fl but not E2F2 or E2F3 to induce apoptosis.
HEL fibroblasts were transfected with the marked siRNA 24 hours prior to infection with
AdCon or AdE7 at an MOI of 1000. Cells were harvested and apoptosis detected at 96
hpi. Error bars represent standard deviation of experiment performed in triplicate. 102
Con E2F1 p53
actin
Ser15
actin
Ser20
actin
Figure 4.22. HPV-16 E7 expression results in p53 accumulation and modifeation similar to those observed following expression of E2F1.
HEL fibroblasts were infected with AdE2FI at an MOI of250, or AdCon and AdE7 at an
MOI of 1000. Cells were harvested and lysates generated at 24 hpi. Western blot analysis was performed to determine the levels of total p53 protein and the levels of the phospho- serine 15 and phospho-serine 20 forms ofp53. Actin blots are shown as protein loading controls. 103
(Figure 4.23). This increase in p53 protein was likely due to activation of the
pI4 /Mdm2 pathway by E2F2 and/or E2F3, while the increase in the phospho-serine 15 form of p53 is likely due to the ability of E2F2 , and possibly other E2Fs, to activate
Atm (Figure 4. 12 and Figure 4.22 B). However, we found that E2Fl was required for E7 to induce the phosphorylation of p53 at serine 20 and to increase the levels of Chk2 protein (Figure 4.23). Additionally, we found that E7 expression resulted in an increase in
Chk2 mRNA levels (Figure 4.24) and this could be attributed to E2FI (Figure 4. 24).
Having found that E2F I is required for E7 to induce C h k 2 expression and
phosphorylation of p53 at serine 20, we next determined if Chk2 was required for E7 to induce this modification. We found that an siRNA directed against Chk2 was able to
block E7-induced phosphorylation of p53 at serine 20, while total p53 levels and the levels of the phospho-serine 15 form of p53 remained unaffected by addition of this siRNA (Figure 4.23). These results demonstrate a requirement for E2FI in Chk2 induction and kinase activation following Rb inactivation by HPV - 16 E7 as measured by an increase in the phospho-serine 20 form of p53. Taken together , apoptosis resulting from inactivation of Rb family members is dependent specifically on E2F 1 and its ability to induce Chk2 expression. 104
Con E2F1
Chk2
actin
Con E2F1
Atm
Ser1981 Atm
Figure 4.23. HPV-16 E7 expression results in an inerease in Chk2 protein levels and levels of the phospho-serine 1981 form of Atm, while leaving total Atm protein levels unehanged.
(A) HEL fibroblasts were infected with AdE2FI at an MOI of 250, or AdCon and AdE7 at an MOI of 1000. Cells were harvested and lysates generated at 24 hpi. Western blot analysis was performed to determine Chk protein levels. Actin blot is shown as protein loading control. (B) HEL fibroblasts were infected as in (A). Cells were harvested and lysates generated at 24 hpi. Western blot analysis was performed to determine Atm protein levels and levels of the phospho-serine 1981 form of Atm. ... . ,.
105
Con
tJ tJ
00 00
p53
E2F1 \':)i n 'fi"-
actin - llij l'tm
Ser20
actin
Ser15
Chk2
actin LJ, rJi
Figure 4.24. HPV-l6 E7 requires E2Fl and Chk2 to inerease the levels of the phospho-serine 20 form of p53.
HEL fibroblasts were transfected with the marked siRNA 24 hours prior to infection with
AdCon or AdE7 at an MOI of 1000. Cells were harested and lysates generated at 24 hpi.
Western blot analysis was performed to determine the levels of total p53, the levels of the phospho-serine 15 and phospho-serine 20 forms of p53 , and levels of Chk2. Actin blots are shown as protein loading controls. + '
106
c:c:0 u.N 00 0 00 ' c: u. c: + o N I' o I'UJ
Chk2
GAPDH
Chk2. Figure 4.25. HPV- l6 E7 requires E2Fl to induee
HEL fibroblasts were untransfected or transfected with the marked siRNA 24 hours prior to infection with AdE2FI at an MOI of 250, or AdCon and AdE7 at an MOI of 1000. Chk2 RNA was isolated at 24 hpi. Northern blot analysis was done to determine levels of
RNA. GAPDH shown as loading control. .:'
107
H. Diseussion
In this chapter we describe a pathway involving the E2F I-specific induction of
Chk2 expression that links loss of proliferation control to an apoptotic pathway with
some similarity to the apoptosis pathway induced by DNA double strand breaks. Unlike
the DNA damage signals that activate p53 in the absence of de novo gene expression
(178), E2F I-mediated apoptosis requires induction of Chk2 expression and possibly other
components of apoptosis signaling (283) to fully activate p53 and kil cells. The results
presented here suggest that deregulated E2F 1 function induces apoptosis by activation of
an AtmlNbsllChk2/p53 pathway following disruptions in the Rb/E2F proliferation
pathway (Figure 4.25).
To model deregulation ofE2F proteins, we expressed the HPV- I6 E7 protein, and
determined the requirement for E2F family members and the Atmlbs lIChk pathway
for signaling to p53 and apoptosis induction. It has been suggested that E2FI is
resonsible for a portion of the phenotypes associated with E7 expression, including
disruption of normal cell cycle control , interfering with cellular differentiation, and
apoptosis induction (54 , 286). Supporting the involvement of E2FI in E7-induced
apoptosis, transgenic mice that express E7 in the lens exhibit increased apoptosis in the
fiber cell compartment that is dependent on E2FI (261 , 304). The results presented here
suggest that apoptosis induction by HPV- I6 E7-mediated Rb family inactivation in
primary human fibroblasts relies primarily on the ability of E7 to activate E2FI and the
I.; AtmlbsllChk/p53 pathway (Figure 4.25).
..1 108
Viral Oncoprotein Inactivation
Rb/E2F E2F1 Chk2 Chk2 p53 Apoptosis
Atm/Nbs1
Figure 4.26. Model of p53 activation following deregulation of E2F1.
Deregulation of the Rb/E2F proliferation pathway results in activation of an apoptotic pathway with some similarities to the apoptosis pathway induced by DNA double strand breaks. Apoptosis induction associated with deregulated E2F activity is dependent on
E2FI and the ability ofE2FI to induce Chk2 and to signal through Atmlbsi resulting in activation of p53 and apoptosis. 109
In this study, we examined phosphorylation of the serine 20 residue on p53 as a marker for Chk2 activation. In normal, AT, and NBS cells, while we observed an increase in total p53 levels, only in normal cells did we observe an increase in the phospho-serine 20 form of p53 , a substrate for active Chk2 kinase. Inhibition of Chk2 activity by a dominant negative construct or by siRNA targeting resulted in a failure to phosphorylate the serine 20 residue following expression of either E2FI or E7 demonstrating the specificity of this modification by Chk.
While we describe the involvement ofE2FI in apoptosis resulting from Rb family inactivation, E2F I also plays a role in apoptosis induction following exposure of cells to
DNA damaging agents (235). We speculate that activation of E2FI by DNA damage
ARF leads to increased pI4 expression resulting in increased pools of p53 protein. E2FI is
also able to activate Atm kinase activity and induce Chk2 expression leading to increased p53 activation and E2FI activity. E2FI activation following DNA damage would therefore act to amplify DNA damage signals converging at p53 to result in apoptosis. 110
CHAPTER V
E2Fl INDUCES Mrell FOCI FORMATION AND BLOCKS CELL CYCLE PROGRESSION IN HUMAN FIBROBLASTS 111
A. Introduetion , we next determined what Having found that Chk2 induction is specific to E2FI upstream signals are specifically activated by E2F1. Activation of p53 following DNA , such as Atm, Atr, and damage involves both activation of DNA damage response kinases , 285 , 372). Chk2, as well as relocalization of DNA damage recognition proteins (I
Among the DNA damage recognition proteins that relocalize to sites of DNA breaks are
Mre11 , Rad50, and NbsI , components of the MRN DNA damage recognition complex
(47 285). Activation of certain S phase cell cycle checkpoints also requires the activity of
Atm (373). The Atm triggered S phase checkpoint requires, among other proteins
functional Nbs I , a component of the MRN complex that is also required for E2F
induced apoptosis and recognition of certain forms of DNA damage (412). In addition to
MRN relocalization following DNA damage, the DNA damage sensors 53BPI and
yH2AX also relocalize to sites at or near DNA breaks (205 , 217, 426). Because of the , we requirement for DNA damage response proteins for E2FI-induced apoptosis
hypothesized that E2FI may induce relocalization of some or all ofthese DNA sensors. 112
B. E2Fl specifieally eauses MRN reloealization
The MRN complex functions in checkpoint signaling by relocalization to discrete , we nuclear foci following certain types of DNA damage (47, 66, 255 , 285). Therefore
examined the cellular localization of the MRN complex following expression of E2F
proteins by examining the Mre II component of the complex. We found that expression
of E2F I induces relocalization of Mre II to discrete nuclear foci, while no foci were
observed following expression of E2F3a (Figure 5. 1 A). MreII foci were observed in
98% of cells following expression of E2FI , but at levels similar to control following
expression ofE2F3a (Figure 5.1 B). Formation of Mrel I foci correlated with an increase
in the phospho-serine 20 form of p53 (Figure 5. 2) and with apoptosis induction (Figure
3). Expression of E2F3a resulted in an increase in the phospho-serine 15 form of p53
(Figure 5.2), a target of the Atm kinase, but not with an increase in the phospho-serine 20
(Figure 5.2) form ofp53 or with apoptosis induction (Figure 5. 3).
C. E2Fl induees irradiation indueed foei (IRIF)-tike formation
The formation of MRN foci has been described under a number of cellular
conditions (241 , 254, 255 , 285 , 437). To determine the type of MRN foci formed
following expression of E2F I , we sought to further characterize the Mre II foci. MRN
foci formation requires the Nbs I component of the complex (47). Similarly, we found
that E2FI-induced Mrell foci fail to form in cells lacking Nbsl (Figure 5.4). DNA
damage foci have also been shown to form independently of Atm (267). Likewise, we ...
113
DAPI Mre11 Merge % Cells Con Virus MOl with Foci Con 2500 ..1%
E2F1 1000 98% E2F1 E2F3a 1000 ..1% E2F3a 1500 ..1% E2F3a 2000 ..1% E2F3 E2F3a 2500 ..1%
Figure 5.1. E2F! specifcally induees Mrell foci formation.
(A) Normal human dermal fibroblasts were infected with AdCon, AdE2FI , or AdE2F3a at an MOl of 1000. Cells were fixed and stained for MreII at 24 hpi. (B) Normal human dermal fibroblasts were infected with AdCon, AdE2FI , or AdE2F3a at the indicated
MOls. Cells were fixed and stained for MreII at 24 hpi. Cells were counted to determine the percentage of cells with MreII foci. A minimum of300 cells per well was counted. 114
p53
actin
Ser15
actin
Ser20
actin
Figure 5.2. Expression of E2Fl, but not E2F3a, results in an increase in the phospho-serine 20 form of p53.
Normal human dermal fibroblasts were infected with AdCon or AdE2FI at an MOI
1000, or AdE2F3a at MOIs of 1000, 1500, 2000, or 2500. Cells were harvested and lysates generated at 24 hpi. Western blot analysis was done to determine levels of total p53 protein, and levels of the phospho-serine 15 and phospho-serine 20 forms of p53.
Actin blots are shown as protein loading controls. 115
Con
Figure 5.3. E2Fl, but not E2F3a, induces apoptosis in human fibroblasts.
Normal human dermal fibroblasts were infected with AdCon or AdE2FI at an MOI
1000, or AdE2F3a at MOIs of 1000, 1500 , 2000, or 2500. Cells were harvested and apoptosis detected at 96 hpi. Error bars represent standard deviation of experiment performed in triplicate......
116
Wildtype NBS DAPI Mre11 Merge DAPI Mre11 Merge
con...
E2F1
Figure 5.4. E2Fl fails to induces Mrell foci in NBS cells.
Normal human dermal fibroblasts or NBS fibroblasts were infected with AdCon or
AdE2FI at an MOI of 1000. Cells were fixed and stained for MreII at 24 hpi. 117
found that E2F I is able to induce the formation Mre II foci in cells lacking Atm (Figure
5). To confirm the involvement of Nbsl in the MreII foci observed following expression of E2F I , we examined the colocalization of Nbs I and Mre II. We found that
Nbsl and MreII co localize to nuclear foci following expression ofE2FI (Figure 5.6 A).
In addition to relocalization of the MRN complex, DNA damage also induces relocalization of other DNA damage response proteins, including yH2AX and 53BPI (9,
205 338 353 426). We found that expression ofE2FI induces relocalization ofyH2AX and 53BPI to nuclear foci, and this correlates with the relocalization of MreII foci
(Figure 5.6 B and Figure 5.6 C). Taken together, these results suggest that ectopic expression of E2F I induces the formation of foci similar to the irradiation induced foci
(IRIF) seen following DNA damage.
MRN complex relocalization has also been observed during normal S phase progression, when no extrinsic DNA damaging agent has been present (254). Since E2FI and E2F3a have been described to induce S phase in rodent fibroblasts (76, 183, 211), we determined if the observed foci could be attributed to S phase progression. A hallmark of the reported S phase MRN foci is their localization at or near sites of BrdU incorporation.
When we examined the subset of cells that incorporate BrdU following expression of
E2FI , we found no colocalization of Mre11 and BrdU (Figure 5.7). Indeed, we oberved
very few BrdU positive cells following expression of E2F1. The rare cells that show
BrdU incorporation following expression of E2FI show no co localization of Mrell and
BrdU (Figure 5.7 bottom panels)......
118
Wildtype DAPI Mre11 Merge DAPI Mre11 Merge
Con
E2F1
Figure 5.5. E2Fl induces Mrell foci formation in AT cells.
Normal human dermal fibroblasts or NBS fibroblasts were infected with AdCon or
AdE2FI at an MOl of 1000. Cells were fixed and stained for Mrell at 24 hpi......
119
eon ..1I1i
E2F1
eon
E2F1
eon ..8.
E2F1
Figure 5.6. E2Fl induces relocalization of DNA damage response proteins.
Normal human dermal fibroblasts were infected with AdCon or AdE2FI at an MOl of
1000. Cells were fixed and stained for Mrell and Nbsl (A), MreII and yH2AX (B), or
MreII and 53BPI (C) at 24 hpi. Merged images are also shown......
120
DAPI BrdU Mre11 Merge
Con
E2F1
E2F1
Figure 5.7. Expression of E2Fl induces Mrell foci that do not correlate with BrdU incorporation. an MOI of Normal human dermal fibroblasts were infected with AdCon or AdE2FI at
1000. Cells were fixed and stained for Mrell and BrdU at 24 hpi. Merged images are also shown...... '- ,
121
DAPI Mre11 Merge
con
E2F1
E2F1 283
Figure 5.8. E2Flt-283 induces Mrell foci formation similar to full length E2Fl.
Normal human dermal fibroblasts were infected with AdCon, AdE2FI , or AdE2Fb-283 at an MOl of 1000. Cells were fixed and stained for MreII at 24 hpi. 122
DAPI M re 11 Merge
Con
E2F1
E2F1
Figure 5.9. E2Fl 132 induces Mrell foci formation similar to E2Fl.
Normal human dermal fibroblasts were infected with AdCon, AdE2FI , or AdE2FII-283 at an MOI of 1000. Cells were fixed and stained for MreII at 24 hpi. 123
It is possible that an interaction between E2FI and Nbsl (254) may provide a mechanism for the observed MRN relocalization following expression of E2FI. The MRN complex has also been shown to relocalize to sites that contain E2F binding elements near origins of DNA replication (254). To examine whether the interaction of E2FI and Nbsl is required for MRN relocalization, we used an E2FI mutant, E2FlJ-283, that lacks the Nbsl interaction domain (254). We found that expression of E2FlJ-283 results in MRN relocalization similar to expression of full length E2FI (Figure 5. 8). Additionally, expression of a DNA binding mutant of E2FI , E2FIe13, was found to induce relocalization of Mrel I (Figure 5.9), suggesting that E2FI is not responsible for targeting the MRN complex to specific sites on DNA. Taken together, these results suggest that expression of E2F I induces foci consistent with IRIF, and not foci correlative of S phase induction.
D. E2Fl blocks eell eycle progression in human fibroblasts, roles for Nbsl and
53BPl
The observed formation ofMRN foci following expression ofE2FI suggests that
E2FI may be activating a cell cycle checkpoint. However, the importance of E2FI and
E2F3a in promoting the transition from G to S phase of the cell cycle has been well documented. Ectopic expression of E2FI or E2F3a in rodent fibroblasts has been shown to promote S phase entry, even under seru deprivation (76, 183 210). However, ectopic
expression of E2FI in human cells has been found to be insufficient for S phase entry,
and can lead to senescence in normal human fibroblasts (83 , 240, 265 379). Following '#
124
Q) :e :J L. J!
Con E2F1 E2F3a
Figure 5.10. E2Fl, butnot E2F3a, bloeks cell eycle progression.
Serum starved HEL fibroblasts were infected with AdCon, AdE2FI , or AdE2F3a at an
MOI of 250. Immediately following infection, cells were returned to normal growth medium. Cells were fixed and stained for BrdU incorporation at 24 hpi. A minimum of
300 cells per well was counted. 125
Con E2F1 E2F3a Ser1981 Atm
Figure 5.11. Expression of E2Fl and E2F3a results in increased levels of the phospho-serine 1981 form of Atm.
HEL fibroblasts were infected with AdCon, AdE2FI , or AdE2F3a at an MOI of 250.
Cells were harvested and lysates generated at 24 hpi. Western blot analysis was performed to determine the levels of the phospho-serine 1981 form of Atm. 126
DNA damage, relocalization of DNA damage response proteins, including the MRN complex and 53BPI , can lead to activation of a cell cycle checkpoint, blocking cell cycle progression (84, III , 185 384 394 423 450). We therefore tested the ability ofE2FI block cell cycle progression. When normal growth medium was added to starved cells at the time of E2F expression, we found that E2FI blocks cells from entering the cell cycle while expression of E2F3a allowed cell cycle progression as measured by BrdU incorporation (Figure 5.9). A block in cell cycle progression is commonly observed following DNA damage when a cell cycle checkpoint is activated to prevent replication of damaged DNA (49). Firing of an S phase checkpoint can involve activation of Atm and downstream signaling proteins (191). We found that expression ofE2FI or E2F3a able to activate Atm, as measured by an increase in the phospho-serine 1981 form of Atm
(Figure 5.10).
Since Atm activation was not unique to E2FI and did not correlate with the observed block in cell cycle progression, we examined the roles of DNA damage signaling proteins Nbsl and 53BPI in preventing cell cycle progression. Nbsl and 53BPI are important in the Atm-dependent S phase checkpoint, and loss of either Nbsl or
53BPI hypersensitizes cells to ionizing radiation and abrogates activation of DNA damage induced cell cycle checkpoints (66, 84, 111 423 427). To determine the roles of
Nbsl and 53BPI in blocking cell cycle progression following expression of E2FI , we used siRNAs to reduce the levels of Nbsl or 53BPI in cells (Figure 5. 11 A). We found that transfection of cells with siNbsi or si53BPI prior to expression of E2FI partially alleviated the block in cell cycle progression (Figure 5. 11 B). Transfection of siNbsi and : '
127
53BP1 Nbs1
actin : actin
siNbs1 a siC on si53BP1 siNbs1 a siCon si53BP1
Con E2F1
Figure 5.12. Roles for Nbsl and 53BPI in blocking eell eycle progression.
(A) HEL fibroblasts were transfected with siRNAs targeted to 53BPI or Nbs1. Cells were
harvested and lysates generated at 48 hpi. Western blot analysis was performed to
determine levels of 53BPI or Nbsl protein. (B) HEL fibroblasts were transfected with
marked siRNAs, serum deprived for 48 hours, and infected with AdCon or AdE2FI at an
MOI of 250. Immediately following infection, cells were returned to normal growth
mediurh Cells were fixed and stained for BrdU incorporation at 24 hpi. A minimum of
300 cells per well was counted. ;,) \) ') " \) ') ,, \)
128
Con E2F1
co ,, co ') co '\') \) 10 ') hpi: \) b- co ,, \) b-co '\')
p21
actin
Figure 5.13. Expression of E2Flieads to aecumulation of p21 protein.
HEL fibroblasts were infected with AdCon or AdE2FI at an MOl of 250. Cells were harvested and lysates generated at indicated hpi. Western blot analysis was performed to
determine levels of p2I protein. Actin blot is shown as protein loading control. ..;,-, " () '"
129
w "en O p21
actin !J'Jlr"'-il"._w
siCon sip21 a siCon sip21a Con E2F1
Figure 5.14. Role for p21 in blocking eell eycle progression.
(A) HEL fibroblasts were transfected with siRNAs targeted to p2I. Cells were harvested
and lysates generated at 48 hpi. Western blot analysis was performed to determine levels
of p2I protein. (B) HEL fibroblasts were transfected with marked siRNAs, serum
deprived for 48 hours, and infected with AdCon or AdE2FI at an MOI of 250.
Immediately following infection, cells were returned to normal growth medium. Cells
were fixed and stained for BrdU incorporation at 24 hpi. A minimum of 300 cells per
well was counted. "- (; " ".""" """' ;::
130
Can E2F1 C Cr r: (' o 1.a.U 0 w Ow " p53 p21 " ,
actin lli actin F"'-"" Q) CJ :J CJ Q) ef
siCon sip53 siCon sip53 Con E2F1
Figure 5.15. Role for p53 in blocking eell cycle progression and p21 aeeumulation.
(A) HEL fibroblasts were transfected with an siRNA targeted to p53. Cells were
harvested and lysates generated at 48 hpi. Western blot analysis was performed to
determine levels of p53 protein. (B) Cells were transfected with marked siRNAs, serum
deprived for 48 hours, and infected with AdCon or AdE2FI at an MOI of 250.
Immediately following infection, cells were returned to normal growth medium. Cells were fixed and stained for BrdU incorporation at 24 hpi. A minimum of 300 cells per
well was counted. (C) Cells were transfected with the marked siRNA 24 hours prior to
infection with AdCon or AdE2Fl at an MOI of 250. Cells were harvested and lysates
generated at 24 hpi. Western blot analysis was performed to determine levels of p2I protein. -=,'.
131
si53BPI together showed no effect on cell cycle progression above transfection of the
single siRNAs (data not shown). This data suggets that Nbsl and 53BPI playa role in
blocking cell cycle progression following expression of E2F I.
E. Roles for p53 and p21 in E2Fl-indueed eell cycle bloek
In the DNA damage response pathway, Nbsl and 53BPI function upstream of
p53 activation(185, 423). We therefore determined ifp53 played a role in the block in
cell cycle progression. We found that treatment of cells with sip53 prior to expression of
E2FI partially alleviated the observed block in cell cycle progression (Figure 5.14 B).
Activation of p53 following certain forms DNA damage can result in activation of a p2I-
dependent cell cycle checkpoint (23 98). We therefore determined ifp2I played a
role in the block in cell cycle progression observed following expression of E2F1. We
found that expression of E2F I in randomly cycling cells resulted in a rapid accumulation
ofp2I protein (Figure 5. 12). Additionally, we found that reducing p2I protein levels in
cells with an siRNA targeted to p2I (Figure 5.13 A) partially alleviated the block in cell
cycle progression observed following expression of E2F I (Figure 5. 13 B). Treatment of
cells with sip21 together with siNbsi and/or si53BPI showed no effect on cell cycle
p2I is a known transcriptional progression above sip2I alone (data not shown). Since
target of p53 (97, 98), we determined if p53 was responsible for the observed induction 14 A) ofp21. We found that treatment of cells with an siRNA targeted to p53 (Figure 5.
reduced the ability ofE2FI to promote accumlation ofp2I (Figure 5.14 C). Treatment of
cells with sip53 together with sip2I , siNbsI , or si53BPI showed no effect on cell cycle 132
progression above sip2I alone (data not shown). Together, these observation suggest that expression of E2F I blocks cell cycle progression in part through its ability of activate p53 resulting in accumulation of p2I protein.
The observation that expression of E2F 1 resulted in a rapid accumulation of p2I protein and a block in cell cycle progression suggests that E2FI is activating a cell cycle checkpoint prior to the induction of apoptosis. To test whether checkpoint activation by
E2FI results in a delay in the apoptotic response, we reduced the levels of 53BPI or p2I in cells with siRNAs and examined the ability of E2FI to induce apoptosis. We found that treatment of cells with si53BPI or sip2I prior to expression of E2FI resulted in an accelerated induction of apoptosis (Figure 5. 15). Taken together, these results suggest that E2Fl is activating a cell cycle checkpoint that delays the induction of apoptosis. 133
70 -
-Can siCon E2F1 siCon C) 40 -Can si53BP1 c: E2F1 si53BP1
Q) 20 -Can sip21 E2F1 sip21 Q)
hpi: 24
Figure 5.16. Reduced levels of 53BP1 of p21 leads to enhanced E2F1-induced apoptosis.
HEL fibroblasts were transfected with the marked siRNA 24 hours prior to infection with
AdCon or AdE2FI at an MOl of 250. Cells were harvested and apoptosis detected at indicated times following infection. Error bars represent standard deviation of experiment performed in trplicate. 134
E. Discussion
In this chapter we describe E2FI-induced relocalization of the DNA damage recognition MRN complex into discrete nuclear foci characteristic of foci observed
following DNA damage. MreII relocalization is found to correlate with relocalization of
the DNA damage response proteins yH2AX and 53BP1. We also find that expression of
E2F I results in a block in cell cycle progression, at least partially dependent on activation
of a cell cycle checkpoint pathway involving NbsI , 53BPI , p53, and p21. Expression of
E2FI results in an increase in p2I protein levels that correlates with the observed block in
cell cycle progression. These data suggest that initially following expression of E2F I , a
growth arrest pathway consisiting ofNbsI , 53BPI , and p2I predominates (Figure 5.
left). The finding that reducing p2I and 53BPI protein levels accelerates apoptosis
suggests that continued expression of E2F I or inactivation of this cell cycle checkpoint
pathway favors activation ofthe apoptosis pathway (Figure 5. 16 right)
Our data show that formation of Mre II-containing foci following expression of
E2FI correlates with relocalization of the DNA damage signaling proteins yH2AX and
53BP1. However, we observe incomplete overlap of these proteins. Colocalization ofthe
DNA damage response proteins MreII and yH2AX has been observed following y-
irradiation (51 , 309). However, these studies examined foci formation at later time points
following y-irradiation when the majority of lesions have been repaired (377). Using a
chromatin extraction protocol, Petrini and colleagues found that MreII and yH2AX foci
do not colocalize, but remain adjacent throughout the course of DNA repair (267, 268).
This is not surprizing given that y H2AX foci can be observed not only at sites of ..
135
Initial E2F1 Activation Sustained E2F1 Activity
E2F1 E2F1
Nbs1 53BP1 Nbs1 53BP1
p53 p53 .. p21
GfO\N\n GroWth p.."ffes\ Arrest
Figure 5.17. Model ofE2Fl-indueed apoptosis and growth arrest.
(Left) Initial expression ofE2FIleads to a block in cell cycle progression through NbsI
53BPI , p53 , and p21. (Right) Continued expression ofE2FIleads to activation of the
apoptosis program. Reduction of 53BPI or p2I protein levels acts to reduce the ability of
E2F I to promote growth arrest and favors the apoptosis pathway. 136
damaged DNA, but also over large portions of a chromosome surrounding DNA damage
(337). Therefore, our data showing incomplete overlap ofE2FI-induced foci is consistent with foci observed following y-irradiation. The question remains as to why DNA damage-like foci form following expression of E2F1. We speculate that ectopic E2FI activity may lead to DNA breaks either directly or as a result of slowing or preventing
DNA repair. The MRN complex and other DNA damage signaling proteins may then recognize these breaks in a manner similar to the DNA damage response, and initiate a signaling pathway resulting in activation of a cell cycle chekpoint or apoptosis.
Our data describe a partial alleviation of the E2FI-induced block in cell cycle progression by siRNA targeting of the p21 checkpoint pathway. While it appears that the p21 pathway is only partially required for this checkpoint, we cannot rule out the possibility that, due to incomplete knockdown in gene expression, that the observed checkpoint is totally dependent on the p2I pathway. However, this seems unlikely due to the undetectable levels of p2I protein remaining after siRNA treatment, and only a modest alleviation of the cell cycle block. It remains unclear as to what additional components may be required for mediating this block. It is possible that p I4AR may be contributing to the observed block in cell cycle progression. pI4ARF has been found to playa role in E2FI-induced senescence (83). However, the role of pI4AR in the E2FI- induced senescence pathway is dependent on p53 function (83). We observe only a partial increase in cell cycle progression following treatment of cells with sip53 suggesting that an additional pathway(s) that are p53 independent may be contributing to the observed growth arrest. One such pathway may involve activation of the cyclin 137
dependent kinase inhibitor pI6ink4a. pI6 plays roles in both growth arrest and senescence by inhibiting Rb phosphorylation (7, 189, 381). The potential role of pI6 in E2FI- mediated growth arrest remains to be determined. :.
138
CHAPTER VI
DISCUSSION
I:i, 139
A. Thesis overview
The goal of this study was to determine the pathway(s) by which deregulation of the Rb growth control pathway results in p53-dependent apoptosis. It had been well established that inactivation of Rb by mutation, gene deletion, or binding by viral oncoproteins results in apoptosis that is often p53- dependent. Mouse models and in vitro evidence suggest that E2FI is the primary mediator linking Rb inactivation to p53 activation and apoptosis (246, 305, 316 408). The prevailing hypothesis ofE2FI-induced apoptosis proposed a central role for the pI9 /Mdm2 pathway in activation ofp53 and apoptosis following deregulation of cellular growth control (25 , 189).
The data presented in this thesis demonstrate that pI ~RF is not required for p53- dependent apoptosis following expression ofE2F1. Instead, we found that E2FI-induced apoptosis requires covalent modifications of p53 by a caffeine sensitive kinase(s). The
DNA damage kinases Atm and Chk2, both of which are sensitive to caffeine, as well as the DNA damage response protein Nbs I are required for E2F I and HPV - 16 E7 to induce
apoptosis. Upregulation of Chk2 is specific to E2FI , and correlates with p53 activation and apoptosis. The data also show that E2FI specifically induces the relocalization of
DNA damage recognition and response proteins including MreII , NbsI , 53BPI , and yH2AX prior to apoptosis. The relocalization of DNA damage sensors correlates with a block in cell cycle progression that is mediated, in part, by activation of a p2I-dependent cell cycle checkpoint. These result suggest that deregulation of E2F I results in activation
of a cell cycle checkpoint prior to the induction of apoptosis. I' '
140
B. E2Fl is the eentral mediator of apoptosis following loss of Rb growth eontrol.
E2F I has been reported to play dual roles as both an oncogene and a tumor suppressor. It has been proposed that the ability of E2F I to act as a tumor suppressor is suppressive mainly due to activation of the apoptosis program (408). The tumor properties of E2FI are revealed in mice lacking E2FI which exhbit defects in apoptosis and display an increase in tumor formation (113 , 449). The role of E2FI in the DNA damage pathway, discussed below, also support the role ofE2FI as a tumor suppressor.
The role of E2FI as an oncogene have been attributed mainly to the ability of E2FI to promote re-entry into the cell cycle. Overexpression of E2F I and activated Ras
in vivo can also primar cells can result in transformation (182). Overexpression ofE2FI
result in an increase in tumor formation and loss of E2F I leads to reduction in tumor
growth in Rb +/- mice (317, 448).
Our data support the roles of E2FI as both an oncogene and a tumor suppressor.
Overexpression of E2FI or inactivation of Rb by the HPV- I6 E7 protein results in
apoptosis, consistant with the role of elevated levels of E2F I acting to suppress
tumorogenesis. Specifically, the data in chapter IV demonstrate that E2FI , and not the
other activator E2F family members, acts as the central mediator of apoptosis induction
in response of loss of Rb growth control. However, our studies also indicate how E2FI
may also function as an oncogene. Overexpression ofE2FI in human fibroblasts leads to
relocalization of DNA damage recognition and response proteins , suggesting that
elevated E2FI activity may lead to an increase in mutation rate and/or genomic
instability, possibly by an accumulation of DNA breaks. 141
It appears as though p53 also plays dual roles in the E2FI signaling pathways.
Accumulation of p2I protein following expression of E2F I is likely due to
transactivation of p2I expression by p53. Indeed, targeting of p53 with an siRNA reduced the ability of E2FI to increase p2I protein levels. Induction of apoptosis by
E2FI also requires activation of p53 through covalent modification by Atm, Chk2, and possibly additional kinases. It remains to be determined if there are differences in p53 activation by E2FI that mediate either a growth arrest or apoptosis.
It is concievable that in normal cells, E2F I has the ability to respond to loss proper growth control and induce a block in cell cycle progression associated with redistribution of DNA damage repair molecules. If proper cell cycle regulation is not returned, sustained E2FI activity would then promote apoptosis. In human cells, the inactivation ofRb by cellular or viral oncoproteins would lead to increased E2FI activity.
E2F I would then activate p2I resulting in activation of cell cycle checkpoint to prevent inappropriate proliferation and tumorogenesis. Activation of p2I could further inhibit
E2FI activity by blocking cellular cyclin-Cdk activt and additional Rb phosphorylation.
However, sustained E2FI activity may bypass the cell cycle arrest, and promote
apoptosis. Therefore, E2F I may act as a transducer of signals resulting from deregulation
of Rb growth control, promoting a block in cell cycle progression to allow for restoration
of proper cell cycle regulation or apoptosis to eliminate the potential for subsequent
transformation of cells. It remains unclear as to why E2F I blocks cell cycle progression
in human fibroblasts but is able to induce S phase in rodent cells, even in the presence of
increased p2I protein levels (77). It is possible that rodent E2FI fails to activate the p53- 142
independent checkpoint pathway suggested in Chapter V. This would be consistent with the observations that rodent fibroblasts fail to undergo senescence as efficiently as human fibroblasts (346, 376).
The importance of the E2F family, and E2FI specifically, in responding to loss of
E2F appropriate cellular proliferation control suggests that genes may be an attractive target for mutation in tumorogenesis. However, while mutations in the Rb growth control pathway are common in tumors, mutations in E2F family members are rarely found in
human tumors. E2F expression has been reported to be either reduced or amplified in some different tumor types (99 106 110 157 177). However, the infrequency ofE2FI mutations identified in human tumors supports the dual roles of E2FI demonstrated our findings. We speculate that mutations resulting in loss of E2F function would be disadvantageous for tumor formation possibly due to a loss in proliferative capacity.
Amplification of E2FI would be similarly disadvantageous due to the ability of E2FI to induce apoptosis when deregulated.
c. Pathways tinking E2F! to p53
1. Role of ARF in E2F signating
Our data presented in chapter III clearly show that distinct pathways mediate p53 accumulation and p53-dependent apoptosis following ecotopic expression of E2FI in rodent fibroblasts. However, in MEFs, mouse models, and human cells pI ~RF AR is dispensible for apoptosis induction by E2FI (236, 340, 342 401 , 409). While pI9 AR not required for E2F I-induced apoptosis, it is likely that activation of the p I9 /Mdm2 ,j .
143
AR in negative pathway influences E2FI signaling. Evidence points to a role for pI9 AR and human pI4AR both physically regulation of cellular growth control. Mouse pI9
interact with E2FI , inhibit E2FI transactivation function, and promote its degradation ARF , expression ofpI9 is able to inhibit (107 251 256). In addition to inhibition ofE2FI
deregulated proliferation associated with expression of the cellular oncoproteins Ras and AR also Myc as well as the viral oncoproteins EIA and E7 (73 , 303 , 306, 463). pI9
physically interacts with DPI , the hertodimeric E2F binding partner (68). The interaction AR and D P , suggesting that between p I9 I is inhibited by coexpression of D P I and E2F I negative DPI-E2FI complexes that exist during the G1- S boundary are resistant to AR (68). Taken together ectopic regulation by p I9 , induction of p I9 , either by
expression of E2F or expression of HPV - 16 E7, would provide negative feedback on the
growth promoting activity ofE2FI.
However, there is a species-specific difference in the activity of mouse pI9 AR in regards to p53 stabilization and cellular growth control. The results and human pI4
presented in chapter III describe the majority of p53 accumulation following expression
of E2FI is dependent on pI9 , while in human fibroblasts, using an siRNA to target (236). The pI4AR had little effect on p53 stabilization following expression of E2FI
human I4AR protein lacks the C-terminal fort amino acids that are present in the mouse
pI9AR protein (25). These C-terminal amino acids have been proposed to be important AR and the peroxisomal targeting chaperone protein for an interaction between p I9 AR may reduce the growth suppressive PexI9p (390). The interaction ofPexI9p with pI9 AR in mouse cells by sequestering pI9AR in the cytoplasm (421). Human activity ofpI9 144
ARF pI4 lacks the C-terminal PexI9p interaction domain, and so has been proposed to
have enhanced growth suppressive properties (421), possibly through mechanisms
independent of p53 (46, 428)
The data presented in chapter V describe a block in cell cycle progression
following expression of E2FI in normal human fibroblasts. The block in cell cycle
progression is due, in part, to the activation of a NbsI- and 53BPI-dependent checkpoint
pathway. Activation of this pathway results in a rapid induction ofp2I protein following
expression of E2F1. Due to the enhanced growth suppressive properties of human pI4 , it is concievable that induction of pI4AR protein following expression of E2FI
could also contribute to the S phase block that is observed in normal human fibroblasts and not in rodent fibroblasts.
The cyclin-dependent kinase inhibitor p I6ink4a may also playa role in the block in cell cycle progression observed following expression of E2F I. p 16 prevents activation of
Cdk4/6, which is thought to be an essential step in Rb inactivation and crucial for the entry into S phase (108, 156). Upregulation ofpI6 protein levels by E2FI would act to maintain Rb in its growth inhibitory state, blocking additional E2F activity and preventing the transition from G to S phase. The importance of the p 16 pathway in controlling deregulated proliferation is suggested by the finding that most, if not all human tumors contain mutations in the cyc1in D/p I6/Rb pathway or in proteins that control this pathway (366). Similar to the function proposed above for ARF, pI6 may also provide negative feedback on the growth promoting activity of E2F I. 145
2. IRIF -like foei formation
Data presented in chapter IV and chapter V suggest that ectopic expression of
E2FI or Rb inactivation by HPV- I6 E7 activates an apoptotis program with similarities to the apoptosis pathway activated by DNA double strand breaks. Both expression of
E2FI and DNA double strand breaks result in relocalization of DNA damage repair and
checkpoint signaling proteins to discrete nuclear foci. In response to y-irradiation or
certain genotoxic agents, proteins, including yH2AX, 53BPI , and the MRN complex
relocalize to sites of actual DNA breaks. MRN and yH2AX were though to be responsible
for the initial recognition of DNA breaks, with the MRN complex functioning to initiate
repair and yH2AX functioning to recruit other DNA repair factors as well as 53BPI to
activate a cell cycle checkpoint. However, recent evidence suggests that the mediator of
DNA damage checkpoint protein I (Mdcl) is required for MRN, yH2AX, and 53BPI foci
formation (127 , 242, 271 , 385, 445) as well as for effcient Chk activation following y-
irradiation (242 , 311 , 445). Inhibiting Mdc I expression with an siRNA results in
decreased p53 stabilization and a failure to activate a cell cycle checkpoint following
irradiation (127 242 385).
The mechanism of MRN, yH2AX, and 53BPI foci formation following
expression of E2F I remains unclear. It has been proposed that E2F I may target the MRN
complex to DNA by virtue of an interaction with the Nbsl component of the complex
(254). This scenerio seems unlikely due to the foci formation observed following
expression of E2F 1 283, an E2F I mutant lacking the Nbs I interaction domain.
Additionally, no interactions between E2FI and either yH2AX or 53BPI have been 146
described. It is possible that E2FI could direct MRN, yH2AX, and 53BPI foci formation through interaction with Mdc I , if such an interaction exists. Another possibility of how
E2FI induces DNA damage foci is that ectopic expression ofE2FI prevents the repair of
DNA damage that may arise during replication or induces DNA damage by an unown mechanism prior to activation of the apoptosis program. E2FI-induced DNA damage
would then recruit DNA damage repair and checkpoint signaling proteins resulting in p53
activation and either apoptosis or the growth arrest described in chapter V.
The possibility exists that expression of E2FI could result in DNA breaks
indirectly through the induction of reactive oxygen species (ROS). Accumulation ofROS
can lead to the induction of apoptosis through several mechanisms, including direct
Myc caspase activation (41). Expression of the oncogene results in ROS accumulation
that contributes to the DNA breaks and MreII relocalization observed following
expression of c-Myc (413). Together with the observations that expression of E2FI can
induce ROS accumulation and that c-Myc induced apoptosis is compromised in E2Fr
MEFs suggests that c-Myc induced accumulation ofROS, and subsequent DNA damage
may be a consequence of E2FI activation (228, 397). Therefore, it is possible that ROS-
induced DNA damage contributes to the observed relocalization of DNA damage sensors
following expression of E2F!. The failure of expression of E2F3a to result in
relocalization of DNA damage sensors may then be due to a failure to cause ROS
accumulation. The contribution of ROS accumulation to E2FI-induced apoptosis, and
E2F signaling, remains to be determined. 147
The findings in chapter V that expression of E2F I in human cells results in a block in cell cycle progression coupled with the ability of E2FI to induce DNA replication in rodent cells, suggests that E2FI may be activating a DNA replication fork arrest. Replication fork arrest occurs in response to perturbations in many aspects of
DNA replication including insufficient pools of dNTPs, polymerase inhibition, DNA modifications, or DNA breaks that may arise during replication (300). It is possible that constitutive expression of E2FI induces DNA damage as a consequence of stalled replication forks, and results in recruitment of recognition and repair complexes. This seems unlikely, as E2F3a induces inappropriate S phase but fails to induce foci formation or checkpoint activation. Additionally, stalled replication forks activate p53 through an
Atr/ChkI-dependent pathway (22), contrary to the Atm/Chk2 pathway required for E2FI to activate p53.
3. Role of Atm in E2F signating
Atm serves as the primary link between DNA damage sensors and activation of cell cycle regulatory proteins. In undamaged cells, Atm exists as an inactive dimer.
Rapidly following DNA double strand breaks Atm undergoes intermolecular autophosphorylation at the serine 1981 residue resulting in dimer dissociation and an increase in Atm kinase activity (17). Atm phosphorylation is thought to be triggered by chromatin changes associated with an influx of DNA repair factors to sites of DNA damage (17). It remains unclear as to how Atm is activated following expression of
E2FI , E2F2, E2F3a, or HPV- I6 E7. We speculate that expression ofE2F proteins or E7 148
results in chromatin changes associated with induction of S phase or activation of DNA damage response proteins. In the case of HPV - 16 E7, activation of Atm may be a result of chromosomal structural changes and DNA breaks that occur following E7 expression
(86). However, since activation of Atm by E2FI results in apoptosis, we canot rule out the possibility that the different E2F family members activate Atm by distinct mechanisms.
Interestingly, Atm was found not to be required for apoptosis resulting from Rb inactivation in murine brain choroid plexus epithelium (233). In this model, apoptosis resulting from Rb inactivation is also dependent on E2FI and p53 , and occurs in the
absence of pI ~RF (408 , 409). While it is not apparent why this apoptosis is Atm- independent in the choroid plexus epithelium , p53-dependent apoptosis that is Atm- independent has been described in certain cell types (21 , 151). Alternatively, these observations suggest that there may be a species-specific bias for Atm in the E2FI-
I- mediated apoptosis pathway. Indeed, the reduction in apoptosis observed in atm MEFs is not as dramatic as that seen in human dermal fibroblasts following E2FI expression.
We speculate that in the murine system other signaling pathways such as Atr/ChkI may
compensate for the loss of Atm, whereas a more stringent requirement for Atm in human
dermal fibroblasts is observed. Therefore, there may be both cell-type and species-
specific requirements for Atm in apoptosis induction, and it is possible that other Atm-
related kinases may compensate for the loss of Atm function in some cells. --
149
viral oncoproteins Rb Inactivation DNA damage
E2F3a Rb/E2F E2F2 S Phase -- L E2F1
;iF
Apoptosis Growth Arrest
Figure 6.1. Model of E2F signaling network.
Inactivation of Rb durng a normal cell cycle leads to activation of E2FI , E2F2, and
E2F3a resulting in cell cycle progression (shown in green). Inapproprite Rb inactivation by mutation or viral oncoproteins can lead to activation of two distinct, E2FI-specific pathways. The apoptosis pathway (shown in red) signals to p53 through NbsI , Atm, and Chk resulting in p53 covalent modification and apoptosis. The checkpoint pathway
(shown in blue) signals, in part, through NbsI , 53BPI , p53, and p21. The checkpoint pathway also involves a signaling pathway independent ofp53 and p2I through an, as of yet, undefined mediator. AR may playa role in E2FI signaling by increasing pools of p53 protein. 150
4. Aetivation of p53 for the induction We have defined a requirement for covalent modification ofp53 of apoptosis following expression of E2F1. Activation of p53 by covalent modification mouse) and serine 20 in specifically phosphorylation of serine 15 (serine 18 in the response to DNA damage, has been a topic of great interest due to the location of these residues within the Mdm2 interaction domain of p53. Mutational analysis of p53 sites serine 20, have been known to be covalently modified, including serine 15 and anyone of these sites for inconsistent regarding the requirements for phosphorylation at 441). The observation p53 dependent apoptosis in response to DNA damage (13 411
that expression of E2FI , 2, and 3a results in phosphorylation of p53 at the serine
residue suggests that some p53 modifications may occur as a result of ectopic induction
of S phase. Chromatin alterations that occur as a consequence of DNA replication may be 15 form ofp53. sufficient to activate Atm, leading to an increase in the phospho-serine
This initial modification of p53 at the serine 15 residue during the onset of S phase would
be insufficient for activation of the apoptosis program, but may prime p53 for additional , occur modifications should errors, including DNA damage or stalled replication forks
during DNA replication.
While our study examined the phosphorylation status of two N-terminal residues
on p53 known to be phosphorylated following DNA damage, we have not established
that either of these modifications are causal in p53 mediated apoptosis, nor have we ruled
out the involvement or importance ofmodification(s) to other residues on p53 following
E2FI expression. However, our finding that apoptosis is reduced when E2FI is 151
coexpressed with a p53 mutant lacking many of the known phosphorylation sites demonstrates a contribution by potential p53 phosphorylation targets to E2FI-induced apoptosis.
C. Model of E2F! apoptosis pathway
Expression ofE2FIleads to relocalization of DNA damage repair and recognition proteins, including the MRN complex and 53BPI , to discrete nuclear foci. The molecular events leading to relocalization of these components remains to be determined.
Activation of Atm by autophosphorylation is proposed to occur following relocalization of DNA damage sensors by, as yet, an unkown mechanism (17). Activated Atm is then able to phosphorylate p53 at the serine 15 residue. However, expression of E2F I , E2F2 and E2F3a results in Atm activation and phosphorylation of the serine 15 residue on p53.
Unique to E2FI is the ability to induce Chk2 mRNA and proteins levels. Chk is able to phosphorylate the serine 20 residue, and this modification is found to correlate with
E2FI-induced apoptosis. Chk2 may be limiting in this pathway, and when provided in trans Chk enhances E2FI-induced apoptosis and allows E2F2 to induce apoptosis. It is
ARF possible that E2FI , E2F2 , or E2F3a could also signal throughpI4 /Mdm2 to increase p53 protein levels and that the increased pools of p53 would provide more substrate for
Atm andChk (Figure 6. 1). 152
D. Summary
The dual functions of E2F I in checkpoint activation and apoptosis identified in this dissertation provide novel insight into the function of Rb/E2F growth control both in normal cells and in cells containing mutations in the Rb/E2F growth control pathway.
The genomic integrity of mamalian cells is subject to constant attack, either by external damaging agents, or internal mutagens such as errors in DNA replication. If mutations arise that compromise proper Rb-mediated growth control, our results suggest that E2FI may initiate a signaling cascade to activate a cell cycle checkpoint. If proper growth control cannot be restored, continued E2FI activity may lead to apoptosis, preventing an accumulation of mutations that may lead to transformation. The importance ofE2FI as a signal transducer of loss of proper Rb-mediated proliferation control can be observed in
mouse models. Loss of E2F I in Rb mouse models results in a decrease in S phase and a
decrease in apoptosis in the central nervous system and choroid plexus, suggesting an
important role for the E2FI apoptotic pathway in preventing tumor formation (305 408).
The obervation that adult mice lacking E2F I develop tumors (449) also suggests that the
E2F I signaling network is important in suppressing tumor formation.
However, our data may point to a role for an E2FI-specific checkpoint activation
E2F I fails in these mouse models. It is possible that loss of Rb function in mice lacking
to activate the E2FI-induced cell cycle checkpoint pathway described in Chapter V.
Since E2F3a, and possibly E2F2, cannot compensate for E2FI in this pathway, cells may
fail to properly block cell cycle progression and this may lead to additional mutations.
Compounding the loss of the E2F I growth control checkpoint would be the failure to /-
153
, loss of E2FI would lead to an activate the E2FI-induced apotosis pathway. Therefore inability to respond to loss of Rb-mediated growt control.
Although we have defined a pathway linking deregulated E2FI activity to p53 Atm/Chk2/p53 and apoptosis, integration of E2FI signaling and activation of the
I in apoptosis pathway also offers a mechanism for the proposed involvement of E2F resulting from DNA damage. Following treatment of cells with DNA damaging agents
E2FI protein accumulates (30, 158, 165 235 266 294), and is phosphorylated at an N- terminal Atm recognition sequence that is unique to E2FI among the E2F family members (235). This phosphorylation of E2FI is largely dependent on Atm and is required for efficient E2FI stabilization following DNA damage (235). Chk2 has also , and this been shown to phosphorylate and stabilize E2FI following DNA damage modification has been shown to be required for E2F I dependent apoptosis following
DNA damage by altering its promoter specificity (383). Additionally, DNA damage E2Fr mice (235), suggesting induced apoptosis is compromised in thymocytes from
that E2FI has multiple roles in DNA damage signaling. We speculate that activation of
ARF E2FI by DNA damage leads to increasedpI4 expression resulting in increased pools
induce Chk2 of p53 protein. E2FI is also able to activate Atm kinase activity and
expression leading to increased p53 activation and E2FI activity. E2FI activation
following DNA damage would therefore act to amplify DNA damage signals converging
at p53 to result in either a growth arrest or apoptosis (Figure 6. 1). 154
E. Future directions While we have made significant advances toward our understanding of E2FI
signaling, a number of questions require further exploration. How does expression of
E2FI result in relocalization of DNA damage response factors? If DNA damage foci
form following DNA damage, what is causing the DNA damage following expression of
E2FI? The finding that E2FI activates both a cell cycle checkpoint and an apoptotic
response suggests that there may be a molecular switch between the divergent outcomes.
How the cell decides which pathway to favor remains unclear. A divergence in E2FI
function was also found between human and rodent cells. It remains to be determined
why E2FI can induce S phase in rodent cells, but blocks cell cycle progression in human
fibroblasts. Given the additional mechanisms of proliferation and checkpoint control
described in this dissertation, E2FI may function in multiple pathways in the cellular
response to DNA damage.
Lastly, the exact mechanisms that control E2F specificity remam to be
determined. The specific induction of Chk2 by E2F I suggests that transcriptional control
of pro-apoptotic , and possibly anti-apoptotic , genes defines the primary difference
between the activator E2F family members in the apoptosis pathway. Specificity for
E2FI-mediated transactivation of the subset of E2F responsive genes involved in
apoptosis would provide an attractive mechanism for the initiation of the apoptosis
program. However, transcriptional activation or derepression, while required for
apoptosis, does not account for all of the observed consequences of E2F I expression. The
difference between E2FI and E2F2 or E2F3a in the formation of DNA damage-like 155
be important for Mre II foci suggests that additional E2F I-specific domains may mediating this response. Specificity for E2F family members inactivation of the apoptotic pathway would therefore depend on multiple domains that are unique to E2FI and serve specific fuctions in activation of components of the pathway. r-,
156
CHAPTER VII
APPENDIX A 157
5 12
ug: pC DNA pCDNA E2F1
Figure AI. E2F! induces apoptosis.
HEL fibroblasts were transfected with the indicated amounts of either pCDNA or pCDNA-E2F1. Cells were harvested and apoptosis detected at 96 hpi. Error bars repesent standard deviation of experiment performed in triplicate. 158
is 2
siCon siChk2b siCon siChk2b
pCDNA pCDNA E2F1
Figure A2. siChk2 inhibits E2Fl-indueed apoptosis.
HEL fibroblasts were cotransfected with the marked siRNA together with either pCDNA or pCDNA-E2F1. Cells were harvested and apoptosis detected at 96 hpi. Error bars represent standard deviation of experiment performed in triplicate. 159
E2F1
E2F1 actin .n8
Figure A3. DNChkl and DNChk2 do not effect E2Fl expression.
Normal human fibroblasts were coinfected with AdDNChkI or AdDNChk at an MOI of
1000 and with AdE2FI at an MOI of 1000. Cells were harvested and lysates generated at
24 hpi. Western blot analysis was performed to determine levels of E2F I protein. Actin blots are shown as protein loading controls. - ,=-'" - ..=.
160
c: 7 2 6
g' 5
0: 2
Con E7 Con E7 Con E7 Con E7 Con E7 Con E7 siE2F3a siE2F3b siCon siE2F1a siE2F2a siE2F2b
Figure A4. Specific requirement for E2Fl in HPV-l6 E7 indueed apoptosis. infectioQ. HEL fibroblasts were transfected with the indicated siRNA 24 hours prior to with AdCon or AdE7 at an MOI of 1000. Cells were harvested and apoptosis detected at
96 hpi. Error bars represent standard deviation of experiment performed in triplicate. 161
CHAPTER VIII
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