Академический Документы
Профессиональный Документы
Культура Документы
3-O-Methyl-Nordihydroguaiaretic Acid
By
November 2006
Dissertation written by
Approved by
Accepted by
ii
TABLE OF CONTENTS
List of figures........ix
List of tables........xiii
List of abbreviations......xiv
Chapters
I. Introduction
3. HPV Virology
3.1 HPV classification...8
3.2 HPV genome structure.....10
3.2.1 HPV E2...13
3.2.2 AP-1.15
3.2.3 Sp1...16
3.3 High Risk HPV Life Cycle...17
3.4 Structures and functions of HPV gene products
3.4.1 HPV E1 protein......22
3.4.2 HPV E2 protein......24
3.4.3 HPV E1-E4 proteins..25
3.4.4 HPV E5 protein......26
3.4.5 HPV E6 protein......27
3.4.6 HPV E7 protein..32
3.4.7 HPV L1 and L2 proteins...40
iii
3.5 Escape of human papillomaviruses from the immune
system......41
1. Reagents..76
2. Cell Culture.77
4. MTT Assay..79
iv
5. Trypan Blue Assay.....79
6. Western Blots......80
8. Transfection Study.....82
9. Caspase Assays.......82
III. Results
v
6. The Caspase-3 Inhibitor ZAD-FMK Rescues lignan treated cells from
the apoptotic pathway.....110
8. The Bax and Puma genes are induced within lignan treated CaSki
cells.....118
IV. Discussion......124
V. References.......132
vi
DEDICATION
ACKNOWLEDGEMENTS
I could not have made it to the finish line with out the unwavering love
support of my husband and better half, Dr. Mathew Lesniewski. I am truly
blessed to find in you not only my soul mate, but a fellow nerd who loves science
as much as I do. I love that you are always available to discuss my work, the
meaning of the universe, and how to create wormholes. I love you with every
ounce of my being.
vii
I would also like to acknowledge my parents, Doug and Jan Allen. Thank
you for your unending help and support, both emotional and financial, and for
always believing in me. You are both my rock and truly encouraged me to see
this endeavor to completion. I am grateful for your encouragement to pursue my
dream and for loving me unconditionally as the bookworm I am. Dad, you
taught me to see the world through different eyes and imparted me with the
steadfastness to never give up. Mom, from you I got my ability to pay close
attention to detail and you taught me to be patient and meticulous in reaching
my goals. These are all qualities that will make me the best scientist I can be. I
love you both.
I would like to thank my sister Amy Allen and my brother Aarin Allen for
their support and encouragement. I am blessed to have you both as part of my
life.
Finally, I would like to extend my gratitude to the pack and the pride. There
is nothing sweeter than a friend who is pleased to see you at the end of a long
day. Your purrs and cuddles kept me sane.
viii
LIST OF FIGURES
Figure
1. The course of events that, over time, can lead to the development of
precancerous and cancerous cervical tumors.....9
ix
16. Intrinsic and extrinsic pathways to caspases activation....50
26. p53 protein levels do not change in C33-A cells treated with 30 M 3-O-
methyl-NDGA...96
28. Structure of the p53-luciferase vector used to transfect HeLa cells with
p53.........99
x
30. 15 M 3-O-methyl-NDGA stabilizes p53 within CaSki cells transfected
with p53-luciferase....102
38. Lignan treated cells are undergoing apoptosis as assayed using flow
cytometry and annexin V-FITC and propidium iodide staining...113
43. Western blot analysis shows that bax protein is induced by 30 M 3-O-
Methyl-NDGA in CaSki cells.118
xi
41. Western blot analysis shows that bax protein is induced by 30 M 3-
O-Methyl-NDGA in HeLa cells.....120
42. Western blot analysis shows that both PUMA protein is induced by 30 M
3-O-Methyl-NDGA in HPV DNA containing cells.....121
xii
LIST OF TABLES
Table
6. Cell lines used in this study and their HPV and p53 statuses....78
xiii
LIST OF ABBREVIATIONS
xiv
xv
CHAPTER ONE
INTRODUCTION
pathogens and are the leading causative agent of sexually transmitted disease
stranded DNA viruses. HPVs are members of the Papillomaviridae family and
are in the genus Papillomavirus which contains more than 100 types of the virus
subgenomic HPV amplicons (de Villiers et al., 2004). HPVs are ubiquitous and
infect a variety of hosts, however, humans and bovines are the most extensively
studied.
viral types and wide variety of lesions HPV infection can generate are illustrated
in table 1. The papillomaviruses that cause the common warts that grow on
hands or feet (plantar warts) differ from the HPVs that cause growths in the
throat or genital regions. All HPVs trigger proliferation of the cells they infect,
but only a few types are associated with the risk of developing cancer.
Sexually transmitted HPVs are divided into low-risk and high-risk types
associated with high-risk HPV types 16 and 18; nevertheless, the majority of
in the prevention of cervical cancer and other anogenital tumors occurred with
the advent of a vaccine against the HPV types responsible for a majority of these
tumors. The tetravalent vaccine Gardasil (Merck) against high risk HPV-16 and -
18 and HPV-6 and -11, the types responsible for a majority of genital warts, has
recently been approved for use by the FDA. Glaxosmithkline also has a vaccine
against HPV-16 and -18 in late stage clinical trials. Results from vaccine trials
have shown that the three-dose vaccine protects against persistent HPV infection
2
Table 1. HPV types in benign and malignant disease.
Skin warts
Plantar warts 1 2,4,63
Common warts 2,27 1,4,7,26,28,29,57,60,65
Flat warts 3,10 2,26,27,28,29,41,49
Anogenital lesions
Condyloma acuminata 6,11 2,16,30,40,41,42,44,45,54,55,61
CIN, VIN, VAIN, PIN, 16,18,31 6,11,30,34,35,39,40,42-45,51,52,
PAIN 56-59,61,62,64,66,67,69
Malignancies
Cervical cancer 16,18,31,45 6,10,11,26,33,35,39,51,52,55,56,
58,59,66,68
Other anogenital cancers 6,16,18 11,31,33
Laryngeal cancer 6,11 16,18,35
Oral cancer 16,18 3,6,57,16
Subungual, digital
3
vaccines will not eliminate the need for cervical cancer screening since they will
not protect the millions of women who are already infected with HPV who have
Cancer of the uterine cervix represents the second most common cancer
al., 2006). In 2006, 9,710 women were diagnosed with cervical cancer and 3,700
Worldwide, morbidity and mortality rates are far higher with an incidence of
490,000 new cases and 273,000 deaths occurring in 2005, according to the World
High-risk HPV infection is the primary risk factor for the development of
uterine cervix and at least 50 percent of other anogenital tumors contain high-
risk HPV DNA (zur Hausen 2001). However, infection with high-risk HPV does
not guarantee a patient will develop cervical cancer because most infections clear
within 12-18 months, and there have been very rare instances where women
have developed cervical cancers that are HPV-negative (Avrich et al., 2006).
4
those patients who do respond positively to chemotherapy have tumors that can
recur (Alexander et al., 2005; Dreyer et al., 2005; Lindeque 2005; Tewari and
Monk 2005). Therefore, the primary goal of this study is to identify novel
oncogenes.
as skin or the mucosal surfaces of the genital tract, anus, mouth, or upper
respiratory tract (Doorbar 2005). HPVs can be divided into cutaneous skin tropic
viruses and mucosal skin tropic viruses. HPV infections trigger the benign or
variety of lesions including common warts, plantar warts, flat warts, laryngeal
papillomas, genital condylomas, and cancerous tumors of the uterine cervix and
anus, and also the head and neck (zur Hausen 1999).
Low-risk cutaneous HPV infections are ubiquitous and are usually spread
papillomas. These include HPV-1 and HPV-2 which cause plantar warts on the
5
soles of the feet and common warts on the hands, respectively (Akgul et al.,
2006). Common warts and plantar warts appear as raised lesions that have a
and typically emerge and regress randomly over a period of weeks to months
(Fox and Tung 2005). Recurring cutaneous warts appear in about 10 percent of
adults as well. Low-risk HPVs can also cause subungual and periungual warts
that form under and around the fingernails and cuticles, or flat warts that can
emerge on the arms, face, forehead, or trunk. A few cutaneous HPVs, such as
HPV-5, can cause persistent subclinical infections that persist for a lifetime
associated with the lesions described above are not usually linked with an
and are transmitted through sexual contact. However, HPV types that have a
tendency to elicit genital warts are not the same ones that can cause cancer. For
this reason, sexually transmitted HPVs are divided into two groups, those
precancerous and cancerous lesions of the anogenital tract such as types 16, 18,
6
31, 33, 35, and 45 (high-risk) (Stanley et al., 2001).
women will become infected with one or more of the sexually-transmitted HPV
types during her lifetime (Baseman and Koutsky 2005). Genital or anal warts
genital HPV infections. HPV-6 and HPV-11 are responsible for a majority of
cases but other HPV types are known to cause this type of infection (Greer et al.,
1995). Nevertheless, other HPV types that infect the genital tract do not cause
sexually transmitted HPV infections clear the virus rapidly without ever
important to note that these people are able to pass the virus to others even if
development of cancer and are termed high-risk HPVs. High-risk HPV infection
that is not cleared by the immune system can lead to malignant transformation of
high-risk HPV types, especially uterine cervical tumors, kill several hundred
7
thousand people each year and represent a major disease burden in women
worldwide (Schiffman and Castle 2003). Persistent HPV infection is critical for
percentage of these tumors do not contain HPV DNA (Walboomers et al., 1999;
infections also develop other types of anogenital cancers or malignant head and
neck tumors. Known high-risk HPVs include types 16, 18, 31, 33, 35, 39, 45, 51,
52, 56, 58, 59, 68, 69, and possibly a few others (zur Hausen 2000, 2002).
Progression from normal epithelium to a CIN lesion to cancer takes decades and
factors (smoking, HIV infection, oral contraceptive use, and family history). The
typical time course of events that leads to the development of CIN and cervical
3. HPV Virology
8
High Risk HPV Persistance
Additive epigenetic events (oncogenes)
Progression
Regression
HPV Clearance
Figure 1. The course of events that, over time, can lead to the development of
takes decades and is preceded by the development of early and late precancerous
9
the order of discovery. HPV types differ from one another by at least a
10%difference in the gene sequences of E6, E7, and L1. Subtypes, or variants,
sequence from the primary type by less than 2-5% (DeVilliers et al., 2004).
base pairs in length. A map of the HPV genome is shown in figure 2. The
genome is divided into three segments, an early region, a late region, and a long
control region (LCR) (Zheng and Baker 2006). The three regions are separated
by two polyadenylation (polyA, pA) sites (see figure 2). The early region of the
HPV genome contains six open reading frames (ORFs): E1, E2, E4, E5, E6, and E7.
There is no E3 gene. Each ORF codes for an individual protein with the
corresponding name. The late region contains 2 ORFs, L1 and L2, that encode
the major and minor capsid proteins, respectively. A linearized map of the
10
Long control region (LCR)
TATA Signal
1,2
Poly A Signal 2
E6
65-556
E7
544-859
L1
5528-7153
HPV-16 E1
7904 base pairs 859-2811
L2 E4
4135-5655 3333-3617 E2
2726-3850
E5
3809-4098
Poly A Signal 1
Figure 2. Circular diagram of HPV-16 genome and open reading frames.
The three separate regions of the HPV genome along with early genes E1-E7 and
late genes L1 and L2 are shown. (Adapted from zur Hausen 1999).
11
HPV-16 Genome Organization
URR
E7 E1 E5 L2
E6 E6 L1
E4
3333 3617
12
organization and regulation of the HPV life cycle. The LCR region is about
850base pairs long and it contains the origin of replication as well as binding sites
for multiple transcription factors that regulate viral gene expression (Bernard
illustrated in figure 4. Both viral and cellular transcription factors, such as Sp1
controls early gene expression (prior to viral DNA replication) and the P670
promoter that controls late gene expression (following viral DNA replication).
The P97 promoter is active in both basal and differentiated keratinocytes and is
tightly regulated by upstream cis elements located in the LCR (Zheng and Baker
2006). These cis elements include four HPV E2 binding sites and binding sites for
host cellular transcription factors such as Sp1 and AP-1. Some of the
transcription factors that bind to the early promoter are depicted in figure 4 and
3.2.1 HPV E2
13
Human papillomavirus Long Control
Region (LCR)
transcription factors. Binding sites of viral and cellular transcription factors are
14
genes (Zimmerman et al, 2003). A dimeric -barrel motif in the DNA binding
The DNA of all HPVs contains multiple E2 binding sites. The E2 DNA
binding site sequences all contain a conserved but variable 4 nucleotide sequence
(N4 spacer). The makeup of this sequence determines E2 binding affinity. For
than 5TTAA (8-fold lower) or 5AATT (33-fold lower) (Zimmerman et al, 2003).
3.2.2 AP-1
subunits c-jun and c-fos. AP-1 binds to the DNA consensus sequence
seventh amino acid in an alpha helix stabilize the c-jun/c-fos interaction as well as
binding to DNA. DNA binding of AP-1 also involves ionic interactions between
DNA and groups of arginine residues located within each subunit. AP-1 controls
15
3.2.3 Sp1
It has been postulated that the tissue specificity of HPVs may, in part, be
due to the fact that the required set of transcription factors are only present in
Early mRNA transcripts that arise from the early promoter are
polyadenylated and each contains three exons and two introns that are capable
unique mRNA species that each have different coding potential. Viral proteins
are translated from mRNA via ribosomal scanning (Sen et al., 2004; Deng et al.,
2003).
The P670 late promoter that controls transcription of the L1 and L2 genes is
only active late in the life cycle of the virus and solely in differentiated
16
P670 promoter controls expression of the L1 and L2 genes whose gene products
skin, or the mucosal surfaces of the genital tract, anus, mouth, or airways, and
are not known to infect any other tissue type (Doorbar 2005). HPVs stimulate the
expansion of the infected cell population but HPV infections do not result in the
lysis and death of the host cell. Thus, papillomaviruses utilize an alternative
mechanism for transmission. The virus resides in, replicates in, and stimulates
the host cell to divide and differentiate. The mature HPV virions are then
(koilocytes) and this serves as the vector of transmission (Ackgul et al., 2006).
HPV- infected epithelium and provides an overview of the viral life cycle.
attaches to a receptor on the surface of a host cell, which has been identified as
17
Normal Squamous Epithelium HPV Infected Epithelium
Stratum
Corneum Keratin Release of
Envelopes mature virions
Stratum
Granulosum Keratins
Accumulate Capsid synthesis
and virion
Keratin assembly
Stratum Synthesis and
Spinosum Nuclear Late promoter
Breakdown active; genome
amplification
Activation of viral genes keeps cells dividing for genome amplification and viral
assembly and mature virions exit as dead cells are sloughed off. (Adapted from
18
Sloughing of virus
HPV Infection laden cells from
upper epithelial
layers
Normal Keratinocyte
Papilloma
Formation
Episomal HPV
DNA in nucleus
of infected cell
Figure 6. Illustration of HPV life cycle. HPV DNA copy number is low in
basal keratinocytes. A lesion develops and viral copy number increases in cells
that are closest to the surface. Mature virions are assembled in these cells and are
sloughed from upper epithelial layers creating the vector for transmission.
19
an integrin (Yoon et al, 2001). The virus then penetrates the cell membrane.
Inside the cell, the virus uncoats and viral DNA is released from the capsid.
The viral DNA is delivered to the nucleus of the infected cell. The closed,
circular, double stranded genome encodes eight open reading frames (ORFs)
Once the viral DNA reaches the nucleus, cellular transcription factors interact
with the non-coding viral regulatory region (LCR) and transcription of two
transforming early genes, E6 and E7, begins (Zheng and Baker 2006).
The viral E1 and E2 genes are expressed concomitantly with the E6 and
E7 genes and function to regulate viral DNA replication and transcription. Both
in duplex DNA and controls transcription of E6 and E7, each of which have
multiple functions that stimulate growth of infected cells (Zheng and Baker
2006). The virus replicates in a rolling circle replication mode. The progression
of the viral replication and maturation are closely linked with the differentiation
of the infected basal keratinocytes. As the basal cells containing HPV divide and
begin to migrate toward the surface, the daughter cells carry multiple copies of
the HPV genome. During the early phase of infection, the viral genome persists
20
extrachromasomally as a slowly replicating plasmid, and the viral copy number
per cell is kept relatively low at fewer than 100 copies per cell (DiMaio and Liao
2006).
expressed, including E4 and E5. The E4 protein is believed to have a role in the
activation of the productive phase of the viral life cycle and may have a role in
viral genome amplification (Hebner 2006). Though little is known about its
infected cell (Straight et al., 1993; Gu and Matlashewski 1995; Hebner 2006).
and L2 that encode viral capsid proteins, are transcribed from the P670 late
promoter. At this point of the viral life cycle, the viral copy number per cell is
greatly increased from several hundred to several thousand per cell. L1 and L2
proteins self assemble into virus particles by encasing a viral genome (Sapp et al.,
1995; Finnen et al., 2003). Little is currently understood regarding how HPV
DNA is packaged into the virion or the process by which the mature virus is
released from the cell. Infected cells approach the surface of the skin and are
sloughed off, and in the process, newly synthesized HPV virus particles are
21
released into the surrounding area where the virions have the potential to infect
The structures and key functions of HPV proteins are reviewed in table 2.
Among all HPV types, the HPV E1 and E2 gene products are highly
conserved (Ustav and Stenlund 1991; Ching et al., 1993; Sverdrup and Khan 1994;
Hebner et al., 2006). Both proteins are absolutely essential for replication of the
viral genome (Ustav and Stenlund 1991). The E1 gene codes for an
(residues 125-170) that serves as a link between the N and C termini (Hughes and
Romanos 1993; Hebner et al., 2006). The N terminal region functions in DNA
binding and the C-terminal has enzymatic functions (Sverdrup and Khan 1994).
22
Table 2. Functions of HPV Early and Late Proteins
Early Proteins
Late Proteins
23
The E1 protein binds to AT rich viral DNA regions close to the start site of
early gene transcription. E1 also forms protein complexes with HPV E2 protein
that bind to HPV DNA at sequence specific sites located in the vicinity of the AT
rich sites. Once it is bound to DNA, the E1 protein forms a hexameric ring
structure around the viral DNA and begins to unwind the viral DNA for
phosphorylated via interaction with cyclin/CDK complexes in the host cell. This
essential for efficient viral replication (Ma et al., 1999; Deng et al., 2004).
The ~50 kDa HPV E2 protein has a role in viral replication and in the
domain and the C terminal region contains both E1 protein and DNA binding
DNA in a beta barrel structure (Hegde et al., 1992; Antson et al., 2000; Kim et al.,
2000; Hebner et al., 2006). The protein binds to the HPV DNA consensus
homodimers to these sequences can both activate and repress transcription of the
24
HPV early genes from the P97 promoter, depending on the amount of E2 protein
especially critical role in the control of E6 and E7 gene expression, because the
The term E1^E4 describes several proteins that are gene products formed
by the alternative splicing of mRNA sequences from the HPV E1 gene and the E4
ORF. These proteins are the most highly expressed of the HPV proteins and
their expression occurs throughout the lifecycle of the virus (Hebner et al., 2006).
The E1^E4 proteins localize in the infected cells cytoplasm and may form
multimers (Sterling et al., 1993; Pray and Laimins 1995; Roberts et al., 1997; Wang
et al., 2004). The exact function of these gene products is not currently known.
Some studies suggest the proteins may have a role in the exit of the mature HPV
virion from the infected cell, since E1^E4 has been shown to bind to cellular
cytokeratins (Doorbar et al., 1991; Roberts et al., 1993; Wang et al,, 2004). It has
also been proposed that the proteins may influence genome amplification by
blocking the infected cells entry into mitosis (Hebner et al., 2005). Recent studies
25
show that overexpression of E1^E4 protein causes arrest at the G2-M cell cycle
host cell. Like the E1^E4 proteins, the functions of this gene product are not
entirely clear at this time and most of the speculated functions currently
attributed to the protein come from the results of overexpression studies. For
2006). The E5 protein is known to target the epidermal growth factor receptor
(EGF), and affect other cell signaling pathways. (Straight et al., 1993; Crusius et
al., 1997; Crusius et al., 1998; Hebner et al., 2006). Studies show cells
26
overexpressing E5 have increased amounts of cell surface phosphorylated EGFR.
E5s alterations in the endosomal pathways have been predicted to alter host cell
the Golgi apparatus and impeding transport of the MHC-peptide complex to the
cell membrane (Ashrafi et al., 2005). There is evidence for a direct interaction of
E5 and HLA I heavy chain (HC) through the first hydrophobic domain of E5. E5
CD95 death receptor on the cell surface. This is yet another mechanism the virus
uses to overcome host defenses and favors propagation of the viral infection
The functions of the E6 protein will be described in the context of high risk
HPV infection, since the protein is critical for the malignant transformation of the
infected cell by high risk HPVs. Low risk HPV E6 protein does not have the
ability to transform infected cells. High risk HPV-16 E6 protein is 106 amino
27
acids in length and contains two C-X-X-C zinc finger domains (Hebner et al.,
2006). The protein is found in both the nuclei and the cytoplasm of HPV-infected
cells. E6 has a variety of functions, though its ability to bind to the p53 and
target it for proteasomal degradation is certainly the most characterized one, and
may be the most critical of its functions for induction of carcinogenesis (Werness
protein that controls both the G1/S and G2/M checkpoints of the cell cycle. It
induce cell cycle arrest upon cell stress or genotoxic assault, and allows for either
damage repair or induction of apoptosis (figure 7). High risk E6 protein binds to
p53 in a heterotrimer with the cellular E6-Ap protein and the complex acts as a
p53 specific ubiquitin ligase that facilitates the degradation of p53 via the
the half-life of the p53 tumor suppressor protein in infected cells and p53-
mediated regulation of the cell cycle is lost. E6 decreases the half-life of p53 in
vitro from several hours to less than 20 minutes. The E6-mediated loss of p53
removes restrictions on DNA synthesis in the infected cell and allows HPV DNA
28
G1 arrest
DNA damage Cyclin/
MDM2
Cell stress CDK
Transcriptional repression
Bax
FAS Apoptosis
IGP-BP3
arrest and apoptosis. p53 activates some genes and represses others when
the protein is stabilized. Stabilized p53 can initiate the processes of growth
29
expression of E6 protein facilitates uncontrolled proliferation of the infected cell
table 3). The vast majority of the proteins E6 binds to contain domains called
PDZ domains. PDZ domains, or post synaptic density protein domains, are
amino acids in length that are found in proteins involved in signal transduction
that E6 binds to have roles in the cell cycle and tumor suppression, cell adhesion,
and cell signaling pathways (James et al., 2006; Hebner et al., 2006). The
variability of the sequence of amino acids that lies directly in the peptide-binding
tumor suppressor pathways can interfere with a cells control over the cell cycle
and cell division. In a study involving transgenic mice, the PDZ domain binding
30
Table 3. Cellular Targets of HPV E6 Oncoprotein
Target Effect
Bak Antiapoptotic
31
Another key role for E6 in carcinogenesis is the ability of the protein to
continuous rounds of cell division, the DNA telomeres shorten until the resulting
chromosomal instability inhibits the cell from undergoing further divisions and
lengthening of the host cells telomeres, the E6 protein inhibits senescence and
prolongs the life cycle of the cell for the perpetuation of progeny viruses.
HPV E7 protein is 107 amino acids in length. Like E6, it contains 2 Cys-
X-X-Cys zinc finger domains. E7 has three conserved regions: the CR1
domain in the N terminal region, the middle CR2 domain which contains the
protein (pRB), and the C terminal CR3 domain that contains the zinc fingers
(Hebner et al., 2006). The protein also has phosphorylation sites, where the
protein can be modified during the G1 and S phases of the cell cycle, that are
ability to bind to and inactivate Retinoblastoma protein (pRB) and related family
member proteins p107 and p130 (Felisani et al., 2006; Ying and Xiao 2006). E7
32
forms a covalent complex with the active hypophosphorylated form of pRB and
promotes its proteolytic destruction (Munger et al., 1989; Felisani et al., 2006).
When functional pRb is transfected into tumor cells with a mutant pRB gene, the
result is growth arrest, indicating that the function of pRB as a tumor suppressor
is to restrict cell proliferation (Felisani et al., 2006). The most relevant target of
pRB is the cellular transcription factor E2F. E2F binds to specific DNA sequences
responsive genes (Giacinti and Giordano 2006). Active E2F is a potent stimulator
of cell proliferation and supports entry of the cell into the S phase of the cell
cycle. Three general classes of genes are under E2F control. These include the
following: genes such as cyclins A and E, c-myc, pRb, and p107, all of which are
upregulated at the G1/S transition; genes that are required for DNA replication
during S phase, such as DNA polymerase and PCNA; and genes involved in
the induction of apoptosis such as p19ARF (Felisani et al., 2006). In normal cells,
E2F sensitive genes (figure 8). As the cell cycle proceeds, phosphorylation of pRB
33
P P
pRb
P
P P
pRb
G1 P
P
Cyclin A
Cdk2
pRb P
M S
P
P
pRb
P
G2 P
P
PP1
P
P P
P pRb P
P P
P
resulting in its release from the E2F transcription factor. PP1 protein regenerates
34
is upregulated (Felisani et al., 2006). pRB is phosphorylated by serine/threonine
kinases called cyclin dependent kinases (CDKs), which associate with regulatory
with E2F, and, once pRB is dissociated from E2F, transcription of E2F active
Later in G1, cyclin E associates with CDK 2 and this enzyme complex
Cyclin A associates with CDK2 in the S phase of the cell cycle and
hyperphosphorylates pRB.
Giordano 2006).
35
P
P
pRb
P
Cyclin D P
Cdk2/4 P
P
pRb P
X
Cyclin E
E2F DP Cdk2 E2F DP
TTTCGCGC TTTCGCGC
36
HPV E7 overrides checkpoint control of the cell by pRB by inducing degradation
E2F promotes unchecked DNA synthesis and cell division. The process by which
protein can also bind to and inactivate the cyclin-dependent kinase (CDK)
inhibitors p21/Cip1 and p27/Kip1 and inhibit their ability to induce cell growth
arrest (Westbrook et al., 2002). E7 protein can modify the cell cycle in other
ways via its ability to bind to cyclin-kinase complexes, such as the cyclin A/cdk2
and cyclin E/cdk2 complexes, and cyclin dependent kinase inhibitors (CDKIs),
and also through its ability to increase cyclin A and cyclin E levels within HPV-
infected cells. Since both cyclins A and E act to phosphorylate pRB, the ability of
the viral protein to inhibit cyclin destruction and increase cyclin levels is another
cells exhibit a variety of abnormal karyotypes, such as tetrasomy, and also are
37
Transcriptional E7
Repressor E7
Complex E2F
pRb
E2F
pRb
G1 Transcriptional
E2F activator
PO4 PO4
pRb M S pRb
PO4 PO4
G2
PO4
pRb
PO4
complex interferes the binding of pRB with E2F. As a result, the E2F
38
Table 4. Cellular Targets of HPV E7 Oncoprotein
Target Effect
39
known to have abnormal numbers of centrosomes. Observations of
lesions and malignant tumors of the uterine cervix (Hebner et al., 2006).
Little is known about the products of the HPV late promoter. HPV late
proteins L1 and L2 make up the components of the viral capsids. Both proteins
are expressed late in the life cycle of the virus; L1 and L2 self assemble into viral
capsids that capture nascent HPV genomes to form complete progeny virions.
The mature virus particles exit the cell when it dies (Finnen et al., 2003; Zheng
and Baker 2006). The 600 Angstrom HPV capsids are composed of 72 pentameric
Each capsomer contains 5 L1 major capsid protein monomers (~55 kDa) and 12
L2 minor capsid proteins (~74 kDa). The exact structural interactions between
the proteins are unknown, although extensive studies of the structures of virus-
like particles (VLPs) have shed light on the approximate structure of native
L1 and L2 proteins have been studied for use as targets for HPV
vaccine studies, although their expression late in the viral life cycle may make
40
them less useful than early gene products for this purpose (Tomson et al., 2004).
or low risk, establish long term latent infections within the stem cells of the basal
layers of the skin. Most often, the host immune system is capable of clearing the
virus after persistence of the infection for 12-18 months, although in some cases,
the infection may never be fully eliminated. The few patients who are unable to
resolve the infection are the ones who are believed to be at high risk for
developing cancer (Stanley 2006). It is believed that the ability of the virus to
expressed, CTLs appear to no longer recognize infected cells. Indeed, the virus
41
has evolved a number of mechanisms by which it can successfully evade and
the innate and acquired immune systems is related in part to all of the following
processes:
above impair the ability of the infected cell to present viral antigens to
HLA-B, the molecules that are used by the cell to present viral peptides
infected cells by CTLs, the cells that would normally slaughter cells
immune system.
42
Infected cells have the ability to ignore apoptotic stimuli stemming
The term carcinogenesis defines the process by which normal cells are
transformed into malignant tumor cells. Cancer is the loss of cellular control
over cell division. At the cellular level, two types of genes, positive regulators, or
in protecting cells from becoming cancer cells and killing the host. p53 is
perhaps the most important tumor suppressor protein. p53 and the molecules it
4.1 p53
10,000 journal papers are published each year related to p53. The p53 tumor
43
suppressor gene (TP53) is the most commonly mutated gene in human cancers;
more than 50 percent of all cancers contain a mutation in this gene (Yee and
Vousden 2005; Bouchet et al., 2006). The TP53 gene codes for a 53 kilo Dalton
The binding domains of p53 are illustrated in figure 11 (Yee and Vousden 2005).
(Vousden and Lu 2002; Manfredi 2003; Meek 2004; Sengupta and Harris 2005).
The p53 tumor suppressor protein normally remains in the cell at low levels in an
inactive form. An autoregulatory feedback loop keeps p53 protein at low levels
because Mdm2 protein binds to p53 protein and targets it for degradation via
stress. Unlike other proteins, the increase in p53 protein levels and its activation
during times of cellular stress are most often a result of post translational
44
Nuclear
Oligimerization
Export
Signal
1 97 300 393
N C
Figure 11. Diagram of the domain structure of p53 protein. The N terminus
contains the transactivation domain and a proline rich region required for
apoptosis. The N terminal region also contains the nuclear export signal,
phosphorylation sites, and the binding site for Mdm2. The central region
contains the DNA binding domain and binding sites for BCL2 family member
45
p53 degraded by
p53 UBQ proteolysis
Mdm2
Proteosome
Mdm2
p53
Mdm2
Mdm2
Nucleus
p53
Figure 12. Mdm2 control of p53 levels in normal cells. p53 levels are kept low
in normal cells because Mdm2 protein binds to p53 and shuttles it to the
46
modification of the p53 protein by phosphorylation or acetylation rather than
which inhibits its interaction with Mdm2 and subsequent degradation. Once p53
Of all its functions, the most well defined role of p53 is its operation as a
makes life or death decisions about the fate of a cell. Hundreds of p53 target
genes have been identified, and a partial list of these genes and their functions is
al, 2006).
47
p53
MDM2
ATM p300/pCAF
P
P
MDM2 p53
P Acetyl
p53 stabilized via
posttranslational modification
NOXA
P21 PUMA
BAX IGFR FAS/APO1 -Binds to mitochondria
14-3-3-
ROS/PIGs BCL-2 DR5 -Modulates BH3 proapoptotic
Cyclin G
p53AIP1 Survivin PIDD proteins ie., BAX
PTGF-
GROWTH
ARREST
APOPTOSIS
Figure 13. A partial list of the many roles of p53. Both transcriptionally
growth arrest and apoptosis are shown (Yu and Zhang 2006).
48
Table 5. Cellular Targets of p53 in the Activation of Apoptosis
Target Effect
49
augments binding to specific DNA targets and upregulates transcription of p53
result in two cellular processes, growth arrest and apoptosis. The first target
gene of p53 to be discovered was p21 (Cip1/Waf1). Upregulation of the p21 gene
by p53 results in the inhibition of CDKs and causes growth arrest (Foijer and
te Riele 2006). This allots the cell time for DNA damage repair or activation of
the pathway to apoptosis. The upregulation of certain genes by p53 is critical for
overexpression (Erster and Moll 2005). p53 can modulate the extrinsic pathway
to apoptosis by upregulating the expression of the death receptors Fas, DR4, and
DR5 (Yu and Zhang 2006). In the intrinsic apoptotic pathway, p53 controls the
expression of pro- apoptotic genes such as Bax, and genes that function upstream
repressed by p53 include BCL2, BCL-XL, and survivin. The role of suppression
50
of these molecules by p53 likely prevents the anti-apoptotic function of these
proteins from interfering with the pathway to apoptosis, but the mechanisms by
It is known that p53 fragments that lack the DNA binding domain retain
(Yee and Vousden 2005). It is now known that p53 has a number of
family that contain sequence regions called Bcl-2 homology 3 (BH3) domains.
Though it does not contain a BH3 domain itself, p53 is capable of binding to
BCL-2 and BCL-XL. By binding to these proteins and abrogating their pro-
survival roles, p53 shunts the cell toward apoptosis (Vousden 2005).
(PUMA) by nuclear p53 is essential for the function of cytoplasmic p53 (figure
14). Activation of the TP53 gene results in the translation of p53 protein that
51
Pro-Apoptotic Events
BCL-2
bax
cytochrome c release
p53 caspase activation
Nucleus
Mitochondria
p53
PUMA p53
BCL-XL
BCL-XL
Figure 14. PUMA protein and the activation of cytoplasmic p53. Stabilization
of nuclear p53 results in its activation of the PUMA gene. In the cytosol, PUMA
protein facilitates the release of cytoplasmic p53 from the grip of the anti-
52
accumulates in both the nucleus and cytosol of the cell. Cytosolic p53 is bound
cytosolic p53 from the grip of BCL-XL in the so that it can activate the chain of
events in the cytoplasm that trigger apoptosis. These events affect the
Bax protein, cytochrome c release, and caspase activation (Vousden 2005). The
overall picture of p53 and molecular players in apoptosis is shown in figure 15.
4.1.3 Decision between cell cycle arrest versus apoptosis by p53 and its targets
How p53 determines the fate of a cell when it is stabilized and activated
remains an intensely studied topic. The choice between growth arrest versus
particular time. There are 2 models of how activation of p53 leads to the choice
of cell cycle arrest over apoptosis. The first model, the p53 dumb model, states
that activation of p53 always results in the same cellular response, which is a
induced survival signals resulting in cell growth arrest, or in other cases, the
53
dATP
cyt. C
Procaspase-9
apaf -1
Procaspase -3
Apoptosome Caspase-9
APOPTOSIS
Caspase-3
Cytochrome C
BCL-XL
GROWTH
BCL-2 pRb ARREST
Bax
cdk
p53
p21
Nucleus
54
induction of apoptosis by different independent signals. The second model, the
p53 smart model, suggests that p53 is able to govern the cellular response to its
growth arrest versus cells destined to die via apoptosis (Vousden 2005). Studies
have revealed that p53 levels themselves can affect the response of cells to p53
activation; low levels of p53 tend to favor growth arrest whereas high p53 levels
activate apoptosis. Promoter binding of p53 may also be affected by p53 post-
2005). Moreover, the presence of PUMA protein may be required for p53 to
can be initiated in several ways. For example, mutations in the TP53 gene can
binding, or accelerated loss of p53 protein can occur via proteasomal degradation
55
4.2 Apoptosis
bodies, which are then ingested and removed by phagocytes (Cereghetti and
Scorano 2006). The process involves a number of signaling pathways and can be
abnormal rates of apoptosis, be they to rapid or too slow (Alirol and Martinou
2006).
Apoptosis can be broken down into three phases, each of which are
56
intermediates, TNF-, ceramide, intracellular calcium pathway
(2) Effector phase - Key signals commit the cell to die in the effector phase.
In addition to being divided into phases, apoptosis can occur via 2 unique
pathways, the intrinsic pathway and the extrinsic pathway. The extrinsic
pathway is triggered by signals from cell surface TNF-family receptors which are
dependent upon signaling from activated p53 protein. p53 activates a number of
down stream signals such as the release of cytochrome C from the mitochondria.
This, in turn, triggers the activity of initiator caspase-9. Once the initiator
caspases are activated, the pathways converge upon the same effector caspase-3,
the process of apoptosis (Pollack and Leeuwenburgh 2001). The intrinsic and
57
extrinsic apoptotic pathways are depicted in figure 16.
proteins from the BCL-2 family, such as Bcl-2 and BCL-XL, block the pathway to
the signaling from the pro-apoptotic BCL-2 family protein Bax which translocates
initiator caspase-9, and ATP to form a structure called the apoptosome. Effector
initiates the destructive caspase cascade and the irreversible final degradation
4.3.1 Survivin
58
Ligand
Survivin, a member of the inhibitor of apoptosis family of proteins (IAP), is a
INTRINSIC EXTRINSIC
PATHWAY p53 PATHWAY
Fas
Mitochondrion
Cytochrome C release
Figure 16. Intrinsic and extrinsic pathways to caspase activation. The p53
59
inhibits apoptosis. The IAP family is a group of proteins that have at least one
BIR domain (a protein fold that binds zinc ion) and suppress apoptosis when
overexpressed in cell lines (Reed et al., 2001). Some IAP family members have
the ability to bind to and inhibit caspases. Studies have shown that survivin
may have the ability to bind to and block caspase-3 activity, but comparisons of
the structure of survivin with other known direct caspase inhibitors such as XIAP
do not show a direct structural explanation for how this may be possible (Reed et
al., 2001).
because its expression is highly tumor specific. Most normal adult cells do not
tumors express high levels of the survivin protein. This indicates that the
60
including as gene expression, mitosis, differentiation, and cell survival versus
apoptosis decisions (Pearson et al., 2001). The p38 MAP kinase pathway can be
activated by cellular stress. The result of p38 MAP kinase activation varies
depending on the tissue type; in many cells, the pathway triggers apoptosis,
whereas in other tissues, its activation promotes survival. In some cases, MAP
that during the course of viral infection, the HPV genome randomly becomes
integrated into the hosts genome (Jeon et al., 1995; Corden et al., 1999). If the
integration of the viral genome results in disruption of the viral E2 gene, then
there is a loss of its control over expression of viral oncoproteins E6 and E7.
interfere with functions of tumor suppressor proteins which control both the cell
(Steger and Corbach 1997). E6 protein binds to p53 via the E6-Ap protein and
(Keen et al., 1994; Scheffner 1998). This dramatically decreases the half-life of the
61
p53 tumor suppressor protein in infected cells, and p53-mediated regulation of
the cell cycle is lost (Scheffner 1998). Additional evidence for deregulation of the
p53 pathway by HPV E6 is that HPV-positive cell lines are resistant to p53-
also interferes with the functions of the cellular tumor suppressor protein
retinoblastoma (pRB). Viral E7 protein binds to pRb and inhibits the interaction
of pRB with E2F and other factors associated with the cell cycle (Ying and
integration of the viral genome into host DNA. The process by which this,
since there is a large discrepancy between the occurrence of HPV infection and
the incidence of cervical cancer. Upon its integration into host chromosomes,
HPV DNA frequently breaks in the E1/E2 gene region. In the episomal state, the
62
E7 proliferation E6
Ubiquitin
E6 Proteolysis
p53
E6
E7 p53
X
E5 immortalization
E6Ap
E6
Growth Arrest
Apoptosis
Loss of cell polarity,
E6 DNA damage
Metastasis
Distant Organs
63
E2 gene controls both the upper regulatory region (URR), which governs viral
transcription and replication, and also the E6 and E7 oncogenes, the two viral
oncogenes. Integration that interrupts the E2 gene locus also causes a loss of E2
High risk E6 and E7 proteins are usually coexpressed within infected cells.
tumor suppressors p53 and pRb, high-risk HPV E6 and E7 proteins immortalize
and transform human cells, setting the stage for cancer progression. As
previously stated, E7 interferes with the cellular tumor suppresser pRb and its
by constitutively active E2F in infected cells. Since E2F has the ability to
upregulate p14ARF to stabilize p53, the function of high risk E6 protein is also
64
important to cell transformation and carcinogenesis. HPV E6 oncoprotein
induces the rapid degradation of p53 via the ubiquitin proteasome pathway by
interacting with E6-AP, a host cell protein/ ubiquitin ligase (Barbosa et al., 1989;
Most cervical tumor cells that have integrated HPV generally retain wild
type TP53 and RB genes (Denk et al, 2001; Scheffner et al, 1991). As the viral
oncogenes and proteins efficiently and effectively interfere with the functions of
cellular tumor suppressor proteins, p53 and pRB, there is seldom any secondary
induced tumors (Crook and Vousden 1994). The wild type status of p53 in HPV-
Treatment options for HPV positive uterine cervical tumors vary with the
stage of disease at diagnosis, the health of the patient, and desire for bearing
children, but in all cases involves surgical removal of the malignant tumor
(Moore 2006). In early stages (see appendix A), tumors are curable by surgical
65
intervention alone. Procedures such as the loop electrosurgical excision
patient to bear children in the future (Schlaerth and Abu-Rustum 2006). In more
and in the most advanced stages, radical surgery called pelvic exenteration,
which includes removal of all pelvic organs (bladder, rectum, cervix, and
More advanced cases and cases of recurrent cervical cancers may require
standard first line treatment for invasive cervical cancer is usually cisplatin
addition to radiation. Recurrent and late stage cervical cancers are often treated
injection, or in pill form. Side effects often accompany treatment and may be
severe; they can include nausea, vomiting, diarrhea, and leukopenia (low white
blood cell count). Most of these side effects are a result of drug toxicity to
healthy tissues such as skin, hair follicles, and epithelial cells that line the
66
digestive tract (Stewart and Viswanathan 2006). Patient tumors can develop
resistance to these therapies as well (Muggia et al, 2004). Hence, there is the need
to develop alternate, safer, less toxic, and more specific therapies for UCC.
Some of the most successful currently used anticancer drugs are extracts
that come from the yew tree (Taxus baccata ), and the vinca alkaloids (Vincristine,
mithramycin, which are all compounds derived from fungi. These compounds
affect the growth of all rapidly dividing cells, whether normal or malignant, and
like other drugs, are associated with significant toxicity and side effects.
products with limited side effects that may be used as effective antitumor agents.
dietary plants (Wilson and Danishefsky 2006; Tan et al., 2006). Examples of such
67
compounds are resveratrol, indole-3 carbinol, quercetin and other catechin
compounds called lignans that may be potent antiviral and anticancer agents
(Hwu et. al, 1998; Gnabre et. Al, 1996; McDonald et al, 2001). Three such
creosote bush, larrea tridentata. The structures of these compounds are depicted
in figure 19. It has been suggested that NDGA or its derivatives may be an
effective treatment for tumors as well as other diseases (Artega et al., 2005).
Some of the potential anticancer properties of the lignan are described as follows.
68
OH
CH 2OH
HO
N
H
OH
Indole-3-Carbinol
Resveratrol
R5
OH O R4 R6
OH O
R1
R7
OH
HO O R3
OH R2 O
Quercetin Flavonoids
O
O O
O
OH
O OH
Azaphilones
anticancer properties.
69
HO HO
HO HO
OH OMe
OH OH
MeO
MeO
OMe
OMe
Tetra-O-Methyl-Nordihydroguaiaretic Acid
Figure 19. Chemical structures of lignans derived from the creosote bush
NDGA).
70
enzyme, NDGA blocks the synthesis of inflammatory mediators such as
such as 5-HETE, are shown to be elevated in cancers and also stimulate tumor
NDGA is known to prevent leukocyte infiltration into tissues and to stop the
release of reactive oxygen species (ROS) (Bhattacherjee 1988). NDGA can also
interfere with the platelet derived growth factor receptor and the protein kinase
includes the observations NDGA induces apoptosis in tumor cells and tumor
xenografts and inhibits the growth of tumors in vitro and in animal models. One
potential benefit of its use as an antitumor drug is that NDGA is fairly nontoxic
to normal cells. Because of these results, there is interest in the compound for
clinical study. However, other studies of NDGA indicate it may have dose
71
are required for efficacy. Hence, the discovery of more potent analogues of
herpes simplex virus (HSV), and SV-40 (Hwu et al., 1998; Gnabre et al., 1995). 3-
HPV E6/E7 gene expression within HPV DNA containing cervical cancer cells
(Craigo et al., 2000 and unpublished data). Previous work in our lab has
compound but also an anticancer agent against HPV induced cancers and
precancerous lesions of the uterine cervix. Since both viral oncogenes E6 and E7
within the HPV transformed cervical tumor cell can induce cell cycle arrest or
72
apoptosis (Hietanen et al., 2000). Based on these observations, it is possible that
the lignans that inhibited the episomal P97 promoter would also inhibit E6
protein expression from the integrated P97 promoter in HPV transformed cervical
tumor cells and interfere with oncogene control over cellular tumor suppressor
genes, which would likely result in a return of cellular control over p53 stability.
If functional p53 can be reactivated within these cells then a cascade of events for
either cell cycle arrest or apoptosis would be set in motion meaning that the
cancers of the uterine cervix. The central hypothesis of this research study is the
genes and apoptosis, and its action is mediated via an interference with the
the correct, it would represent a novel mechanism of inducing tumor cell death
73
HO
HO
3-O-methyl-NDGA
OMe
OH
PPrrooccaassppaassee--99
B
BCCLL--22
BBaaxx
E66//EE77 pp5533
PPUUMMAA
PPrrooccaassppaassee--3
C
Caassppaassee--99
C
Caasspaassee--33
APOPTOSIS
lignan interferes with E6/E7 oncogene activity and results in the stabilization of
p53 protein. Stabilized p53 is active in inducing pro-apoptotic bax and caspase
74
HPV-positive cells, treatment of precancerous and UCC lesions with this lignan
the therapy while, at the same time, reducing side effects associated with
standard treatments.
hypothesis.
(1) The ability of the plant lignan to induce cell cycle arrest in a variety of
75
CHAPTER TWO
1. Reagents
a gift from Dr. R. Huang (Gnabre et. al, 1995, 1996; Hwu et. al, 1998).
percent dimethylsulfoxide (DMSO, Sigma) and the stock solution was diluted to
adding to cell cultures. All other compounds were dissolved in 100 percent
76
Final DMSO concentrations were 0.2 percent or less. Untreated vehicle controls
2. Cell Culture
Cervical carcinoma cell lines used in this study were purchased from the
American Type Culture Collection (ATCC). C33-A, SiHa, and HeLa cells were
grown in DME, purchased in powder form from Sigma, containing 0.075 percent
bovine serum (FBS) and 50 g/mL gentamycin sulfate (Sigma). CaSki cells were
grown in 4.5g/L glucose DME from Gibco supplemented with 10 percent FBS
HPV content and p53 status of these cell lines is outlined in table 6.
Cells were plated at a density of 7.5 x 103 cells per well in a 96 well plate.
500 nM to 500 M. Cells were incubated for 20 hours after which 20 microliters
of alamar Blue Reagent was added to each well. Cells were incubated with
77
Cell Line HPV Status p53 Status tissue
Table 6. Cell lines used in this study and their HPV and p53 statuses.
78
each well at 560 and 600 nM were read on a plate reader (Molecular Devices).
controls and error due to antioxidant activity of the lignan on alamar Blue was
Cells were plated at a density of 7.5 x 103 cells per well in a 96 well plate.
500 nM to 500 M. Cells were incubated for 21 hours after which media
containing lignan was replaced with fresh media. 100 microliters of MTT [3-(4,5-
was added to each well and the plate was incubated for three hours at 37 C.
Cells were cultured and treated with various concentrations of the lignan on a
six well plate. Following treatment, cells were trypsinized and placed in a micro-
79
centrifuge tube and combined with 100 microliters of Trypan Blue dye. Cell
suspension was then placed on a hemacytometer and cells were counted as either
blue or clear using a phase contrast microscope. Data was reported as viability
percent of control.
5. Western Blots
Cells were plated at a density of 1 x 106 cells per 10 cm2 plate, treated with
drug compounds, and cell lysates were prepared using radio immune
precipitation assay (RIPA) lysis buffer (50mM Tris-HCl pH7.4, 1 percent Nonidet
were determined utilizing the Quant-Pro protein assay kit from Sigma. 10-20
micrograms of protein sample was loaded onto a SDS-page gel (Cambrex) and
were blocked in blotto (5% Carnation nonfat dry milk in 0.1 percent Tween-20 in
TBS) overnight at 4Celsius. After blocking, blots were incubated with mouse
monoclonal primary antibodies (Santa Cruz) to p53, p21, bax, beta actin, survivin
(Abcam), or, in the case of PUMA, rabbit polyclonal antibodies (Abcam), for one
80
hour at 25 Celsius, followed by incubation with anti-mouse or anti-rabbit
at 25 Celsius. The internal standard used for protein loading was beta actin.
Bands were visualized using the chemiluminescent detection kit (ECL-plus) from
CaSki cells were grown at a density of 4x 104 cells per well on an eight
chambered slide (Labtek). Following drug treatment, cells were washed twice
with PBS. Cells were fixed with 4 percent paraformaldehyde for 15 minutes.
Cells were blocked for 20 minutes in blocking buffer (5 percent glycine, 5 percent
FBS, 0.2 percent Triton X-100, 0.1 percent NaN3 in PBS). Cells were incubated
with mouse anti-p53 (Santa Cruz, San F) in blocking buffer for 1 hour at 37
Celsius. Wells were washed with PBS three times for 5 minutes. Cells were then
washed again with PBS three times for 5 minutes and allowed to dry. Slides
were taken.
81
7. Transfection Studies- to measure p53 transcriptional activity
HeLa, CaSki, and SiHa cells were analyzed for the activation of a p53-
transcriptional activity of stabilized p53. 5 x 105 cells were seeded on a six well
plate. Eight hours later, the cells were transfected with 1 microgram of the
reporter vector and/or the positive control vector pFC-p53 using the
Lipofectamine method. DNA was removed from cells three hours later and and
NDGA. Cells were analyzed for luciferase activity the following day using a
colorimetric assay kit from Calbiochem. Caspase-3 activity was measured using
the CaspACE colorimetric caspase-3 assay kit from Promega. Both assays make
pNA for caspase-3, LEHD-pNA for caspase-9). Cleavage of the substrate by the
specific cellular caspase yields free pNA that can be read by a spectrophotometer
82
amount of pNA product detected spectrophotometrically. Typically, C33-A,
CaSki, and HeLa cells were seeded at a density of 1 x 106 cells on a 10 cm2 plate.
Cells were allowed to attach overnight and were then treated with drug
compounds for amounts of time ranging from 3-12 hours. Following drug
treatment, cells were washed once with PBS, scraped off the plate, resuspended
in the lysis buffer from the kit and lysed via 3 cycles of freezing/thawing on dry
ice. Equal volumes of cell lysates were then assayed for caspase-3 or caspase-9
caspase assays, the protein concentration for each sample was measured using
the Quant-PRO protein assay from Sigma. Enzyme activity was divided by
apoptosis via flow cytometry (Beckman) utilizing the Annexin V-FITC Apoptosis
Detection Kit from Calbiochem. CaSki cells were plated as described above,
83
treated with 30-60 M lignan or 50 nM Actinomycin D for 3-12 hours. Cells were
and propidium iodide (PI). For each sample, 10,000 events were cataloged on a
fluorescence versus cell number plot and the percentage of Annexin V-FITC
positive cells was determined. Apoptosis was defined as the percentage of cells
that appeared in quadrant A1 (high Annexin V-FITC signal, low PI signal, early
apoptosis) compared to the total number of cells analyzed. Cells that appeared
in quadrant A2 (high Annexin V-FITC signal, low PI signal) were excluded from
the analysis due to the inability to distinguish cell death by apoptosis from cell
death by necrosis in those cells. Throughout the study, common settings for
these were determined by analysis of singly dyed and undyed controls. All
confirmed using the TdT-mediated dUTP nick end labeling (TUNEL) assay. The
84
DeadEnd Fluorometric TUNEL System kit used in this study was purchased
from Promega. Cells were seeded at a density of 35,000 cells per well on 8-
chambered slides (Lab Tek). Cells were then treated with 15 M to 80 M lignan
for 18 hours. Control cells were treated with 0.2 percent DMSO only. Following
PBS for 25 minutes. Cells were permeabilized using 0.2 percent Triton X-100 in
Apoptotic cells are reported as a percentage of total cells in the bright field
Cayman Chemical. Cell lysates are not compatible for use with this screening
method. A range of concentrations of both the methylated lignan and its parent
lipoxygenase enzyme was added to each well and then the reactions were
85
initiated by the addition of substrate. The reaction was allowed to proceed for 8
minutes after which 100 L of Chromagen was added to stop the reaction. The
absorbance of each well at 495 nm was then read on a plate reader (Molecular
86
CHAPTER THREE
RESULTS
selective toxicity to cells containing HPV DNA and wild type p53 protein.
Taken together, the CD50 values obtained from the alamar Blue assay,
MTT assay, and Trypan blue assays suggest that the lignan has some selective
toxicity to cells that contain both HPV DNA and wild type p53. While overall
CD50 values varied somewhat with respect to the cell proliferation assay used,
the overall trend of toxicity values shows that the compound is more toxic to
cells that contain HPV DNA and p53 that is wild type. Representative individual
results from the Trypan Blue assay and MTT assay in CaSki cells are shown in
figures 21 and 22. The assays were repeated for a variety of other cell lines (data
not shown). Data obtained from the alamar Blue assay for multiple cell lines is
shown in figure 23. The lignan showed a dose dependent inhibition of cell
87
3'-O-Methyl-NDGA toxicity in Caski cells
at 24 hours as determined by MTT assay
120
100
Growth Percent Control
80
60
40
20
0
0 25 50 100 200 400
[3'-O-methyl-NDGA] in M
treatment as determined by the MTT assay. (Error bars represent +/- standard
deviation).
88
3'-O-Methyl-NDGA toxicity in Caski cells
at 24 hours as determined by Trypan Blue Assay
120
100
Viability percent control
80
60
40
20
0
0 50 100 150 200
[3'-O-methyl-NDGA] in M
Figure 22. Trypan Blue viability assay of CaSki cells treated with 3-O-methyl-
NDGA and analyzed at 24 hours post treatment. (Error bars represent +/-
standard deviation).
89
Toxicity of 3'-O-Methyl-NDGA in Cancer Cells
at 24 hours determined by alamar Blue assay
100
Growth Percent Control
80
60
40 CaSki Cells
MRC-5 cells
20 C33-A Cells
HT-3 Cells
HeLa Cells
0
0 50 100 150 200
[3'-O-Methyl-NDGA] in M
Figure 23. Toxicity results for 3-O-methyl-NDGA in multiple cancer cell lines
obtained from the alamar Blue assay. Toxicities were calculated as a percentage
of the absorbance of DMSO treated controls and error due to antioxidant activity
of the lignan was subtracted. (Error bars represent +/- standard deviation).
90
growth in all cell lines tested. HPV DNA containing cells appeared to be most
sensitive to lignan treatment and showed the lowest CD50 values. Average CD50
values calculated from data obtained from the three assays used for all cell lines
lines.
of a luciferase reporter gene linked to the P97 promoter of high risk human
lignan may also decrease E6 expression from the integrated HPV promoters
expression within treated cells should result in the stabilization of p53 protein.
In order to assess whether lignan treatment stabilizes p53 protein steady state
levels within treated cells, we measured p53 protein levels found in the HPV-
containing cervical cell lines HeLa and CaSki with and without treatment with
treated controls were analyzed for p53 protein amount by Western blot analysis.
91
CELL LINE CD50 M
HeLa (cervical carcinoma, high risk HPV-18 + wt p53) 83
CaSki (cervical carcinoma, high risk HPV-16 + wt p53) 69
SiHa (cervical carcinoma, high risk HPV-16 + wt p53) 64
HT-3 (cervical carcinoma, low risk HPV-30 + mutant p53) 263
C33-A (cervical carcinoma, HPV-negative, mutant p53) 187
MRC-5 (human immortalized fetal lung fibroblasts) >250
HFK (human foreskin keratinocytes) >80
cell lines. Table lists the corresponding CD 50 values for these cell lines as the
average CD50 values calculated from results of alamar Blue, MTT, and Trypan
Blue assays. Values obtained from each assay were similar (data not shown).
92
The 3-O-methyl NDGA treated cells had increased p53 protein levels from 2 to
12 hours after treatment within CaSki cells (figure 24) and 12 to 18 hours after
treatment in HeLa cells (figure 25). The lignan did not alter the mutant p53
results suggest that the compound is effective in stabilizing wild type p53, but
possible that Western blot is not sensitive enough to detect any changes in
mutant p53 induced by the lignan due to the increased half-life of the mutant
protein and the high levels of p53 protein within the cells tested.
secondary (Santa Cruz) antibody confirmed the increase in p53 protein found in
timepoints of 6, 12, and 18 hours post treatment. The optimal time point for
where the half life of p53 is normal (Bartley and Ross 2002; Moll et al, 1995).
93
CaSki Cells
Control 30 M
3-O-Methyl-NDGA Treated
12h 2h 4h 6h 9h 12h
p53
-Actin
Figure 24. p53 protein is stabilized in CaSki cells treated with 30 M 3-O-
treated CaSki cells. Stabilization of p53 is enhanced over time from 2 to 12 hours
94
HeLa Cells
Control __Treated__
p53
-Actin
Figure 25. p53 protein is stabilized in HeLa cells treated with 30 M 3-O-
with 30 M 3-OMe-NDGA for 12 and 18 hours. p53 levels are elevated in lignan
95
C33-A Cells
Control ________Treated_
3-OMe-NDGA treated _
30 uM
control
hrs 12h
12 6h
6 9h
9 12h
12
p53
C33A Cells
Figure 26. p53 protein levels do not change in C33-A cells treated with 30 M
M lignan treated C33-A cells which contain mutant p53. Hence, the lignan does
96
CaSki Cells
p53p53
Results show a larger amount of p53 staining in lignan treated cells compared to
97
3. The p53 protein stabilized by 3-O-methyl NDGA treatment is
transcriptionally active.
reporter. The p53-luciferase reporter plasmid has p53 response elements linked
to the luciferase gene and the activation of p53 in transfected cells drives
activity compared to the transfected non-treated cells (figure 29) indicating that
HeLa cells were also co-transfected with a pFC- p53 vector (Stratagene)
that has the wild type p53 gene linked to the immediate early CMV promoter as
wild type p53 along with the reporter plasmid into HeLa cells shows that the
reporter vector responds to excess production of p53 within the cells as shown in
figure 29. Similar increases were observed when CaSki cells and SiHa cells were
transfected with the p53-luciferase vector indicating that the stabilized p53 in
98
Figure 28. Structure of the p53-luciferase vector used to transfect HeLa
cells with p53. The vector has the wild type p53 responsive gene linked to
the immediate early CMV promoter. The inducible reporter plasmid contains
the luciferase reporter gene driven by a basic promoter (TATA box) plus a cis-
99
p53-luc Transfection of HeLa Cells
50
40
Specific Activity Luciferase
(cpm/ug protein x 10 )
7
30
20
10
0
p53-luciferase p53-luciferase + p53-luciferase + 15
pFC uM lignan
cells that were treated with the lignan showed a 5-6 fold increase in luciferase
deviation. P values for treated vs. control <<.001 at alpha = .05 for 2 tailed
Student T test).
100
these cells is transcriptionally active as well (figures 30 and 31).
activation of growth arrest pathways. p21 was not induced at 12 or 18 hour time
blot analysis (figures 32 and 33). However, treatment of the same cell lines with
protein (positive control). This indicates treated cells are not undergoing growth
The activation of caspases, which are cysteine proteases, is a signal for the
and CaSki cell lines. The HPV positive cell lines HeLa and CaSki both showed
increased over time and peaked at 12 hours post-treatment (figure 34). The
101
p53-luc Transfection of CaSki cells
40
35
Specific Activity Luciferase
(cpm/ug protein x 10 )
6
30
25
20
15
10
5
0
p53-luciferase p53-luciferase + p53-luciferase +
15 uM lignan pFC-p53
transfected cells that were treated with the lignan showed a 3 fold increase in
deviation. P values for treated vs. control <<.001 at alpha = .05 for 2 tailed
Student T test).
102
p53-luciferase transfection of SiHa cells
14
Specific Activity Luciferase
12
(cpm/ug protein x 106)
10
0
p53-luciferase p53-luciferase p53-luciferase +
+15 uM lignan pFC-p53
each sample and specific activity was calculated by dividing sample activity in
were treated with the lignan showed an 8 fold increase in luciferase activity
active p53 in treated cells. (Error bars represent +/- standard deviation. P values
for treated vs. control <<.001 at alpha = .05 for 2 tailed Student T test).
103
CaSki Cells
Control ____Treated_____
12h 12h 18h AD
p21
-Actin
p21
OMe-NDGA. Cells were treated with lignan and analyzed for p21 expression via
shown. Beta actin was used as an internal control for protein loading for all blots
depicted above.
104
HeLa Cells
Control _Treated__
12h 12h 18h
p21
-actin
OMe-NDGA. Cells were treated with lignan and analyzed for p21 expression via
Western blot. 3-O-Methyl-NDGA failed to induce p21 CaSki cells. Beta actin
was used as an internal control for protein loading for all blots depicted above.
105
Caspase-3 Activity in Cervical Cancer Cells
Treated With 3'-O-Me-NDGA
35
Specific Activity of Caspase-3
30 HeLa Cells *
CaSki Cells
25 *
C33-A Cells
(nmol/h/mg)
20
15
10
5
0
0 3 6 9 12
Time Post Treatment (hours)
containing cervical cancer cells, but not in HPV-negative C33-A cells. Caspase-
HPV-positive HeLa and CaSki cells. C33-A cells do not show caspase-3
activation when treated the lignan. (Error bars represent +/- standard deviation).
106
caspase-3 levels induced with micromolar amounts of lignan were comparable to
the levels found with 50 nM Actinomycin D treatment (data not shown). HPV
negative C33-A cells, which also contain a mutant p53 gene, did not show
over 3 to 12 hours post treatment. C33-A cells contain inducible caspase-3 that
induced in HPV positive cells after treatment with 30 M lignan (figure 36). The
HeLa and CaSki cells, which was about 6 hours before maximal effector caspase-
3 activity was observed. The HPV negative C33-A cells did not shown any
107
Caspase-3 Activity in C33-A Cells
Treated with Camptothecin
Specific Activity of Caspase-3
80
70
60
(nmol/h/mg)
*
50
40
30
20
10
0
0 3 6 9 12 15 18
Figure 35. C33-A cells have an intact caspase-3 pathway. Caspase-3 activity in
treated the lignan but C33-A cells have an intact caspase-3 pathway as shown
deviation).
108
Caspase-9 Activity in Cervical Cancer Cells
Treated with 3'-O-Me-NDGA
45
Specific Activity of Caspase-9
40 HeLa Cells
35 CaSki Cells
30 C33-A Cells
(nmol/h/mg)
25
20
15
10
5
0
0 3 6 9
Time Post Treatment (hours)
containing cervical cancer cells, but not in HPV-negative C33-A cells. The
NDGA induces caspase-9 activity in HPV positive HeLa and CaSki cells but fails
109
6. The caspase-3 inhibitor Z-VAD-FMK rescues lignan treated cells from the
apoptotic pathway.
potent cell permeable inhibitor of all caspases including caspases-3. Cells treated
with a combination of the lignan and the caspase inhibitor showed lower caspase
activity than cells treated with the lignan alone as shown in figure 37. This data
suggests that lignan treatment is acting directly on the cellular path to apoptosis.
However, the data does not distinguish whether the intrinsic pathway or the
pathways to apoptosis.
The induction of caspase-9 and caspase-3 activity within HPV containing cell
lines after treatment with lignan strongly suggests that the cells are undergoing
110
(3)
Caspase-3 Inhibitor Z-VAD-FMK Protects
CaSki Cells From Lignan Induced Apoptosis
50
Percent Cell Death as determined by TUNEL Assay
45
40
35
30
25
20
15
10
Figure 37. Addition of the caspase-3 inhibitor ZADFMK to cells treated with 30
111
programmed cell death, lignan treated cells were assayed for apoptosis by flow
cytometry and the TUNEL assay. We were able to confirm that treated cells
were undergoing apoptosis using flow cytometry and Annexin V-FITC and
propidium iodide staining. Cells staining with Annexin V-FITC were found in
non-synchronous lignan treated cell cultures with maximal cell numbers at 6-9
hours after lignan treatment (figure 38). Apoptosis was defined as the
propidium iodide signal; early apoptosis) compared to the total number of cells
iodide signal) were excluded from analysis due to the fact that it cannot be
determined whether the cells were dying via apoptosis or necrosis. Cells treated
6 and 9 hours post treatment compared to vehicle treated controls (figure 38).
The TUNEL assay confirmed the flow cytometry results. HPV DNA
containing cervical cancer cells had positive TUNEL staining when treated with
the lignan. The TUNEL assay also showed a different sensitivity between the
HPV positive cell lines CaSki and SiHa (figures 39 and 40) as compared to the
HPV negative cervical cancer HT-3 and C33-A cell lines (figures 41 and 42).
HeLa cells were not tested due to difficulty in fixing the cells.
112
Annexin V-FITC/ PI Staining in CaSki Cells
35
*
Percent of Annexin V Only Positive Cells
30
25
*
20
15
10
Control 6h 30 uM 9h 30uM
Figure 38. Lignan treated cells are undergoing apoptosis as assayed using flow
low propidium iodide signal; early apoptosis) compared to the total number of
113
CaSki Cells
Control 30 M 3-OMe-NDGA
3% cell death 50% cell death
positive CaSki cells, which contain wild-type p53, showed an increased number
114
SiHa Cells
Control 30 M 3-OMe-NDGA
<5% cell death >50% cell death
control according to two tailed student T test at alpha = 0.05; p < 0.001).
115
C33-A Cells
Control 30 M 3-OMe-NDGA
<5% cell death 10% cell death
assay. C33-A cells, which contain a mutation in the p53 tumor suppressor, did
not show significant apoptosis and were not nearly as sensitive to lignan
treatment as CaSki and SiHa cells. (No significant difference between treated
116
HT-3 Cells
Control 30 M 3-OMe-NDGA
4% Cell death 9% Cell Death
with wild type p53. 30 M lignan does not induce significant apoptosis in HT-3
cells which contain HPV-30 DNA and mutant p53. (No significant difference
between treated and control according to two tailed student T test at alpha =
0.05).
117
8. The Bax and PUMA genes are induced within lignan treated CaSki cells.
Bax and PUMA. Both proteins are pro-apoptotic and are induced via the
lignan to induce bax and PUMA by examining protein levels by Western blot.
CaSki cells and HeLa cells treated with 30 M 3-O-methyl NDGA showed
treated CaSki cells 6 to 18 hours post treatment (figure 45). The elevations in
both Bax and PUMA protein levels are consistent with induction of the intrinsic
Western blot analysis shows that CaSki cells treated with 30 M lignan
have a reduction in HPV E7 protein levels over time. E7 protein levels decreased
over time from 9 to 18 hours post treatment (figure 46). This correlates with the
loss of full length E6/E7 mRNA as determined by real time PCR (D. Tschantz and
118
CaSki Cells
Control Treated
12h 12h
Bax
-Actin
Figure 43. Western blot analysis shows that bax protein is induced by 30 M
following treatment with 30 M lignan. Western blot analysis shows that bax
protein levels are higher in lignan-treated cells versus control. The internal
119
HeLa Cells
Control Treated
12h 12h
Bax
-Actin
Figure 44. Western blot analysis shows that bax protein is induced by 30 M
following treatment with 30 M lignan. The internal standard used for protein
120
CaSki Cells
Control Treated____________
12h CPT AD 6h 9h 12h 18h
Figure 45. Western blot analysis shows that both PUMA protein is induced by
in CaSki cells after treatment with the lignan for 9-18 hours and were compared
121
CaSki Cells
0.2%DMSO 30 M 3-O-methyl-NDGA
E7
Figure 46. E7 protein levels decrease when CaSki cells are treated with 30 M
lignan. The decrease in E7 protein correlates with the observed loss of E6/E7
mRNA in CaSki cells treated with the lignan (Tschantz and DeLucia, manuscript
in preparation)
122
with mRNA loss suggests that the lignan may work at the transcriptional level.
123
CHAPTER FOUR
DISCUSSION
from the creosote bush interfered with HPV type 16 early promoter, P97 , activity
(Craigo et al., 2000). This led to the prediction that such lignans could inhibit the
transcription of the HPV genome including the oncogenes E6 and E7. Most
cervical tumor cells that have integrated HPV generally retain wild type p53 and
RB genes (Denk et al., 2001; Scheffner et al., 1991). Viral oncogenes and proteins
proteins, p53 and pRB, so there is seldom any accumulation of further mutations
to maintain the transformed state. Since HPV tumors contain wild-type p53, it is
plausible that the protein could be reactivated if the functions of the HPV
stabilization and accumulation of active p53 protein within the HPV transformed
cervical tumor cell can induce cell cycle arrest or apoptosis (Ying and Xiao 2006).
124
Since E6s primary function is to shuttle endogenous p53 to the
tested the natural plant lignan 3-O-methyl-NDGA for the interference with HPV
p53 stability and the stabilized p53 protein was transcriptionally active as shown
by luciferase reporter assay and the induction of p53 responsive genes. We also
Upon treatment of HPV positive cervical cancer cell lines with the lignan,
quickly established within 2 hours post-treatment with lignan. The early and
rapid appearance of increased p53 levels and the lack of concomitant increases in
translation of stabilized p53 mRNA. The increased p53 protein levels observed
in HPV positive cell lines does not infer by itself that the lignan is acting through
an HPV specific factor nor does it establish that p53 is functional. Typical signals
125
that are considered important for p53 protein stabilization work through the
disruption of p53 regulation by MDM2 (Momand et al., 1992; Picksley and Lane
p53 regulation in HPV-positive cervical tumor cells due to the presence of E6.
The high levels of p53 stabilization found in lignan treated cells likely depend on
is the level of E6 protein itself that is being affected or the function of E6 protein
that is being interfered with by the action of the lignan. It is also possible that the
lignan modulates the interaction of E6, E6-AP, and p53 thereby stabilizing p53 in
treated cells. The functional status of p53 stabilized by the lignan was
PUMA and bax in lignan-treated cells, and indirectly by the activation of the p53-
HPV positive cervical tumor cells did not show an increase in p21 protein
treated cells appear to follow a pathway directed at apoptosis rather than growth
arrest. One possible explanation for the absence of p21 may be that the lignan is
interfering with Sp1 transcription factor levels, and the transcription of p21 is
126
highly Sp1 dependent. It has been suggested previously that the lignan is
capable of interfering with Sp1, and the interference with Sp1 may also explain
the ability of the compound to block gene expression from the P97 promoter,
since P97 also contains sever Sp1 binging sites. It is also possible that we failed to
observe the induction of p21 at the time points tested. p21 is known to be a
substrate for active caspase-3, and the induction of caspase-3 may have
controls and HPV negative cervical cancer cell lines, lignan-treated HPV positive
apoptotic pathway. They are maintained in a quiescent state within cells until
they are activated (Cereghetti and Scorano 2006). The p53 dependent intrinsic
apoptotic pathway begins the cascade of proteolytic destruction within the cell
127
by first activating procaspase-9 which, in turn, cleaves and activates the other
caspases and terminates with the formation of the apoptosome and the activation
with the lignan and levels peaked between 6 and 9 hours. The effector caspase-3
12 hours. The kinetics of caspase activations provides strong evidence for the
induction of the intrinsic apoptotic pathway by the active p53 at 2-3 hours post-
treatment since caspase activation coincided with the observed increases in p53
This data also shows that induction of cell death by the plant lignan is
likely occurring through the intrinsic apoptotic pathway. That endogenous wild
type p53 was responsible for the caspase activation was further supported by the
lack of caspase-9 and caspase-3 activation within C-33A cells, a cervical tumor
cell line that lacks HPV DNA and also has a mutant p53 gene, but has normal
induction of apoptosis after lignan treatment and stabilization of p53 protein was
further confirmed by both flow cytometry using Annexin V and the counter stain
128
asynchrony of the cells under the experimental conditions.
protein. Increasing p53 protein levels in the nucleus activates expression of the
PUMA gene, which then increases its protein levels after translation of increased
PUMA mRNA. PUMA protein then interacts with a p53-BCL-XL complex in the
cytosol, and results in the release of p53 protein from the grip of BCL-XL,
allowing p53 to interact with the Bax protein stationed at the surface of the
mitochondria. This starts the cascade for the intrinsic apoptotic pathway. The
induction of PUMA protein was observed in lignan treated cells at time points
following the stabilization of p53 protein. Bax protein was also shown to be
treatment are explained by the cell initiating and carrying out the intrinsic
Other data in our lab shows that lignan treatment induces a significant
reduction in mRNA coding for both E6 and E7 proteins (Tschantz and DeLucia,
129
only E7 protein levels could be measured following lignan treatment. A decrease
in E7 protein levels was observed in lignan treated CaSki cells and this is
be interfering with the activity of the endogenous P97 promoter in these cells.
Since most currently used chemotherapy drugs target all rapidly dividing cells,
cancer.
The observations found in this work coupled with previous work permit
Upon entry of the compound into the cell, the lignan overwhelms the viral E6
protein control of wild type p53 within the cell. The loss of E6 function is at a
level that permits the stabilization of substantial amounts of p53 within a short
time. The rapid accumulation of transcriptionally active p53 protein induces key
proteins that function in the apoptotic pathway, and end point assays suggest
that the cell death induced by the lignan follows a p53-dependent pathway.
130
passage immortalized cell lines that can interrupt cell death and p53 stabilization
induced by the lignan. This work argues that, as E6 protein is a principle player
lesions, any interference with its function may disrupt the viral induced pathway
to carcinogenesis.
products represent a promising start since their metabolism and uptake within
transformed cells may actually stress them more than normal cells. The lignans
may also be starting points to examine if such products can lower the
131
REFERENCES
132
Beausoleil SA, et al. Large-scale characterization of HeLa cell nuclear
phosphoproteins. Proc Natl Acad Sci U S A. 2004 Aug 17;101(33):12130-5.
Epub 2004 Aug 9.
Bouchet BP et al. p53 as a target for anti-cancer drug development. Crit Rev
Oncol Hematol. 2006 Jun;58(3):190-207.
Buck CB, et. al. Maturation of papillomavirus capsids. J Virol 2005; 79:2839-46.
Cox JT. Epidemiology and natural history of HPV. J Fam Pract. 2006 Nov;
Suppl:3-9.
133
Craigo J, Callahan M, Huang RC, DeLucia AL. Inhibition of human
papillomavirus type 16 gene expression by nordihydroguaiaretic acid
plant lignan derivatives. Antiviral Res. 2000 Jul;47(1):19-28.
Crook T and Vousden KH. Properties of p53 mutations detected in primary and
secondary cervical cancers suggest mechanisms of metastasis and
involvement of environmental carcinogens. EMBO J. 1992 Nov;11(11):3935-
40.
De Villiers EM, Fauquet C, Broker TR, Bernard HU, zur Hausen H. Classification
of papillomaviruses. Virology. 2004 Jun 20; 324(1):17-27.
DiMaio D and Liao JB. Human papillomaviruses and cervical cancer. Adv Virus
Res. 2006;66:125-59.
134
Doorbar J, et al. Specific interaction between HPV-16 E1-E4 and cytokeratins
results in collapse of the epithelial cell intermediate filament network.
Nature 1991;352:824-827.
Doorbar J. The papillomavirus life cycle. J Clin Virol. 2005 Mar;32 Suppl 1:S7-
15.
Doorbar J. Molecular biology of human papillomavirus infection and cervical
cancer. Clin Sci (Lond) 2006; 110(5): 525-41.
Fadeel B. Programmed cell clearance. Cell Mol Life Sci. 2003 Dec;60(12):2575-
85.
Felisani A, Mileo AM, Paggi MG. Retinoblastoma family proteins as key targets
of the small DNA virus oncoproteins. Oncogene. 2006 Aug 28;25(38):5277-
85.
135
Foijer F and te Riele H. Check, double check: the G2 barrier to cancer. Cell
Cycle. 2006 Apr;5(8):831-6. Epub 2006 Apr 17.
Fox PA and Tung MY. Human papillomavirus: burden of illness and treatment
cost considerations. Am J Clin Dermatol. 2005;6(6):365-81.
Giacinti C and Giordano A. RB and cell cycle progression. Oncogene. 2006 Aug
28;25(38):5220-7.
Gnabre JN, Brady JN, Clanton DJ, Ito Y, Dittmer J, Bates RB, Huang RC.
Inhibition of human immunodeficiency virus type 1 transcription and
replication by DNA sequence-selective plant lignans. Proc Natl Acad Sci U
S A. 1995 Nov 21;92(24):11239-43.
Greer CE, Wheeler CM, Ladner MB, Beutner K, Coyne MY, Liang H, Langenberg
A, Yen TS, Ralston R. Human papillomavirus (HPV) type distribution
and serological response to HPV type 6 virus-like particles in patients
with genital warts. J Clin Microbiol. 1995 Aug;33(8):2058-63.
136
Hegde RS et al. Crystal structure at 1.7 A of the bovine papillomavirus-1 E2
DMA binding domain bound to its DNA target. Nature 1992;359:505-12.
Hwu JR, Tseng WN, Gnabre J, Giza P, Huang RC. Antiviral activities of
methylated nordihydroguaiaretic acids. 1. Synthesis, structure
identification, and inhibition of tat-regulated HIV transactivation. J Med
Chem. 1998 Jul 30;41(16):2994-3000.
James MA, Lee JH, and Klingelhutz AJ. Human papillomavirus type 16 E6
activates NF-kappaB, induces cIAP-2 expression, and protects against
apoptosis in a PDZ binding motif-dependent manner. J Virol. 2006
Jun;80(11):5301-7.
137
Lindeque BG. Management of cervical premalignant lesions. Best Pract Res Clin
Obstet Gynaecol. 2005 Aug;19(4):545-61.
Manfredi JJ. p53 and apoptosis: its not just in the nucleus anymore. Mol Cell.
2003;11:552-554.
Maruta H and Burgess AW. Regulation of the ras signaling network. Bioessays.
1994 Jul;16(7):489-96.
Meek DW. The p53 response to DNA damage. DNA Repair. 2004(3):1049-56.
Michalak E, et al. Death squads enlisted by the tumour suppressor p53. Biochem
Biophys Res Commun. 2005 Jun 10;331(3):786-98.
Momand J, et al. The mdm-2 oncogene product forms a complex with the p53
protein and inhibits p53-mediated transactivation. Cell. 1992 Jun
26;69(7):1237-45.
138
Muggia FM. Recent updates in the clinical use of platinum compounds for the
treatment of gynecologic cancers. Semin Oncol. 2004 Dec;31(6 Suppl 14):17-
24.
Padilla LA, Leung BS, Carson LF. Evidence of an association between human
papillomavirus and impaired chemotherapy-induced apoptosis in cervical
cancer cells. Gynecol Oncol. 2002 Apr;85(1):59-66.
139
Reed J. The survivin saga goes in vivo. J Clin Invest. 2001 Oct;108(7): 965-
969.
Sapp M, et al. Organization of the major and minor capsid proteins in human
papillomavirus type 33 virus-like particles. J Gen Virol 1995;76:2407-12.
Scheffner M, Munger K, Byrne JC, Howley PM. The state of the p53 and
retinoblastoma genes in human cervical carcinoma cell lines. Proc Natl
Acad Sci U S A. 1991 Jul 1;88(13):5523-7.
140
Sengupta S and Harris CC. p53: traffic cop at the crossroads of DNA repair and
recombination. Nat Rev Mol Cell Biol. 2005;6:44-55.
Stanley MA. Human papillomavirus and cervical carcinogenesis. Best Pract Res
Clin Obstet Gynaecol. 2001 Oct;15(5):663-76.
141
Tewari KS, Monk BJ. Gynecologic oncology group trials of chemotherapy for
metastatic and recurrent cervical cancer. Curr Oncol Rep. 2005
Nov;7(6):419-34.
Tomson TT, Roden RB, Wu TC. Human papillomavirus vaccines for the
prevention and treatment of cervical cancer. Curr Opin Investig Drugs.
2004 Dec;5(12):1247-61.
Vousden KH. P53 and PUMA: A deadly duo. Science. 2005 Sept 9; 309:1685-
1686.
Vousden KH and Lu X. Live or let die: the cells response to p53. Nat Rev
Cancer. 2002;2:594-604.
Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV,
Snijders PJ, Peto J, Meijer CJ, Munoz N. Human papillomavirus is a
necessary cause of invasive cervical cancer worldwide. J Pathol. 1999
Sep;189(1):12-9.
142
Werness BA, Levine AJ, Howley PM. Association of human papillomavirus
types 16 and 18 E6 proteins with p53. Science. 1990 Apr 6;248(4951):76-9.
Westbrook TF, Nguyen DX, Thrash BR, McCance DJ. E7 abolishes raf-induced
arrest via mislocalization of p21(Cip1). Mol Cell Biol. 2002 Oct;22(20):7041-
52.
Youngren JF, et al. Nordihydroguaiaretic acid (NDGA) inhibits the IGF-1 and c-
erbB2/HER2/neu receptors and suppresses growth in breast cancer cells.
Breast Cancer Res Treat. 2005 Nov;94(1):37-46.
Yoon CS, et al. alpha(6) Integrin is the main receptor of human papillomavirus
type 16 VLP. Biochem Biophys Res Commun. 2001 May 11;283(3):668-73.
143
Zimmerman JM and Maher, III, LJ. Solution measurement of DNA curvature in
papillomavirus E2 binding sites. Nucleic Acids Res. 2003 September 1; (17):
51345139.
144
145