Affiliations of authors: W. Beerheide, Y.-J. Tan, A. E.Ting (Screening for Novel Inhibitors Laboratory), H.-U. Bernard (Papillomavirus Biology Laboratory), A. Ganesan (Medicinal and Combinatorial Chemistry Laboratory), Institute of Molecular and Cell Biology, Singapore; W. G. Rice, Laboratory of Antiviral Drug Mechanisms, Science Applications International Corp., National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD.
Correspondence to: Hans-Ulrich Bernard, Ph.D., Institute of Molecular and Cell Biology, 30 Medical Dr., Singapore 117609, Republic of Singapore (e-mail: mcbhub{at}imcb.nus.edu.sg).
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ABSTRACT |
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INTRODUCTION |
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Infection by HPV16 can persist subclinically or can develop into potential precursor lesions of cervical cancer, which are called "cervical intraepithelial neoplasia" or "squamous intraepithelial lesions" (4). These lesions can regress or progress to invasive cancer, likely as a result of the acquisition of additional mutations in cellular genes as a consequence of HPV gene functions (5,6). Current treatment for HPV16-associated lesions is surgery, while limited success is achieved with immune modulators like interferon against HPV6- and HPV11-associated lesions. Prevention of HPV infection by vaccination and challenge of established HPV infections by immune therapy are under investigation but not yet established. Approximately half a million women die every year of cervical cancer, while a much higher number of patients are exposed to noninvasive disease or genital warts (1-3), and one has to conclude that treatment of these virally caused neoplasias is still inadequate in spite of the long-term establishment of surgical techniques. These statistics suggest a need for alternatives to current treatment approaches. With the detailed knowledge of the molecular biology of HPVs, it is now possible to target individual HPV proteins by chemical compounds that interfere selectively with the tertiary structure and function of viral gene products. Compounds with desirable properties in vitro may lead to the development of drugs to treat HPV infection or HPV-associated neoplasia in situ.
HPVs have circular, double-stranded DNA genomes that are approximately 8 kilobases in size and encode eight genes that are homologous among all genital HPV types. The transforming properties of HPV16 originate from three oncoproteins that are the products of the E5, E6, and E7 genes. These proteins have pleiotropic effects with consequences for transmembrane signaling, regulation of the cell cycle, transformation of established cell lines, immortalization of primary cell lines, and chromosomal stability (1,2). The E6 oncoprotein can form a ternary complex with the cell-cycle regulator p53 and the E6-associated protein (E6AP), resulting in degradation of p53 by the ubiquitination pathway (7,8). E6 can also bind E6-binding protein (E6BP; also known as ERC-55), a calcium-binding protein localized in the endoplasmic reticulum, with possible consequences for intracellular signaling (9). E6 can change cellular morphology by interacting with paxillin and disrupting the actin cytoskeleton (10). E6 has also been described to activate (11,12) or, alternatively, to repress transcription (13), to stimulate telomerase (14), to immortalize primary cell cultures (15), and to interfere with the differentiation of human keratinocytes (8).
The E6 protein of HPV16 consists of 158-amino-acid residues (Fig. 1, A) and contains two hypothetical
Cys-X2-Cys-X29-Cys-X2-Cys (where X represents any
amino acid and the number represents the number
of residues) zinc fingers (16-19).
A Patscan search (20) reveals that this sequence
motif is unique for papillomavirus E6 as well as for E7
proteins and includes numerous specific amino acid residues, highly conserved among all
carcinogenic HPVs, as well as many animal and human papillomaviruses associated with benign
lesions (21,22). The structures of the E6 and
E7 zinc fingers are unique
among all other known zinc-binding proteins (23). The conservation of
the E6 and E7 zinc fingers among distantly related HPV types suggests that this zinc-binding
motif is strictly required for the function of the E6 and E7 oncoproteins. In fact, mutations
affecting the HPV16 and the bovine papillomavirus type E6 zinc fingers interfere with cellular
transformation as well as with complex formation between E6 and E6AP or E6BP (9,24-27). On the basis of these
observations, one can predict similar effects of
chemical compounds that would target and modify these zinc fingers.
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MATERIALS AND METHODS |
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The compounds can be classified into three major groups, i.e., disulfides (including C1, C5,
C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, R1, R2, R6, R7, R8, R9,
R10, R15, R16, R17, R18, and R19), azoics (including C4, R4, R5, R11, R13, and R14), and
nitroso aromatics (including C2 and C3). The chemical names and abbreviations used for the
compounds follow, and structures of representative compounds from each group are shown in
Fig. 1, B.
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Expression of E6, E6AP, and E6BP as Glutathione S-Transferase-Fusion Proteins
E6, E6AP, and E6BP-glutathione S-transferase (GST)-fusion proteins were prepared with the pGEX system (Amersham Pharmacia Biotech AB, Uppsala, Sweden). The HPV16 E6 gene was amplified by polymerase chain reaction and cloned into pGEX4T2 vector as an Not1-Sal1 insert. A clone encoding the C-terminal 210 amino acids of E6BP in pGEX3X vector was a gift from E. J. Androphy (9). E6AP (amino acids 213-865), cloned in pGEX2T vector, was a gift from P. M. Howley (37). These vectors were grown in Escherichia coli AB1899, induced for protein expression for 4 hours with 0.2 mM isopropyl-ß-D-thiogalactopyranoside, harvested and lysed in GST buffer (phosphate-buffered saline [PBS], 50 mM Tris [pH 8.0], and 0.1% Triton X-100) with 5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mg/mL lysozyme, and followed by sonication. After ultracentrifugation at 240 000g for 40 minutes at 4 °C, supernatants of bacterial lysates were incubated at 4 °C on a column of reduced glutathione (GSH)-Sepharose beads (Amersham Pharmacia Biotech AB). Unbound, non-GST-fusion proteins were eliminated by several washes with GST buffer. For direct use of GST-fusion proteins bound on GSH-Sepharose beads in the zinc-release assay, the GSH-Sepharose beads were resuspended in PBS and Tris buffer at 50 mM (pH 8.2). GST-fusion proteins for BIACORE analysis were eluted with elution buffer (10 mM GSH, 50 mM Tris, and PBS [pH 8.2]).
Zinc-Release Assays
Release of zinc from HPV16 E6 was monitored by the change in fluorescence of the zinc-specific fluorophore N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide (TSQ) (Molecular Probes, Inc., Eugene, OR) by a modification of published procedures (31,34,38). In a total reaction volume of 200 µL, 9 µg of recombinant GST-E6 protein (corresponding to a concentration of 1 µM) bound to GSH-Sepharose beads was incubated with 10 µM compound or 0.6% (170 mM) H2O2 in TSQ-assay buffer (10 mM sodium phosphate buffer [pH 7.0] and 10% glycerol) for 2 hours at room temperature in 96-well plates.
Immediately after addition of TSQ to a concentration of 100 µM, the increase
in fluorescence was measured on an SLT Fluostar (355-nm excitation filter and 460-nm emission
filter; Tecan, Salzburg, Austria). Measurements were performed in duplicate, and the values
± the standard deviation obtained for the compound were compared with the values
obtained with the positive control, hydrogen peroxide (H2O2). The
values were standardized with a ZnCl2 solution covering a concentration range of 0-1
µM zinc ion, and positive compounds were arbitrarily identified as those having
values greater than 50% release of zinc from E6. The TSQ-background values for
compounds were less than the equivalent 0.1-µM ZnCl2 value. The
results are presented in Table 1.
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Binding of GST-E6 to GST-E6BP, GST-E6AP, and GST was monitored by surface plasmon
resonance (SPR) on a BIACORE 2000 machine (BIACORE AB, Uppsala, Sweden). SPR occurs
when surface plasmon waves (the calculated quantity of the entire longitudinal wave of a solid
substance's electron gas) are excited by a light source at a metal-liquid interface that in the
BIACORE 2000 occurs on a chip with buffer flowing over it. Light is reflected from the side of
the surface not in contact with sample coupled to the chip, and SPR causes a change in the
reflected light intensity at a specific combination of angle and wavelength. Thus, the binding of
molecules (termed "analytes") to the molecules coupled to the chip (termed
"ligands") causes changes in the refractive index at the surface layer, which are
measured as changes in the resonance signal and are presented as resonance units (RU). The
purified ligand (GST, GST-E6AP, or GST-E6BP) was covalently amine coupled to a CM-5
sensor chip by use of the amine-coupling kit from BIACORE AB. Briefly, the carboxyl groups
on the dextran matrix of the sensor chip surface were activated with a 1 : 1 mixture of 50
mM N-hydroxysuccinimide (NHS) and 200 mM
N-ethyl-N'-(3-diethyl-aminopropyl)-carbodiimide for 6 minutes. Thirty
microliters of ligand,
GST, GST-E6AP, or GST-E6BP (30 µg/mL in sodium acetate buffer [pH 4 to pH
5]), was then injected across the activated surface and followed by injection of 35 µL
of 1 M ethanolamine-hydrochloride (pH 8.5), which deactivated any residual NHS
esters on the sensor chip surface. All the steps were performed at 5 µL/minute. Typically,
6000-10 000 RU of GST, E6BP, and E6AP were immobilized on three different flow
cells.
Aliquots of purified HPV16 GST-E6 (7 µM in 10 mM GSH and 50
mM Tris-PBS buffer [pH 8.2]) were incubated with either 400
µM compound or 5 mM EDTA or 0.6% (170 mM)
H2O2 for 2 hours at room temperature. The 10 mM GSH was
present in the
aliquots of GST-E6 because GSH was required to elute GST-E6 from GSH-Sepharose beads.
Then 10 µL of sample was injected at 1 µL/minute over the three immobilized ligands
by use of the sequential flow mode. Interactions between GST-E6 and the ligands GST,
GST-E6AP, and GST-E6BP were monitored by the change in signal measured in RU. Between
each sample examined, the surfaces were regenerated with a 1-minute pulse of 50 mM
NaOH that resulted in complete dissociation of noncovalently bound analyte (GST-E6), leaving
the immobilized GST, GST-E6BP, or GST-E6AP. After 20 cycles of binding and regeneration,
the amount of ligand present on the chip decreased by approximately 18%-19%
and, therefore, reduced the maximal amount of E6 binding. Typically, complex formation
without compound treatment led to signals of 1540-1900 RU and 1150-1400 RU for GST-E6
with GST-E6BP and GST-E6AP, respectively. Absence of a resonance signal, or a reduced
signal, was scored as an active compound because it indicated the failure to form complexes.
Oxidation of GST-E6 sulfhydryl groups by H2O2 or chelating of zinc
ions by EDTA eliminated complex formation. Also, GST-E6 had little binding to a CM-5 sensor
chip containing no ligand (data not shown) or GST, excluding in the latter case the nonspecific
interaction of the N-terminal GST residues. The results are given in Fig. 2, A and B.
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The open-reading frame of HPV16 E6, cloned into the HindIII and PstI
sites of the pSP64 plasmid (39), was translated
in vitro with
[35S]cysteine by use of the TNT-SP6 Coupled Reticulocyte Lysate
System as recommended by the manufacturer (Promega Corp., Madison, WI). All washing and
binding reactions were performed with the E6BP-binding buffer as described (9) but without DTT. Forty microliters of the lysate
containing in vitro
translated E6 plus 360 µL of E6BP-binding buffer was incubated for 2 hours at room
temperature with test compounds at concentrations from 0 to 1 mM (dissolved in
DMSO at 1%) or 5 mM EDTA or H2O2 at 0.3%
(85 mM). The samples were then passed over columns containing GSH-Sepharose
beads with bound GST, GST-E6, GST-E6BP, or GST-E6AP proteins. The beads were washed
twice with E6BP-binding buffer. Finally, the beads were heated to 95 °C in 50 µL of
Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA) with 2.5%
2-mercaptoethanol, and the proteins solubilized in the sample buffer were subjected to
electrophoresis on a 15% polyacrylamide gel, fixed, stained, and autoradiographed.
Interference with complex formation between in vitro translated E6 and GST, GST-E6,
GST-E6BP, or GST-E6AP identified reactive compounds. Densitometric quantification was
performed with a Bio-Rad/GS700 imaging densitometer. The results are given in Figs.
3 and 4
.
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All cell lines were obtained from the American Type Culture Collection (Manassas, VA)
unless otherwise noted. SiHa (human cervical epithelial tumor line, HPV16-positive), CaSki
(human cervical epithelial tumor line, HPV16-positive), HaCat (immortalized human skin
epithelial cell line, HPV-negative), HeLa (human cervical epithelial tumor line,
HPV18-positive), 444 (hybrid of HeLa and fibroblast, HPV18-positive; obtained from Eric
Stanbridge, University of California, Irvine), MCF7 (human mammary epithelial tumor cell line,
HPV-negative), HT3 (human cervical epithelial tumor cell line, HPV-negative; a line that has
been in culture in the laboratory of H. U. Bernard), and HepG2 (human liver epithelial tumor cell
line, HPV-negative) were grown in Dulbecco's modified Eagle medium supplemented
with 10% fetal calf serum, 100 U penicillin, and 1000 U streptomycin. Cells were allowed
to attach to the surface of microwell dishes overnight and subsequently incubated with medium
containing the zinc-ejecting compounds at the concentrations (10-100 µM) shown
in the legends to Figs. 5-7. The viability of the cells
was scored by measurement of the absorption of the
tetrazolium salt WST1 (Roche Molecular Biochemicals, Mannheim, Germany) with the use of
the plate reader (Tecan) at a wavelength of 450 nm and a reference wavelength of 630 nm.
Results are given in Figs. 5
and 6
.
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Cells (106) (see Fig. 7
legend for details) were
plated on 10-cm Petri
dishes with 10 mL of medium; after attachment overnight, they were treated with C16 (final
concentration, 100 µM) or DMSO (final concentration, 0.5%) for 1 day. At
the time of cell harvest, most C16-treated cells were still attached to the plate. Cells were
harvested with the use of a rubber policeman and lysed in 10 mM HEPES buffer (pH
7.2), 150 mM NaCl, 0.2% Nonidet P-40, and 1 mM PMSF, followed by
centrifugation at 10 000g for 20 minutes at 4 °C. A total of 20 µg of
protein
was loaded onto a 12% polyacrylamide gel containing sodium dodecyl sulfate and
transferred to a nitrocellulose membrane, and the membrane was blocked with 5% nonfat
dry milk in 20 mM Tris-Cl (pH 7.6), 150 mM NaCl, and 0.05% Tween
20 overnight at 4 °C. The membrane was then probed with primary antibodies against p53
(Santa Cruz Biotechnology, Santa Cruz, CA), ß-actin (Sigma Chemical Co., St. Louis, MO),
or PARP (C2-10; Centre de Research du Chul, Quebec, Canada) and followed by incubation with
horseradish peroxidase-conjugated secondary antibody (Pierce Chemical Co., Rockford, IL).
Finally, the blot was treated with an enhanced chemiluminescent detection substrate
(SuperSignal; Pierce Chemical Co.) and autoradiographed. Results are given in Fig. 7.
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RESULTS |
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The structure and function of the HPV16 E6 oncoprotein depend on the integrity of two zinc fingers, in which the thiolates of eight cysteine residues serve as metal-chelating residues. Chemical alteration of these thiolates should lead to passive zinc release and corresponding structural and functional changes, as observed for the HIV-1 zinc-finger protein NCp7 (30-32,34). On the basis of these assumptions, we established an assay to monitor zinc release from E6. This release was measured as an increase in the fluorescence of the zinc-selective fluorophore TSQ (34,38,40) in the presence of E6 protein and presumed thiolate-reactive compounds.
Thirty-six compounds were selected for the screen based on previous studies on HIV-1 NCp7
(30-32) or based on their potential to be
able to participate in disulfide
exchange and other redox reactions. We refer to these compounds as C1 to C19 and R1 to R19
(see "Materials and Methods" section). These compounds can be
classified into three major groups, i.e., disulfides, azoics, and nitroso aromatics; an example of
each is shown in Fig. 1, B. Of the 36 compounds
tested for zinc release
from bacterially
expressed E6-fusion protein (GST-E6), nine compounds (i.e., C4, C13, C14, C16, R2, R15, R16,
R18, and R19) produced relative fluorescent unit values, the indicator of zinc release, of
50%-75% compared with the value of the positive control for zinc release,
H2O2 (Table 1
). No
compound was as effective as the H2O2 in releasing bound zinc. The
other 27 compounds, although
structurally similar to some of
the active compounds, either were inactive or released only a small fraction of the bound zinc. In
similar experiments (data not shown), we observed that GST-E6 protein derived from HPV18
was also selectively altered by these nine but not by the other 27 compounds.
Inhibition of Formation of E6-E6AP and E6-E6BP Complexes by Zinc-Releasing Compounds as Measured in BIACORE Assays
BIACORE technology allows real time analysis of biomolecular interactions without the need for isotopic or enzymatic labeling (41). This technology is based on optical SPR, a technique that allows for the detection of small changes in the refractive index on the surface of a thin gold film coated with a dextran matrix. Using this technique, we monitored the interactions of E6 with E6AP and with E6BP. GST-E6 treated with compounds was passed over immobilized E6AP or E6BP, and the interaction between the molecules was measured in RU.
We assumed that zinc-releasing compounds would lead to a change in the tertiary structure
of E6 protein and would prevent E6 from forming heterologous complexes. As Fig. 2
demonstrates, two (C16 and R16) of the nine compounds that triggered zinc release from
GST-E6 also interfered with the ability of GST-E6 to form complexes with GST-E6AP or
GST-E6BP. The activity profiles of the compounds for these two cellular proteins were very
similar, reflecting the fact that the domain of E6BP that binds to E6 is also found in E6AP
(42). We conclude that, under the conditions of
these BIACORE assays,
C16 and R16 trigger zinc release, resulting in alteration of the structure of GST-E6 and the
failure to establish the heterologous complexes.
Differential Effects of Zinc-Releasing Compounds on E6-E6BP Heterodimer Interactions and E6-E6 Dimer Interactions Observed With the Use of In Vitro Translation and Binding Assays
Fig 3, A,. shows the outcome of binding
experiments between
GST-E6BP
incubated with in vitro translated E6 (GST pulldown), which
was treated with zinc-releasing compounds. Here, seven of the nine
compounds that led to zinc ejection in the TSQ assays, including C16
and R16, prevented E6 protein from binding to the GST-E6BP. These
results exclude the possibility that binding between GST-E6BP and
GST-E6 protein as measured in BIACORE assays was due to interactions
between GST termini, since GST was not present in the in vitro
translated E6.
We also observed that GST-E6 can bind to in vitro translated E6 protein (Fig. 3,
B),
which is in support of previous evidence that E6 homodimers exist in vitro (43). This
interaction requires E6 to be complexed with zinc, inasmuch as EDTA and several of the
compounds that release zinc inhibit E6 homodimer formation. It is interesting that the activity
profile of these compounds against E6-dimer formation was different from that observed with the
E6-E6BP interaction. The most dramatic difference observed was with the compound C13,
where almost 80% of the E6-E6BP interaction was inhibited, while none was observed for
the E6-dimer formation. These results suggest that functional differences exist between the two
zinc fingers in E6.
We also compared the effect of C16, which had the greatest inhibitory activity in BIACORE
assays, on E6BP and E6AP binding in GST-pulldown tests. As shown in Fig. 4, A and B, C16
inhibits E6 binding to both cellular proteins (E6AP and E6BP), with the concentration range
from 10 µM (as used in the TSQ assay) to 100 µM being required
for inhibitory activity.
Effects of C16 on Viability of Tumor-Derived HPV-Containing Cell Lines
Experiments with antisense RNA and inhibitors of HPV transcription suggest that the E6 and E7 proteins are required for continuous growth of HPV16- or HPV18-containing tumor cell lines (39,44-46) and that repression of E6 and E7 transcription leads to G1 arrest and apoptosis (47). It is therefore conceivable that compounds directed against the E6 protein may not just interfere with the HPV16 life cycle but that they may also arrest HPV16-associated malignancies.
To determine whether the nine compounds that had scored positive in the TSQ assay would
affect cell viability, we examined SiHa and CaSki cells, whose growth depends on the expression
of endogenous copies of HPV16, and HaCat cells, an immortalized human skin epithelial cell
line that does not contain HPV (Fig. 5, A).
Further examination (Fig. 5, B
) demonstrates that
only C16 had strong inhibitory effects on SiHa and CaSki cells and no adverse effect on HT3
cells, an HPV-negative cell line derived from a cervical carcinoma, HaCat cells, or MCF7 cells, a
breast carcinoma. In other experiments, C16 had no effect on the human hepatoma cell line
HepG2 (data not shown). As an additional control, we compared the activity of C16 with that of
C4 (Fig. 5, B
), which causes the ejection of zinc
from HIV-1 NCp7 and is
currently used in
clinical trials for the treatment of AIDS. In addition to the three cell lines targeted by all nine
compounds, C4 and C16 were also applied to HeLa and 444 cells (Fig. 5, B
). Proliferation of
HeLa cells depends on expression of the HPV18 E6 and E7 genes, while 444 cells, which were
derived from HeLa cells by fusion with fibroblasts, show in vivo low expression levels
of HPV18 genes, E6- and E7-independent viability, and lack of tumorigenicity in nude mice
(44,48). Fig. 5, B,
documents that C4 did not
cause growth inhibition in
any of these six cell lines, while C16 specifically reduced the viability of SiHa, CaSki, and HeLa
cells. Microscopic observation (Fig. 6
) clearly
demonstrates the
differential effect of C16 on
E6-dependent cells (SiHa and HeLa) and E6-independent cells (444 and HaCat). The most likely
explanation for these results is that C16 abrogates the function of the HPV16 and HPV18 E6
proteins by ejecting the zinc moiety, with adverse effects on the viability of these
HPV-dependent cells.
Effects of C16 on Intracellular p53 Levels of Cells Containing HPV
The E6 protein forms a heteromeric complex with E6AP and p53,
thereby targeting p53 for degradation by the ubiquitination pathway
(7). To examine whether inhibition of the
E6-E6AP interaction
may interfere with p53 degradation, we treated four cell lines with C16
and examined p53 protein expression. Fig. 7, A,
shows that C16
increased the concentration of p53 80-fold in HeLa cells and sevenfold
in SiHa cells. No increase in p53 levels occurred in HaCat cells, which
carry a mutant p53 gene (49), and only a twofold
increase was
observed in MCF7 cells. Because increases in p53 expression are known
to be associated with apoptosis (50), we
determined whether
the C16-dependent elevated levels of p53 induced apoptosis. This was
examined by monitoring the cleavage of PARP, a hallmark of apoptosis,
in C16-treated cell lines (Fig. 7, B
). PARP
cleavage was observed only
in the HeLa cells incubated with C16 and not in the C16-treated HaCat
cells. These results strongly correlate with the viability measurements
observed in Figs. 5
and 6
.
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DISCUSSION |
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Mutation analysis of the cysteines involved in coordinating zinc has demonstrated that zinc binding is a requirement for E6 interaction with E6AP and E6BP (9,24-27). On the basis of the assumption that chemical alteration of cysteine residues should have similar consequences to mutational alteration, we used BIACORE and GST-pulldown experiments as secondary screens. Among the nine zinc-releasing compounds, C16 and R16 were able to inhibit the heteromeric interactions of E6 with E6AP or E6BP in the BIACORE assay, and the same two compounds and five additional ones were active in the GST-pulldown experiments, the differences most likely originating from different assay conditions. The GST-pulldown results also led to the observation that E6 can form zinc-dependent homodimers. Furthermore, a differential effect of the compounds was observed for this interaction as compared with the heterocomplex formation, suggesting that the different zinc fingers of E6 may play different roles in protein-protein interactions.
Role of GSH in Drug Screens
Reduced GSH is, at concentrations of 1-10 mM in most cell types, the most abundant non-protein intracellular thiol, and it is involved in biochemical reactions that can inactivate pharmaceutical compounds (51,52). Under these conditions, only a few of the compounds, including C16, were capable of releasing zinc (data not shown) in the TSQ assay. One possibility for the different results observed between the TSQ and the BIACORE assays is that the physiologic levels of GSH present in the BIACORE assay may have interfered with the inhibitory activity of seven of the nine compounds that released zinc from E6 in the TSQ assay, which is in the absence of GSH. Increased concentrations of C16 were also required in the GST-pulldown and cell viability assays, possibly to overcome the endogenous levels of GSH in the reticulolysate extracts and cells, respectively. These observations position the TSQ assay as superior when it comes to initially screening compounds that induce zinc release from E6. The zinc-releasing compounds can then be evaluated in the BIACORE, GST-pulldown, and in vivo assays, where physiologic GSH levels are present, to identify compounds that reach intracellular E6 in sufficiently high concentrations and in chemically unaltered form. Similar considerations were encountered in studying the inactivation of HIV-1 NCp7 by C4 (32), which is active on NCp7 as part of the cell-free virion, inhibiting HIV-induced cytopathicity of CEM-SS cells with an EC50 (i.e., median effective concentration) of 37 µM, but fails to be active on intracellular viral zinc fingers. Reduction and inactivation of C4 by intracellular mechanisms are further suggested by the observation that the reduced form of C4, biurea, fails to have an effect on NCp7 in vitro.
Specificity of Zinc-Releasing Compounds in Cell Culture Studies
Zinc-finger proteins are required for the maintenance of cell viability. Therefore, one concern is that the zinc-releasing compounds may have effects on normal cellular functions. While two of the compounds tested were toxic to all the cell lines, the majority of compounds tested had little or no effect on cell growth, suggesting that there is little interference with the endogenous zinc-finger proteins. At concentrations of 50-100 µM, C16 preferentially inhibited the growth of the tumorigenic HPV cell lines SiHa, CaSki, and HeLa, as opposed to a lack of inhibition of the nontumorigenic HPV cell line 444 and the immortalized epithelial HaCat cell line. Furthermore, 50-100 µM C16 did not inhibit cell growth in tumorigenic HPV-negative cervical HT3 cells or tumorigenic MCF7 and HepG2 cells.
These data suggest that the inactivation of the E6 oncoprotein by C16 leads to reduced
viability of cell lines whose continuing growth depends on E6 functions. Decreased cell viability
appears to be connected with the p53 pathway, since we could demonstrate a dramatic increase in
p53 protein levels in the C16-sensitive cell lines HeLa, SiHa, and CaSki but not in the
HPV-negative cell lines HaCat and MCF-7 (Fig. 7, A). The role of p53 in
apoptosis is well
documented (50). Further evidence to support
our model that C16
induces p53-mediated apoptosis is the observation that PARP cleavage (Fig. 7, B
) and increased
caspase 3 activity (data not shown), both indicators of apoptosis, occurred with C16-treated HeLa
cells but not with C16-treated, HPV-negative HaCat cells.
Potential Scopes of Anti-HPV E6 Drugs
Our present knowledge suggests functions of E6 (and E7) in situ in three different pathologic scenarios. 1) In stratified epithelia, uninfected epithelial cells differentiate without further mitosis after they leave the basal layers and become part of the suprabasal layers. HPV E6 and E7 proteins interfere with this repression of mitosis and induce a dedifferentiated and expanded cell population that can progress from a latent infection to a benign intraepithelial neoplasia (2). 2) In these benign lesions, E6 and E7 maintain a high frequency of aberrant mitoses leading to chromosomal aberrations and aneuploidies, raising the chance for the generation of increasingly tumorigenic cellular variants (6). 3) Continuous expression of E6 and E7 may be required for continuous proliferation of malignant tumors (39,45,48). Anti-E6 and anti-E7 drugs may be able to interfere with formation or persistence of HPV-associated lesions at all three levels of carcinogenesis.
Future Objectives
To the best of our knowledge, this article describes for the first time assay systems to identify anti-HPV16 E6 drugs. We believe that similar approaches can target the E7 protein, since it is another zinc-finger protein with the unique Cys-X2-Cys-X29-Cys-X2-Cys motif. It is possible that the different chemical environment of each of the cysteines could lead to identification of compounds that not only are specific for either of the two proteins and their three zinc fingers but also are directed against a singular target cysteine. Nonconserved amino acids surrounding these cysteines may even allow us to identify compounds that are specific for different HPV types.
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NOTES |
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REFERENCES |
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1 Howley PM. Papillomavirinae: the viruses and their replication. In: Field BN, Knipe DM, Howley PM, editors. Field's virology. Philadelphia (PA): Lippincott-Raven Publ.; 1996. p. 2045-76.
2 International Agency for Research on Cancer (IARC). Human papillomaviruses, vol 64. Lyon (France): IARC; 1995.
3 zur Hausen H, de Villiers EM. Human papillomaviruses.Annu Rev Microbiol1994 ;48:427-47.[Medline]
4 Wright TC, Kurman RJ. A critical review of the morphologic classification systems of preinvasive lesions of the cervix: the scientific basis for shifting the paradigm. Papillomavirus Rep1994 ;5:175-82.
5 Stanley M, Sarkar S. Genetic changes in cervical cancer.Papillomavirus Rep1994 ;5:141-7.
6 White AE, Livanos EM, Tlsty TD. Differential disruption of genomic integrity and cell cycle regulation in normal human fibroblasts by the HPV oncoproteins. Genes Dev1994 ;8:666-77.[Abstract]
7 Scheffner M, Huibregtse JM, Howley PM. Identification of a human ubiquitin-conjugating enzyme that mediates the E6-AP-dependent ubiquitination of p53.Proc Natl Acad Sci U S A1994 ;91:8797-801.[Abstract]
8 Sherman L, Jackman A, Itzhaki H, Stoppler MC, Koval D, Schlegel R. Inhibition of serum- and calcium-induced differentiation of human keratinocytes by HPV16 E6 oncoprotein: role of p53 inactivation. Virology1997 ;237:296-306.[Medline]
9 Chen JJ, Reid CE, Band V, Androphy EJ. Interaction of papillomavirus E6 oncoproteins with a putative calcium-binding protein. Science1995 ;269:529-31.[Medline]
10
Tong X, Howley PM. The bovine papillomavirus E6
oncoprotein interacts with paxillin and disrupts the actin cytoskeleton. Proc Natl Acad Sci
U S A1997
;94:4412-7.
11 Sedman SA, Barbosa MS, Vass WC, Hubbert NL, Haas JA, Lowy DR, et al. The full-length E6 protein of human papillomavirus type 16 has transforming and trans-activating activities and cooperates with E7 to immortalize keratinocytes in culture.J Virol1991 ;65:4860-6.[Medline]
12 Lamberti C, Morrissey LC, Grossman SR, Androphy EJ. Transcriptional activation by the papillomavirus E6 zinc finger oncoprotein. EMBO J1990 ;9:1907-13.[Abstract]
13 Etscheid BG, Foster SA, Galloway DA. The E6 protein of human papillomavirus type 16 functions as a transcriptional repressor in a mechanism independent of the tumor suppressor protein, p53. Virology1994 ;205:583-5.[Medline]
14 Klingelhutz AJ, Foster SA, McDougall JK. Telomerase activation by the E6 gene product of human papillomavirus type 16. Nature1996 ;380:79-82.[Medline]
15 Hawley-Nelson P, Vousden KH, Hubbert NL, Lowy DR, Schiller JT. HPV16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes.EMBO J1989 ;8:3905-10.[Abstract]
16 Meyers G, Androphy EJ. The E6 protein. In: Meyers G, Bernard HU, Delius H, Baker C, Icenogel J, Halpern AL, et al, editors. Human papillomaviruses 1995. Los Alamos (NM): Los Alamos National Laboratory; 1995. p. 47-57.
17 Androphy EJ, Hubbert NL, Schiller JT, Lowy DR. Identification of the HPV-16 E6 protein from transformed mouse cells and human cervical carcinoma cell lines. EMBO J1987 ;6:989-92.[Abstract]
18 Grossman SR, Laimins LA. E6 protein of human papillomavirus type 18 binds zinc. Oncogene1989 ;4:1089-93.[Medline]
19 Kanda T, Watanabe S, Zanma S, Sato H, Furuno A, Yoshiike K. Human papillomavirus type 16 E6 proteins with glycine substitution for cysteine in the metal-binding motif. Virology1991 ;185:536-43.[Medline]
20 Dsouza M, Larsen N, Overbeek R. Searching for patterns in genomic data. Trends Genet1997 ;13:497-8.[Medline]
21 Chan SY, Delius H, Halpern AL, Bernard HU. Analysis of genomic sequences of 95 papillomavirus types: uniting typing, phylogeny, and taxonomy.J Virol1995 ;69:3074-83.[Abstract]
22 Meyers G, Bernard HU, Delius H, Baker C, Icenogel J, Halpern AL, et al, editors. Human papillomaviruses 1994. Los Alamos (NM): Los Alamos National Laboratory; 1994. p. II-E6-II-E6-7.
23 Ullman CG, Haris PI, Galloway DA, Emery VC, Perkins SJ. Predicted alpha-helix/beta-sheet secondary structures for the zinc-binding motifs of human papillomavirus E7 and E6 proteins by consensus prediction averaging and spectroscopic studies of E7. Biochem J1996 ;319:229-39.[Medline]
24 Crook T, Tidy JA, Vousden KH. Degradation of p53 can be targeted by HPV E6 sequences distinct from those required for p53 binding and trans-activation.Cell1991 ;67:547-56.[Medline]
25 Dalal S, Gao Q, Androphy EJ, Band V. Mutational analysis of human papillomavirus type 16 E6 demonstrates that p53 degradation is necessary for immortalization of mammary epithelial cells. J Virol1996 ;70:683-8.[Abstract]
26 Nakagawa S, Watanabe S, Yoshikawa H, Taketani Y, Yoshiike K, Kanda T. Mutational analysis of human papillomavirus type 16 E6 protein: transforming function for human cells and degradation of p53 in vitro. Virology1995 ;212:535-42.[Medline]
27 Vousden KH, Androphy EJ, Schiller JT, Lowy DR. Mutational analysis of bovine papillomavirus E6 gene. J Virol1989 ;63:2340-2.[Medline]
28 Mackay JP, Crossley M. Zinc fingers are sticking together. Trends Biochem Sci1998 ;23:1-4.[Medline]
29
Klug A, Schwabe JW. Protein motifs 5. Zinc
fingers.FASEB J1995
;9:597-604.
30 Rice WG, Schaeffer CA, Harten B, Villinger F, South TL, Summers MF, et al. Inhibition of HIV-1 infectivity by zinc-ejecting aromatic C-nitroso compounds. Nature1993 ;361:473-5.[Medline]
31 Rice WG, Supko JG, Malspeis L, Buckheit RW Jr, Clanton D, Bu M, et al. Inhibitors of HIV nucleocapsid protein zinc fingers as candidates for the treatment of AIDS. Science1995 ;270:1194-7.[Abstract]
32 Rice WG, Turpin JA, Huang M, Clanton D, Buckheit RW Jr, Covell DG, et al. Azodicarbonamide inhibits HIV-1 replication by targeting the nucleocapsid protein. Nat Med1997 ;3:341-5.[Medline]
33 Yu X, Hathout Y, Fenselau C, Sowder RC 2nd, Henderson LE, Rice WG, et al. Specific disulfide formation in the oxidation of HIV-1 zinc finger protein nucleocapsid p7. Chem Res Toxicol1995 ;8:586-90.[Medline]
34
Tummino PJ, Scholten JD, Harvey PJ, Holler TP,
Maloney L,
Gogliotti R, et al. The in vitro ejection of zinc from human immunodeficiency virus
(HIV) type 1 nucleocapsid protein by disulfide benzamides with cellular anti-HIV activity.Proc Natl Acad Sci U S A1996
;93:969-73.
35 Rice WG, Hillyer CD, Harten B, Schaeffer CA, Dorminy M, Lackey DA 3d, et al. Induction of endonuclease-mediated apoptosis in tumor cells by C-nitroso-substituted ligands of poly(ADP-ribose) polymerase. Proc Natl Acad Sci U S A1992 ;89:7703-7.[Abstract]
36 Vandevelde M, Witvrouw M, Schmit JC, Sprecher S, De Clercq E, Tassignon JP. ADA, a potential anti-HIV drug [letter]. AIDS Res Hum Retroviruses1996 ;12:567-8.[Medline]
37 Huibregtse JM, Scheffner M, Howley PM. Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53. Mol Cell Biol1993 ;13:775-84.[Abstract]
38 Rice WG, Turpin JA, Schaeffer CA, Graham L, Clanton D, Buckheit RW Jr, et al. Evaluation of selected chemotypes in coupled cellular and molecular target-based screens identifies novel HIV-1 zinc finger inhibitors. J Med Chem1996 ;39:3606-16.[Medline]
39 Tan TM, Ting RC. In vitro and in vivo inhibition of human papillomavirus type 16 E6 and E7 genes. Cancer Res1995 ;55:4599-605.[Abstract]
40 Frederickson CJ, Kasarskis EJ, Ringo D, Frederickson RE. A quinoline fluorescence method for visualizing and assaying the histochemically reactive zinc (bouton zinc) in the brain. J Neurosci Methods1987 ;20:91-103.[Medline]
41 Malmqvist M. Surface plasmon resonance for detection and measurement of antibody-antigen affinity and kinetics. Curr Opin Immunol1993 ;5:282-6.[Medline]
42
Chen JJ, Hong Y, Rustamzadeh E, Baleja JD,
Androphy EJ.
Identification of an alpha helical motif sufficient for association with papillomavirus E6. J
Biol Chem1998
;273:13537-44.
43 Daniels PR, Sanders CM, Coulson P, Maitland NJ. Molecular analysis of the interaction between HPV type 16 E6 and human E6-associated protein.FEBS Lett1997 ;416:6-10.[Medline]
44 Bartsch D, Boye B, Baust C, zur Hausen H, Schwarz E. Retinoic acid-mediated repression of human papillomavirus 18 transcription and different ligand regulation of the retinoic acid receptor beta gene in non-tumorigenic and tumorigenic HeLa hybrid cells. EMBO J1992 ;11:2283-91.[Abstract]
45 von Knebel Doeberitz M, Rittmuller C, zur Hausen H, Durst M. Inhibition of tumorigenicity of cervical cancer cells in nude mice by HPV E6-E7 anti-sense RNA [letter]. Int J Cancer1992 ;51:831-4.[Medline]
46 Steele C, Sacks PG, Adler-Storthz K, Shillitoe EJ. Effect on cancer cells of plasmids that express antisense RNA of human papillomavirus type 18.Cancer Res1992 ;52:4706-11.[Abstract]
47
Desaintes C, Demeret C, Goyat S, Yaniv M, Thierry
F.
Expression of the papillomavirus E2 protein in HeLa cells leads to apoptosis. EMBO
J1997
;16:504-14.
48 Bosch FX, Schwarz E, Boukamp P, Fusenig NE, Bartsch D, zur Hausen H. Suppression in vivo of human papillomavirus type 18 E6-E7 gene expression in nontumorigenic HeLa X fibroblast hybrid cells. J Virol1990 ;64:4743-54.[Medline]
49 Lehman TA, Modali R, Boukamp P, Stanek J, Bennett WP, Welsh JA, et al. p53 mutations in human immortalized epithelial cell lines [published erratum appears in Carcinogenesis 1993;14:1491]. Carcinogenesis1993 ;14:833-9.[Abstract]
50 Polyak K, Xia Y, Zweier JL, Kinzler KW, Vogelstein B. A model for p53-induced apoptosis. Nature1997 ;389:300-5.[Medline]
51 Kearns PR, Hall AG. Glutathione and the response of malignant cells to chemotherapy. Drug Discov Today1998 ;3:113-21.
52 Tew KD. Glutathione-associated enzymes in anticancer drug resistance. Cancer Res1994 ;54:4313-20.[Abstract]
Manuscript received October 19, 1998; revised May 7, 1999; accepted May 27, 1999.
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