U.S. patent application number 14/314095 was filed with the patent office on 2014-10-16 for method of determining susceptibility of a tumor cell to a chemotherapeutic agent: novel use of herpes simplex virus.
The applicant listed for this patent is Medical Diagnostic Laboratories, LLC. Invention is credited to John Blaho.
Application Number | 20140308668 14/314095 |
Document ID | / |
Family ID | 41447910 |
Filed Date | 2014-10-16 |
United States Patent
Application |
20140308668 |
Kind Code |
A1 |
Blaho; John |
October 16, 2014 |
Method of Determining Susceptibility of a Tumor Cell to a
Chemotherapeutic Agent: Novel Use of Herpes SImplex Virus
Abstract
The present invention provides a method of determining if a
tumor cell is susceptible to killing by a chemotherapeutic agent,
comprising: (a) providing a tumor cell; (b) infecting said tumor
cell with a herpes simplex virus or a herpes simplex virus
defective in an immediate early gene selected from the group
consisting of ICP27, ICP4, and ICP22; and (c) determining the
presence of apoptotic killing of said tumor cell, wherein the
presence of apoptotic killing is indicative of susceptibility to
said chemotherapeutic agent. Chemotherapeutic agent may include
doxorubicin, etoposide, paclitaxel, cisplatin, or 5-fluororuacil.
The present invention also provides a herpes simplex virus promoter
construct having a lacZ gene to assess tumor resistance to
chemotherapeutic agents.
Inventors: |
Blaho; John; (New York,
NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Medical Diagnostic Laboratories, LLC |
Hamilton |
NJ |
US |
|
|
Family ID: |
41447910 |
Appl. No.: |
14/314095 |
Filed: |
June 25, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12459332 |
Jun 30, 2009 |
8778331 |
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14314095 |
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61133478 |
Jun 30, 2008 |
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Current U.S.
Class: |
435/6.11 |
Current CPC
Class: |
C12Q 1/6897 20130101;
G01N 33/5011 20130101; G01N 33/5026 20130101; G01N 2333/035
20130101; C12N 2710/16631 20130101 |
Class at
Publication: |
435/6.11 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A kit for determining susceptibility of a tumor cell to
apoptotic killing by a chemotherapeutic agent, said kit comprising:
(a) a herpes simplex virus report construct, said construct
comprises: (i) a herpes simplex virus immediate early gene promoter
selected from the group consisting of ICP27, ICP4, and ICP22; and
(ii) a lacZ gene, wherein said gene promoter is operably linked to
said lacZ gene; (b) .beta.-galactosidase to detect gene expression
of said lacZ gene; and (c) an instruction, said instruction
describes the use of said herpes simplex virus report construct to
infect said tumor cell and staining of said .beta.-galactosidase to
detect the gene expression of said lacZ gene, wherein negative
.beta.-galactosidase staining reveals that said tumor cell is
susceptible to chemotherapeutic agent.
2. The kit of claim 1, wherein said chemotherapeutic agent is
selected from doxorubicin, etoposide, paclitaxel, cisplatin, and
5-fluorouracil.
3. The kit of claim 1, wherein said tumor cell is derived from a
source selected from the group consisting of breast, brain, and
cervix.
4. The kit of claim 1, wherein said tumor cell is derived from
breast.
5. A kit for determining susceptibility of a tumor cell to a
chemotherapeutic agent, comprising: (a) herpes simplex virus
lacking an immediate early gene selected from the group consisting
of ICP27, ICP4 and ICP22; (b) a reagent to determine apoptosis of
tumor cell; and (c) an instruction, said instruction details the
use of said herpes simplex virus in inducing apoptosis of tumor
cells and measuring said apoptosis, wherein the presence of said
apoptosis of tumor cells reveals said tumor cell is susceptible to
a chemotherapeutic agent.
6. The kit of claim 5, wherein said chemotherapeutic agent is
selected from doxorubicin, etoposide, paclitaxel, cisplatin, and
5-fluorouracil.
7. The kit of claim 5, wherein said tumor cell is derived from a
source selected from the group consisting of breast, brain, and
cervix.
8. The kit of claim 5, wherein said tumor cell is derived from
breast.
9. The kit of claim 5, wherein said reagent determines apoptotic
killing by chromatin condensation, fragmentation of nucleic,
membrane blebbing or formation of apopotic bodies.
10. The kit of claim 5, said reagent is a fluorescence probe.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional application of U.S.
application Ser. No. 12/459,332 filed on Jun. 30, 2009 and claims
the benefit of priority under 35 U.S.C. .sctn.119(e) to U.S.
Provisional Application No. 61/133,478, filed Jun. 30, 2008, the
disclosures of which are hereby incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present application relates generally to a method of
determining tumor cell susceptibility to a chemotherapeutic agent;
more particularly, the present invention relates to using a herpes
simplex virus mutant lacking an immediate early gene in determining
tumor cell susceptibility to a chemotherapeutic agent.
BACKGROUND OF THE INVENTION
[0003] Chemotherapeutic agents are frequently used in the clinical
treatment of many forms of tumors. Information regarding whether a
given tumor cell is susceptible (i.e., sensitive) or resistant to a
particular chemotherapeutic agent is critical. Provided in advance,
this information greatly enhances a physician's ability to
implement proper dosages to kill the tumor cells. In addition, such
information permits swift changes in treatment regimes and
therefore avoids toxic side effects of the chemotherapeutic agent
if the tumor cell proves to be chemotherapy resistant. Where a
given tumor is initially sensitive to chemotherapy agents but
develops resistance over the course of treatment, it becomes
necessary to gain information about the susceptibility change.
[0004] There have been several disclosed tests whose goals are to
predict tumor sensitivity to chemotherapy agents. One early test is
based on the observation in 1954 that the ability of chemotherapy
agents to reduce cellular metabolism could be monitored by
measuring tetrazolium blue reduction by fresh tumor biopsy
materials. (Black et al., J. Nat'l Cancer Inst. 14, 1147-1158
(1954)). Most other tests correlate chemo-sensitivity to a
particular intracellular chemical. For example, U.S. Pat. No.
5,366,885 discloses the use of elevated glutathione to predict
tumor drug sensitivity. To overcome false-negative or
false-positive results, however, a four-tiered confirmatory testing
is required. This cumbersome biochemical tests render the approach
undesirable.
[0005] U.S. Pat. No. 5,270,172 discloses an in vitro method that
utilizes estrogen and anti-estrogen and requires quantifying cell
growth inhibition under these culture conditions. U.S. Patent Appl.
No. 2006/0172305 discloses a method of measuring susceptibility via
a glucose transporter. U.S. Pat. No. 6,949,359 discloses
chemosensitivity determination using one marker whose specific
binding capability to phosphatidylserine can be detected. U.S. Pat.
No. 7,344,829 discloses a method for detecting the efficacy of
anti-cancer treatment by comparing growth factor receptor
phosphorylation. While all these assays may provide a measure of
predictability to the question of tumor drug resistance, they often
require long assay duration and lack reliability. Thus, there
exists an unfulfilled need for a predictive assay for drug
resistance, which provides rapid, reliable results for a spectrum
of possible chemotherapy agents.
[0006] A method of determining drug susceptibility profile for a
particular tumor (prior to the administration of chemotherapy
agents) is highly desirable. However, there has been no suggestion
in the art relating to a method of using virus as a means to
determine tumor cell susceptibility to chemotherapy agents. There
has also been no information relating the application of herpes
simplex virus as a vehicle to assess drug susceptibility. The
present inventors have surprisingly discovered that herpes simplex
virus lacking an immediate early gene is a novel and reliable
indicator for use in determining tumor cell susceptibility to
chemotherapeutic agents.
SUMMARY OF THE INVENTION
[0007] The present invention provides a method of determining if a
tumor cell is susceptible to apopotic killing by a chemotherapeutic
agent using a herpes simplex virus lacking an immediate early gene.
Preferably, the herpes simplex virus lacks ICP27, ICP4 or
ICP22.
[0008] In one aspect, the present invention provides a method of
determining susceptibility of a tumor cell to apoptotic killing by
a chemotherapeutic agent, comprising the steps of: (a) providing a
tumor cell; (b) infecting said tumor cell with a herpes simplex
virus-1 lacking an immediate early gene selected from the group
consisting of ICP27, ICP4, and ICP22; and (c) determining the
presence of apoptotic killing of said infected tumor cell, wherein
the presence of apoptotic killing is indicative of susceptibility
of said tumor cell. Preferably, the chemotherapeutic agent is
doxorubicin, etoposide, paclitaxel, cisplatin, or
5-fluororuacil.
[0009] In another aspect, the absence of apoptotic killing of said
infected tumor cell is indicative of resistance of said tumor cell
to a chemotherapeutic agent.
[0010] In another aspect, the tumor cell is derived from a source
of pancreas, colon, prostate, brain, skin, cervix, liver or
stomach. Preferably, the tumor cell is derived from breast, brain
or cervix. More preferably, the tumor cell is derived from
breast.
[0011] In another aspect, the infecting step is performed using a
herpes simplex virus-1 lacking immediate early gene of ICP27.
[0012] In another aspect, the determining step is performed by
analyzing one morphological alternation including cell shrinkage,
membrane blebbing, or chromatin condensation. Preferably,
determining step is performed by analyzing chromatin
condensation.
[0013] In an alterative aspect, the determining step is performed
by measuring death factor including poly(ADP-ribose) polymerase,
caspase 3, or DNA fragmentation factor-45. Preferably, the
determining step is performed by measuring poly(ADP-ribose)
polymerase.
[0014] In yet another aspect, the present invention provides a
herpes simplex virus report construct, comprising: (a) a herpes
simplex virus immediate early gene promoter, said gene promoter is
selected from the group consisting of ICP27, ICP4, and ICP22; and
(b) a lacZ gene, wherein said gene promoter is operably linked to
said lacZ gene.
[0015] In another aspect, the present invention also provides a
herpes simplex virus hosting the reporter construct.
[0016] In another aspect, the present invention provides a method
of using a herpes simplex virus report construct, comprising the
steps of: (a) providing a tumor cell; (b) infecting said tumor cell
with said herpes simplex virus hosting the reporter construct; and
(c) determining lacZ gene activity of the reporter construct.
Preferably, the determining step is performed by
.beta.-galactosidase staining.
[0017] In another aspect, the present invention provides a kit,
comprising: (a) a herpes simplex virus lacking an immediate early
gene selected from the group consisting of ICP27, ICP4 and ICP22;
(b) a reagent used to determine apoptotic killing of a herpes
simplex virus infected tumor cell; and (c) an instruction, wherein
said instruction detailing the use of said herpes simplex virus
lacking said immediate early gene to infect a tumor cell and said
reagent in determining apoptotic killing of said herpes simplex
virus infected tumor cell.
[0018] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description to those skilled in the art. It should be understood,
however, that the detailed description and the specific examples,
while indicating preferred embodiments of the invention, are given
by way of illustration only. Various changes and modifications
within the spirit and scope of the invention are encompassed by the
present disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 depicts phase contrast (top) and Hoechst fluorescence
(bottom) images of Hs578T tumor cells. Hs578T tumor cells were
visualized at 24 hours after either no treatment (control) or
treatment with staurosporine (STS) (40.times. magnification).
Values in the lower right corner of Hoechst panels denote the mean
and standard deviation of the percentage (%) of nuclei containing
condensed chromatin from three (3) independent experiments.
[0020] FIG. 2 depicts immune-reactivities of apoptotic death
factors. Immunoblots for death factors (PARP, DFF-45, and
procaspase 3) were prepared from Hs578T tumor cells 24 hour post
infection with HSV-1(KOS), wtHSV, in the presence (+) or absence
(-) of cycloheximide (CHX). The 116,000 molecular weight uncleaved
and 85,000 molecular weight cleaved apoptotic PARP products are
observed. The anti-DFF-45 and -procaspase 3 recognize the full
length forms of these proteins so loss of reactivity signal
indicates apoptosis.
[0021] FIG. 3 depicts phase contrast (top) and Hoechst fluorescence
(bottom) images of DICP27 infected Hs578T tumor cells. Hs578T tumor
cells were visualized at 24 hours after either no treatment or
infection with DICP27 (40.times. magnification). The values in the
lower right corner of Hoechst panels denote the mean and standard
deviation of the percentage (%) of nuclei containing condensed
chromatin from three (3) independent experiments.
[0022] FIG. 4 depicts immune reactivities of apoptotic death
factors. Immunoblots for death factors (PARP, DFF-45, and
procaspase 3) were prepared from Hs578T tumor cells 24 hours post
Mock infection or infection with DICP27. The 116,000 molecular
weight uncleaved and 85,000 molecular weight cleaved apoptotic PARP
products are observed. The anti-DFF-45 and procaspase 3 recognize
the full-length forms of these proteins so loss of reactivity
signal indicates apoptosis.
DETAILED DESCRIPTION THE INVENTION
Definitions:
[0023] As used herein, the term "a" or "an" means one or more. As
used herein, the term "apoptosis" refers to a process of programmed
cell death that occurs in multi-cellular organisms. Apoptosis
involves a series of biochemical events such as appearance of cell
death factors (e.g., poly (ADP-ribose) polymerase, caspase 3, DNA
fragmentation factor-45 and the like). These biochemical events are
associated with characteristic cell morphology and cell death.
Specifically, morphological changes of apoptotic cells include cell
shrinkage, membrane blebbing, membrane asymmetry, nuclear
fragmentation, and chromatin condensation. Apoptosis is distinct
from necrosis. The term "apoptotic killing" refers to killing of a
target cell (e.g., tumor cell) by apoptosis. For purposes of this
invention, apoptotic killing is mediated by infecting a target cell
with a herpes simplex virus lacking an immediate early gene.
[0024] As used herein, the term "death factor" refers to one of the
several cellular factors, generally protein based, which facilitate
the apoptotic process, such as caspase 3, DFF, and PARP. In
addition, "death factor processing" refers to the detection of
proteolytic cleavage of the death factor protein as determined by
immunoblotting methods. As such, detection of death factor
processing represents validation that apoptotic cell death,
apoptosis, has occurred in the cells.
[0025] As used herein, the term "poly(ADP-ribose) polymerase"
(PARP) refers to a protein involved in a number of cellular
processes involving mainly DNA repair and apoptosis. PARP-1 is the
principal member of the PARP enzyme family and is an abundant
nuclear protein in mammalian cells. PARP-1 catalyses the formation
of poly (ADP-ribose) (PAR) polymers using NAD as substrate. Upon
DNA damage, PARP-1 binds rapidly to a DNA single-strand break and
catalyses the addition of negatively charged PAR chains to itself
(automodification) and other proteins. Thus, PARP is crucial in
repairing of single-strand DNA nicks. As used herein, the term
"caspase 3" is a caspase protein which interacts with caspase 8 and
caspase 9. Caspase 3 protein is a member of the cysteine-aspartic
acid protease (caspase) family.
[0026] As used herein, the term "chemotherapeutic agent" refers to
a chemical agent that is used in a chemotherapy treatment in a
patient that has a tumor cell. A chemotherapeutic agent generally
includes alkylators, anthracyclines, radionucleotides, enzyme
inhibitors, aromatase inhibitors, biphosphonates, cyclo-oxygenase
inhibitors, estrogen receptor modulators, folate antagonists,
inorganic arsenates, microtubule inhibitors, modifiers,
nitrosoureas, nucleoside analogs, orthoclase inhibitors,
platinum-containing compounds, retinoid, topoisomerase 1
inhibitors, topoisomerase 2 inhibitors, or tyrosine kinase
inhibitors.
[0027] As used herein, the term "tumor cell" includes, but is not
limited to, tumor cells derived from a source of breast, prostrate,
colon, pancreas, brain, liver, skin, stomach, cervix, uterus, or
the like.
[0028] As used herein, multiplicity of infection (MOI) is the ratio
of infectious agents (e.g. herpes simplex virus) to infection
targets (e.g., tumor cells). For example, when referring to a group
of cells inoculated with infectious virus particles (plaque-forming
units; pfu), the multiplicity of infection or MOI is the ratio
defined by the number of infectious virus particles divided by the
number of target cells present. MOI often ranges from 0.1-10.0
pfu/cell.
[0029] As used herein, the term "immediate early gene" is defined
as a virus gene immediately express upon virus infection of a
target. This includes ICP4, 22 and 27. HSV-1 genome has been
sequenced and publically available in Genbank (NC.sub.--001806
(Locus: HE1CG; Accession numbers: X141112, D00317, D00374, S40593).
ICP27 has a Gene name of UL54; Locus tag HHV1gp079,
protein_id="NP.sub.--044657.1." ICP4 has a Gene name of RS1; Locus
tag HHV1gp084, protein_id="NP.sub.--044662.1" ICP22 has a Gene name
of US1; Locus tag HHV1gp085, protein_id="NP.sub.--044663.1."
[0030] As used herein, the term "operable linked" refers that a
first gene element (such as a promoter) to be in operable linkage
with a second gene element (such as a lacZ gene) in a manner that
the first gene element modulates the expression of the second gene
element (increasing or decreasing expression, as appropriate). The
present invention therefore provides an immediate early promoter of
viral genes (e.g., ICP4, ICP22 or ICP27) "operably linked" to a
reporter gene element (e.g., lacZ gene). Specifically, lacZ gene
was genetically engineered into a recombinant plasmid DNA construct
in such a way that it is biologically expressed and functional when
introduced into a mammalian cell in culture. When lacZ is operably
linked to a gene expression promoter region, the DNA tract contains
the necessary sequence information required for its association
with relevant cellular transcription factors.
[0031] The present invention provides a method of determining tumor
cell susceptibility to chemotherapeutic agents with the use of
herpes simplex virus as well as herpes simplex virus lacking an
immediate early gene (e.g., ICP27, ICP4 or ICP22). The present
inventor discovered that tumor cells that are resistant to
HSV-induced apoptotic killing are also resistant to
chemotherapeutic agent-mediated apoptotic killing. In other words,
tumor cells that are susceptible to HSV-mediated apoptotic killing
could be killed by chemotherapeutic agents, such as doxorubicin,
etoposide, and the like.
[0032] Without being bound by any particular theory, we hypothesize
that the susceptibility of tumor cells to HSV-dependent apoptotic
killing requires that these cells may possess a functional ability
to undergo apoptosis. As such, tumor cells may require the
machinery of the mitochondrial-dependent apoptotic cascade. The
present invention provides that a HSV-dependent apoptosis
technology that can be used to monitor cellular signaling and
metabolic pathways and therefore, to determine tumor cell
susceptibility to anti-tumor drugs (i.e., chemotherapeutic agents,
such as doxorubicin, etoposide, paclitaxel, cisplastin, 5-FU and
the like).
[0033] The present invention provides a novel approach of using
herpes simplex virus lacking an immediate early gene to induce
tumor cell apoptotic killing and correlate it with chemotherapeutic
resistance. It is contemplated that viruses for the present
invention will be those that are sufficient to induce tumor cell
apoptotic killing. An exemplary virus includes the herpes simplex
virus, which in turn encompasses herpes simplex virus-1 and herpes
simplex virus-2. Protein synthesis inhibitor is required when
herpes simplex viruses (i.e., HSV-1 and HSV-2) are used. Exemplary
protein synthesis inhibitor includes cycloheximide, puromycin, and
the like. Optimal doses of cycloheximide or puromycin can
conveniently be determined to prevent protein synthesis in tumor
cells. Preferably, a dose of cycloheximide of 10 .mu.g/ml is
used.
[0034] It is also contemplated that modified herpes simplex viruses
are included. The modification, deletion of an immediate early
gene, results in a replication defective HSV (rdHSV). These
modified HSV cannot replicate in cells after infection, due to its
inability to complete its replication cycle while inducing
apoptotic killing of the infected tumor cell.
[0035] In one embodiment, the present invention encompasses HSV
deleted for ICP27; other preferred virus is one that has been
deleted in ICP4 or IC22. Viruses deleted in one or more of the
genes listed above will also be particularly useful for inducing
apoptotic killing in tumor cells in the present invention. When
rdHSV is used, protein synthesis inhibitor is not required in order
to induce apoptotic killing in tumor cells.
[0036] In one embodiment, the present invention provides a method
of using an amount of a herpes simplex virus to infect tumor cells
(i.e., effectively induce apoptotic killing in tumor cells).
Preferably, the MOI for infecting tumor cells ranges from 0.1
pfu/cell to 10.0 pfu/cell. Preferably, MOI ranges from 1 pfu/cell
to 5 pfu/cell. More preferably, MOI is 5 pfu/cell.
[0037] Preferably, a tumor cell is derived from a mammal.
Preferably, the mammal is a human. In one embodiment, tumor cells
may be surgically excised and put into culture media (e.g.,
DMEM+10% FBS) for a brief period of time (i.e., <24 hours). The
obtained tumor cells may then be infected by HSV-1 mutants (e.g.,
HSV-1 lacking an immediate early gene) and apoptotic killing may be
determined in accordance with the protocols described herein.
[0038] In specific embodiments, the mammal has a tumor of a tissue
or organ derived from brain, lung, liver, spleen, kidney, blood
cells, pancreas, colon, breast, cervix, prostate, skin, and the
like. Preferably, the tumor cell is derived from breast, colon and
cervix. More preferably, the tumor cell is derived from breast. In
addition, the tumor cells may have a defective p53 (e.g., colon
tumor cells).
[0039] Tumor cell susceptibility towards HSV-1 mutant (e.g.,
.DELTA.ICP4, .DELTA.ICP22, or .DELTA.ICP27) is evaluated. If there
is HSV-mediated apoptotic killing, it is concluded that the tumor
cell may also be susceptible towards chemotherapeutic agents.
Relying on the present inventive assay, a physician may
conveniently determine if a particular cancer patient would respond
to a chemotherapeutic agent. The present invention thus provide a
rapid, non-invasive and reliable assay in determining if a cancer
patient may respond to chemotherapy. Another advantage of the
present invention is that the assay assists a physician to tailor
particular needs for a cancer patient (i.e., personal
medicine).
[0040] The chemotherapeutic agent includes, but is not limited to
cisplatin, 5-fluororuacil, mitomycin, etoposide, camptothecin,
actinomycin-D, doxorubicin, verapamil, podophyllotoxin,
daunorubicin, vincristine, vinblastine, melphalan cyclophosphamide,
tumor necrosis factor, taxol and bleomycin. Preferably, the
chemotherapeutic agent is doxorubicin, etoposide, paclitaxel,
cisplatin, or 5-fluororuacil.
[0041] One clear advantage of this aspect of the invention involves
treatment of tumors in which some cells are p53-positive while
others are p53-negative. Apoptotic killing is hypothesized to act
through two major pathways. One is mediated via a death receptor,
such as Fas or tumor necrosis factor receptor. Once the receptor
binds ligand, it then recruits an adaptor molecule that allows the
binding and autocleavage/activation of procaspase-8. Activated
caspase-8 induces a cascade, which includes processing of effector
caspases (executioners) caspase-3 and caspase-7.
[0042] The other is mediated by mitochondria release of cytochrome
C into the cytoplasm, where it associates with Apaf-1 and permits
the recruitment and activation of caspase-9. This, in turn, also
leads to the cascade of events culminating in the activation of the
executioners of apoptotic killing. In both pathways, caspase
cleavage ultimately leads to the morphological and biochemical
features characteristic of apoptotic killing, including apoptotic
body formation, cell shrinkage, membrane blebbing, chromatin
condensation, and DNA fragmentation. Among the cleavage targets are
the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) and the
DNA fragmentation factor-45 (DFF-45). Thus, the process of
apoptotic killing generally involves the processing of caspase-3,
DFF-45, and PARP.
[0043] Apoptotic killing features include chromatin condensation,
fragmentation of nuclei, membrane blebbing, and the formation of
apoptotic bodies. Assays for monitoring apoptotic killing are well
known to those of skill in the art and include for example,
monitoring cell shrinkage, nuclear condensation, monitoring
appearance of genomic DNA fragmentation ladders; monitoring the
processing of PARP, a 116 kDa protein, which generates an 85 kDa
product which may be detected by the anti-PARP antibody (Aubert et
al., J. Virol. 1999, 73:10359-70); and monitoring apoptosis-induced
processing of DFF (45 kDa) and caspase-3 (32 kDa) as determined by
the loss of reactivity with the anti-DFF-45 and anti-caspase-3
antibodies.
[0044] The method of the present invention is useful for monitoring
the effects of therapeutic agents in treating cancer. In one
embodiment of the invention, there is provided a method for
assessing the efficacy of a therapeutic compound (e.g., 5-FU,
cisplatin and the like) for the treatment of a tumor disease.
Protocols for obtaining tumor cells from a cancer patient (from a
source such as pancreas, colon, cervix, liver, breast and the like)
are established, and a physician can conveniently apply the present
assay using the tumor cells derived from the various tumor
cells.
[0045] The present invention comprises the steps of (i) obtaining a
tumor cell from a cancer patient; (ii) infecting the tumor cell
with HSV-1 or HSV-2 lacking an immediate early gene (i.e., ICP4,
ICP22, or ICP27); and (iii) determining the presence of apoptotic
killing of the tumor cell mediated by the HSV mutants. If there is
presence of apoptotic killing of the tumor cells, it is indicative
of efficacy of a chemotherapeutic compound in treating the patient
of the cancer disease.
[0046] The present invention also provides a herpes simplex virus
report construct, comprising a herpes simplex virus containing (i)
a herpes simplex virus immediate early promoter; and (ii) a lacZ
gene, wherein said herpes simplex virus lacks gene lacking an
immediate early gene selected from the group consisting of ICP27,
ICP4, and ICP22, and said promoter is operably linked to said lacZ
gene.
[0047] The present invention provides a method of using a herpes
simplex virus report construct, comprising the steps of: (i)
providing a tumor cell; (ii) infecting said tumor cell with a
herpes simplex virus report construct having an immediate early
promoter operably linked to a lacZ gene; (iii) determining the
activity of said lacZ gene; wherein the positive activity of said
lacZ gene is indicative of tumor cell resistance to
chemotherapeutic agents, and the negative activity of said lacZ
gene is indicative of tumor cell sensitivity to chemotherapeutic
agents.
[0048] Chemotherapy Resistance Measurement Kit
[0049] In one embodiment, the present invention provides a kit for
screening patient tumors to determine susceptibility to a
particular chemotherapeutic agent.
[0050] Kits of the invention include reagents for assessing
apoptotic killing, herpes simplex virus (including HSV-1 and HSV-2)
and herpes simplex virus that is deficient in immediate early gene
replication (i.e., lacking ICP27, ICP4, and ICP22), instructions
that details the use of the herpes simplex virus-1 in inducing
apoptosis of tumor cells and conditions whereby extent of apoptotic
killing can be measured. In one embodiment, the kit contains
fluorescence probe specific for measuring apoptotic killing of
tumor cells. The kit of the invention may optionally comprise
additional components useful for performing the methods of the
invention, such as devices for use in isolating tumor cells from
blood source of a patient. In addition, the kits may contain
calibration curves, a reference sample (a reference tumor cell and
herpes simplex virus-1) for comparison to a reference value as
described herein. Kits can conveniently be provided in an array
format, for example, in multi-well plates.
Experiments
EXAMPLE 1
Herpes Simplex Virus Induces Apoptotic Killing in Tumor Cells
[0051] a) Tumor Cell Apoptosis
[0052] We have established an in vitro cell model and examined
herpes simplex virus-induced apoptotic killing of tumor cells. In
the first series of studies, we used a standard inducer of
apoptotic killing. A protein kinase inhibitor (i.e., staurosporine)
was used. Mammary tumor cells (Hs578T) were cultured and treated
with staurosporine (0.1-2 .mu.M) for about 12-18 hours.
Staurosporine caused Hs578T cells to undergo morphological
alternations consistent with apoptotic killing (i.e., cell
shrinkage, chromatin condensation, and membrane blebbing) as
compared to untreated cells. (FIG. 1). When this phenotype was
quantified for three independent experiments, the
staurosporine-treated Hs578T cells exhibited 86.+-.14% chromatin
condensation.
[0053] In addition, staurosporine caused drastic death factor
processing [poly(ADP-ribose) polymerase, procaspase 3, and DNA
fragmentation factor-45] in Hs578T cells (FIG. 2). Thus, we
established an in vitro system showing that Hs578T tumor cells are
capable of undergoing apoptotic killing.
[0054] b) HSV-1 Induces Apoptotic Killing in Tumor Cells
[0055] Using Hs578T cells, we next examined whether herpes simplex
virus (HSV) can induce apoptotic killing in these tumor cells.
Standard methods were used to prepare wild-type HSV-1 (i.e.,
wtHSV-1). (Blaho et al.: Herpes Simplex Virus: Propagation,
Quantification, and Storage. Current Protocols in Microbiology.
Wiley & Sons. 14:1-23, 2005). Hs578T cells were infected with
wtHSV-1 and examined for tumor cell apoptotic killing. A range of
wtHSV-1 concentration was used and we observed that one particle of
wtHSV-1 was sufficient to induce apoptotic killing of a tumor cell.
Apoptotic killing was evaluated at 24-hour post infection by
monitoring morphological alternations and presence of death factor
processing as detailed above.
[0056] wtHSV-1 infected Hs578T cells (in the presence of 10
.mu.g/ml cycloheximide) caused drastic death factor processing
(poly(ADP-ribose) polymerase, procaspase 3, and DNA fragmentation
factor-45) (FIG. 2). Not wishing to be bound by any theory, we
observed that the inhibition of protein synthesis (e.g.,
cycloheximide) during wtHSV-1 infection would permit apoptotic
killing to occur. In addition, wtHSV-1 infection caused
morphological alternations consistent with apoptotic killing (data
not shown). Mock-infected Hs578T cells exhibited flat cell shapes
and were well spread-out. The nuclei in the mock-infected cells
exhibited homogenous Hoechst staining (data not shown).
Cycloheximide alone had no apparent apoptotic effects in Hs578T
cells (data not shown).
[0057] These results demonstrate that wtHSV-1 induces apoptotic
killing in Hs578T cells.
[0058] c) HSV-2 Induces Apoptotic Killing in Tumor Cells
[0059] We also examined if a different type of herpes simplex virus
(i.e., HSV-2) may induce apoptotic killing of tumor cells. Standard
methods were used to prepare wild-type HSV-2 (i.e., wtHSV-2).
(Blaho et al.: Herpes Simplex Virus: Propagation, Quantification,
and Storage. Current Protocols in Microbiology. Wiley & Sons.
14:1-23, 2005). Tumor cells (human carcinoma HEp-2) were used and
infected with wtHSV-2. The results indicate that, like wtHSV-1,
wtHSV-2 (in the presence of 10 .mu.g/ml cycloheximide) induces
apoptotic killing of HEp-2 cells. In particular, wtHSV-2 caused
both the morphological alternations and death factor processing
(data not shown). These results with wtHSV-1 and wtHSV-2 are
summarized in Table 1. Thus, infection of Hs578T and HEp-2 cells
with wtHSV-1 and wtHSV-2, respectively, leads to substantial
apoptotic killing of tumor cells.
TABLE-US-00001 TABLE 1 Herpes Simplex Virus Induces Apoptosis in
Tumor Cells Death Factor Processing Morphological Alternations
Poly(ADP-ribose) Cell, shrinkage, Polymerase, Membrane blebbing,
Procaspase 3, and DNA Treatments Condensed Chromatin Fragmentation
Factor-45 Staurosporine + + wtHSV-1 + + + cycloheximide wtHSV-2 + +
+ cycloheximide
EXAMPLE 2
HSVs Lacking Immediate Early Genes Induce Tumor Apoptotic
Killing
[0060] Herpes simplex virus contains a total of five (5) immediate
early genes, of which three (3) are essential for viral replication
(i.e., ICP27, ICP4 and ICP22). We prepared ICP27-, ICP4-, and
ICP22-null recombinant viruses (i.e., .DELTA.ICP27, .DELTA.ICP4,
.DELTA.ICP22) using standard methods. (Sanfilippo et al. ICP0 gene
expression is a HSV-1 apoptotic trigger. J. Virol. 78: 6810-6821,
2006). The characteristics of these deletion mutants are summarized
in Table 2.
TABLE-US-00002 TABLE 2 Herpes Simplex Virus Deletion Mutants HSV
Deletion Mutants Genes Deleted Characteristics .DELTA.ICP27 UL54
Defective in viral DNA synthesis and late gene expression
.DELTA.ICP4 Alpha4 Defective in viral early and late gene
expression .DELTA.ICP22 US1, alpha22 Defective in viral late gene
expression
[0061] a) .DELTA.ICP27 Induces Apoptotic Killing in Tumor Cells
[0062] In this study, we examined whether herpes simplex virus
lacking an immediate early gene (e.g., .DELTA.ICP27) would induce
apoptotic killing of Hs578T tumor cells. Fifty-three percent (53%)
of the Hs578T cells, when infected with AICP27, exhibited membrane
blebbing and chromatin condensation (FIG. 3). Additionally, the
AICP27-infected Hs578T cells displayed PARP cleavage and had lower
levels of DFF-45 and procaspase 3 as compared to mock-infected
cells (FIG. 4). These results demonstrate that infection of Hs578T
cells with HSV lacking an immediate early gene (e.g., .DELTA.ICP27)
sufficiently leads to tumor apoptotic killing. Thus, viruses
deleted for the major viral regulatory protein ICP27 resulted in
apoptotic cell death of the tumor cell.
[0063] b) .DELTA.ICP4 Induces Apoptotic Killing in Tumor Cells
[0064] Infection of HEp-2 cells with .DELTA.ICP4 caused the tumor
cells to undergo apoptotic killing as evidenced by morphological
and biological changes and death factor processing (data not
shown).
[0065] c) .DELTA.ICP22 Induces Apoptotic Killing in Tumor Cells
[0066] Infection of HEp-2 cells with .DELTA.ICP22 similarly caused
the tumor cells to undergo apoptotic killing as evidenced by
morphological and biological changes and death factor processing
(data not shown). In conclusion, these data indicate that HSV
lacking immediate early genes can induce apoptotic killing in tumor
cells. (See, Table 3)
TABLE-US-00003 TABLE 3 Herpes Simplex Virus Deletion Mutants Induce
Apoptotic Killing in Tumor Cells Morphological Alternations Death
Factor Processing HSV Cell, shrinkage, Membrane Poly(ADP-ribose)
Deletion blebbing, Condensed Polymerase, Procaspase 3, and Mutants
Chromatin DNA Fragmentation Factor-45 .DELTA.ICP27 + + .DELTA.ICP4
+ + .DELTA.ICP22 + +
EXAMPLE 3
HSV Induces Apoptotic Killing in Additional Tumor Cells
[0067] a) HSV Induces Apoptosis in Colon, Brain and Breast Tumor
Cells
[0068] So far, we have demonstrated HSV induces apoptotic killing
in Hs578T (mammary cancer cells) and HEp-2 cells (epithelial
carcinoma). To gain further insight into the tumor cell
determinants for susceptibility to HSV-induced apoptotic killing,
we analyzed a selected group of tumor cells. In this study, we used
both wtHSV-1 and AICP27 and representative tumor cells. Experiments
were conducted using conditions described above. Table 4 summarizes
the results using ..DELTA.ICP27:
TABLE-US-00004 TABLE 4 Herpes Simplex Virus Induce Apoptotic
Killing in Several Tumor Cells Apoptotic Killing (measured by
morphological alternations and death HSV Types of Tumor Cells
factor processing .DELTA.ICP27 Colon Tumor HT-29 + RKO + RKO-E6 +
Brain Tumor SK-N-SH + Breast Tumor MCF-7/C3 +
[0069] Similar results were obtained when wtHSV (in the presence of
cycloheximide) was used (data not shown). From these results, we
conclude that certain colon and brain tumor derived cells can
respond to HSV induced apoptotic killing.
[0070] b) HSV Fails to Induce Apoptotic Killing in Some Tumor
Cells
[0071] We discovered that not all tumor cells tested were
susceptibility to killing by HSV. The inability to induce apoptotic
killing does not relate to the absence of immediate early genes in
HSV (e.g., .DELTA.ICP27) because wtHSV-1 also fails to induce
apoptotic killing in these cells (data not shown). These results
suggest that the susceptibility of HSV-induced apoptotic killing
may relate to intrinsic properties of the tumor cells and is not
dependent on the virus. The underlying mechanism is presently not
known and one of ordinary skill in the art would not be able to
predict if a particular tumor cell may be sensitive or resistant to
HSV-induced apoptotic killing.
TABLE-US-00005 TABLE 5 Herpes Simplex Virus Did Not Induce
Apoptosis in Certain Tumor Cells Apoptotic Killing (measured by
morphological alternations and death HSV Types of Tumor Cells
factor processing .DELTA.ICP27 Prostate Tumor PC3 -- Brain Tumor
U373 -- Breast Tumor MCF-7 --
[0072] These results indicate that certain tumor cells such as
PC-3, MCF-7, and U373 are resistant to HSV induced apoptotic
killing.
EXAMPLE 4
HSV-Dependent Apoptotic Killing in Tumor Cells Requires
Mobilization of Mitochondrial Cytochrome C
[0073] There are at least two known apoptotic pathways. The first
pathway is initiated by the extracellular (extrinsic) binding of a
death ligand to its receptor on the cell surface; while the second
pathway integrates intracellular apoptotic signals through a
mitochondrial (intrinsic) route. We showed that HSV-dependent
apoptotic killing may involve the second pathway (i.e.,
mitochondrial apoptotic pathway) in tumor cells. To this end, we
showed that: (i) .DELTA.ICP27 infected HEp-2 tumor cells had
release cytochrome c from mitochondria; and (ii) addition of
caspase-9-specific inhibitor prevented HSV-induced apoptotic
killing in HEp-2 cells (data not shown). Because caspase-9 is
activated by released cytochrome c, these findings suggest that HSV
induces apoptotic killing by stimulating mitochondrial cytochrome c
release.
EXAMPLE 5
Chemotherapeutic Susceptibility in Tumor Cells
[0074] We examined tumor cell killing by chemotherapeutic agents.
In this study, HEp-2 cells were treated with varying amounts of
doxorubicin and etoposide. Apoptotic killing was measured using the
method detailed above. Both doxorubicin (10 .mu.M) and etoposide
(125 .mu.M) induce morphological alternations and death factor
processing (data not shown). Thus, these tumor cells were sensitive
to chemotherapeutic agents.
[0075] During our survey of the sensitivities of patient derived
tumor cells to chemotherapeutic agents, we discovered that certain
tumor cells (e.g., MCF-7) were resistant to drug-induced apoptosis.
Altogether, we have identified chemo-resistant and -sensitive tumor
cells.
[0076] Furthermore, we conducted a survey of the literature and
found a group of tumor cells that are chemo-resistant or
-sensitive. We examined 5-fluorouracil, cisplatin, doxorubicin,
etoposide and paclitaxel because they are classical
chemotherapeutic agents.
[0077] Table 6 summarizes the panel of chemo-resistant and
-sensitive tumor cells from different sources.
TABLE-US-00006 TABLE 6 Tumor Cells That Are Known to Be Resistant
or Sensitive to Chemotherapeutic Agents 5-Fluorouracil Cisplatin
Doxorubicin Etoposide Paclitaxel Melanoma Resistant Resistant
Resistant Resistant Sensitive (A375) Ovarian tumor Sensitive
Resistant Resistant Resistant Resistant (ES-2) Hepatic tumor
Resistant Resistant Resistant Resistant Resistant (Hep3B) Hepatic
tumor Resistant Resistant Resistant Resistant Resistant (SNU182)
Hepatic tumor Resistant Resistant Resistant Resistant Sensitive
(SNU423) Breast tumor Sensitive Resistant Resistant Resistant
Sensitive (R193) Breast tumor Sensitive Resistant Resistant
Resistant Sensitive (SKBR3) Pancreatic tumor Sensitive Resistant
Resistant Resistant Sensitive (181/85P Gastric tumor Resistant
Resistant Resistant Resistant Sensitive (257P)
EXAMPLE 6
HSV Induced Apoptotic killing Correlates with Chemotherapeutic
Sensitivity
[0078] So far, we have established a correlation in certain tumor
cells between HSV-induced apoptotic killing and chemotherapeutic
susceptibility. For example, MCF-7 tumor cells were found to be
resistant to doxorubicin and etoposide and also resistant to
HSV-induced apoptotic killing. On the other hand, HEp-2 cells were
found to be sensitive to doxorubicin and etoposide as well as
HSV-induced apoptotic killing.
[0079] To further establish the correlation, we chose a large panel
of patient derived breast tumor cells. More than twenty (20) tumor
cell lines were obtained from patients or from a commercial source.
We chose the following tumor cell mutants because they possess
defined p53 mutations. We showed that Hs578Ts are sensitive to
HSV-induced apoptotic killing (See, Example 2). These tumor cells
are evaluated for their sensitivity to HSV-induced apoptotic
killing.
TABLE-US-00007 TABLE 7 p53 Mutant Breast Tumor Cells Cell Lines
Types Cell Lines Types BT20 Missense BT-474 Missense Hs578T
Missense MDA-MB-231 Missense MDA-MB-435 Missense MDA-MB-436
Insertion MDA-MB-453 Deletion MDA-MB-468 Missense SK-BR-3 Missense
T-47D Missense
[0080] The following breast tumor cells were specifically chosen
because they possess wild type p53. (See, Table 8). These tumor
cells are evaluated for their sensitivity to HSV-induced apoptotic
killing.
TABLE-US-00008 TABLE 8 Wild type p53 Breast Tumor cells Cell Lines
Types Cell Lines Types MCF-7 ATCC wt MCF-7 PV wt MCF-7 N wt MCF-7 P
wt MCF-7 C3 wt/caspase 3+ MDA-MB-175-VII wt ZR-75-1 wt
[0081] Furthermore, we chose additional breast tumor cell lines,
including HCC1419, HCC1954, MDA-MB-330, and UACC812. Together,
these breast tumor cells establish the tumor cell susceptibility to
HSV-induced apoptotic killing.
EXAMPLE 7
Use of HSV Reporter Constructs to Assess Chemotherapy Resistance in
Tumor Cells
[0082] We observed that tumor cells which are sensitive to
HSV-dependent apoptotic killing undergo cell lysis and therefore do
not allow any viral gene expression from the HSV genome. However,
tumor cells which are resistant to HSV-dependent apoptotic killing,
by virtue of its cell survival, allow gene expression from the HSV
genome. We took advantage of this observation and used a HSV report
construct to assess chemotherapy resistance in tumor cells.
[0083] We prepared a novel HSV report construct. We used an
established protocol (Aubert and Blaho, J. Virol 73:2803-2813,
1999) and prepared .DELTA.ICP27. .DELTA.ICP27 is an ICP27-null
virus that contains a replacement of the ICP27 gene with the
Escherichia coli lacZ gene. .DELTA.ICP27 is then added to tumor
cells that are either sensitive or resistant to chemotherapeutic
agents. After optimal culturing, we stain for .beta.-galactosidase
to assess gene expression from the HSV genome. A positive staining
would reveal the expression of the lacZ gene, which is indicative
of viral gene expression and correlate with its resistance to (i)
HSV-dependent apoptotic killing, and (ii) chemotherapeutic killing.
On the other hand, a negative staining would reveal the lack of
lacZ expression, and indicate susceptibility to HSV-dependent
apoptotic killing and chemotherapeutic agents. Thus, infection of
tumor cells with AICP27 combined with .beta.-galactosidase staining
is a useful tool to identify tumor cells that are resistant to
chemotherapeutic agents.
Protocols and Reagents
[0084] CELLS FOR VIRUS PROPAGATION: African green monkey kidney
(Vero) cells were obtained from the American Type Culture
Collection (Rockville, Md., USA). Vero 2-2 cells represent Vero
cells expressing the ICP27 protein. Vero and Vero 2-2 cells were
cultured in Dulbecco's modified Eagle's medium (DMEM) and
supplemented with 5% fetal bovine serum (FBS). These Vero cells
were used to propagate Herpes Simplex Viruses (see below).
[0085] TUMOR CELLS: All tumor cells used in our studies were
obtained from the American Type Culture Collection (Rockville,
Md.). U373, SK-N-SH, MCF-7, RKO and RKO-E6 cells were maintained in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal bovine serum (FBS). Hs578T cells were grown in DMEM
containing 10% FBS and 0.01 mg/ml bovine insulin. HT-29 and PC-3
cells were grown in 10% FBS-containing McCoys 5a or F12K medium,
respectively. HEp-2 cells were maintained in DMEM with 5% FBS.
[0086] HERPES SIMPLEX VIRUSES: Wild type HSV-1 and HSV-2 were
obtained and prepared as described (Blaho et al.: Herpes Simplex
Virus: Propagation, Quantification, and Storage. Current Protocols
in Microbiology. Wiley & Sons., 14:1-23, 2005). In essence, we
obtained these herpes simplex viruses from patients, and were
propagated and tittered on Vero cells and used subsequently to
infect tumor cells at varying MOI of 1-10. HSV-1(.DELTA.ICP27) is
an ICP27-null virus derived from HSV-1 containing a replacement of
the .alpha.27 gene with the Escherichia coli lacZ gene. .DELTA.ICP4
was derived from the HSV-1 and is an ICP4-null virus which is
deleted for 3.6 kb of the coding region of ICP4 due to having the
Escherichia coli lacZ gene inserted in place of ICP4. .DELTA.ICP22
is derived from HSV-1 and contains a complete deletion of the ICP22
gene.
[0087] PREPARATION OF HSV MUTANT STRAINS: .DELTA.ICP27 is an HSV-1
deletion mutant virus in which the ICP27 coding sequence was
completely removed from the viral genome. Because ICP27 is
essential for virus growth, the .DELTA.ICP27 virus was generated
and propagated with Vero 2-2 cells which express ICP27 and thus
complement the ICP27 deficiency. Preparation of HSV mutant strains
was made by a protocol similar to those described in O'Toole et al.
(Virology 305, pp.153-167, 2003) and Pomeranz et al. (Journal of
Virology 74 (21), pp. 10041-10054, 2000). AICP27 was generated by
homologous recombination between the wild type KOS genome and a
linearized plasmid containing a deletion of ICP27 but maintaining
flanking sequences homologous to the KOS genome. The integrity of
the deletion mutation in .DELTA.ICP27 was validated by Southern
hydridization, PCR, and immunoblotting methods. Similar approaches
were used for the generation of .DELTA.ICP4 and .DELTA.ICP22 HSV
mutant strains.
[0088] INFECTION OF TUMOR CELLS WITH HSV MUTANTS: Approximately
1.times.10.sup.6 cells (i.e., tumor cells) were exposed to 10
plaque forming units (PFU) per ml of .DELTA.ICP27 virus in 1 ml of
5% NBCS for 1 hour at 37.degree. C. After this adsorption step, the
medium was removed and discarded. 3 ml of fresh 5 NBCS was then
added. Cells were maintained at 37.degree. C. for at least 18
hours.
[0089] MICROSCOPIC ANALYSIS OF HSV INFECTED TUMOR CELLS: The
morphology of HSV infected cells was analyzed by phase contrast and
fluorescence microscopy using an Olympus IX70/IX-FLA inverted
fluorescence microscope. Images were acquired using a Sony DKC-5000
digital photo camera linked to a PowerMac workstation and processed
through Adobe Photoshop.
Analysis of Chromatin Condensation of HSV Infected Tumor Cells:
[0090] For visualization of chromatin condensation in live cells, 5
mg Hoechst 33258 (Sigma) per ml was added to the medium and allowed
to incubate at 37.degree. C. for 30 min. The percentage (%) of
nuclei containing condensed chromatin was determined by dividing
the number of brightly stained, small (condensed) nuclei by the
total number of nuclei (uncondensed plus condensed) in a particular
(640) microscopic field. At least 100 nuclei were counted for each
data point. The percentage (%) of chromatin condensation is
represented as the mean.+-.SD of three independent experiments.
[0091] IMMUNOBLOT ANALYSIS OF HSV INFECTED TUMOR CELLS: Whole-cell
protein extract was prepared using lysis buffer (50 mM Tris/HCl, pH
7.5, 150 mM NaCl, 1% Triton X-100,1% deoxycholate, 0.1% SDS)
supplemented with 2 mM PMSF (freshly prepared stock), 1%
Translysol, 0.1 mM L-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone,
0.01 mM
L-1-chlor-3-(4-tosylamido)-7-amino-2-heptanon-hydrochloride.
Protein concentrations were determined using a modified Bradford
protein assay (Bio-Rad Laboratories). Total protein (20 or 50 mg)
was separated on 15% N,N9-diallyltartardiamide-acrylamide gels and
electrically transferred to nitrocellulose. Pre-stained molecular
mass markers were loaded and immunostaining of the actin loading
control was carried out.
[0092] ANALYSIS OF APOPTOTIC DEATH FACTOR PROCESSING OF HSV
INFECTED TUMOR CELLS: Cell membranes were incubated for 1 hour at
room temperature in blocking buffer (PBS containing 5% non-fat,
dried milk) and incubated overnight at 4.degree. C. in primary
antibody. Monoclonal antibodies specific for poly(ADP-ribose)
polymerase (PARP) (PharMingen), procaspase 3 (BD Transduction) and
the control actin (Sigma) and DFF-45 (Santa Cruz) were diluted at a
concentration of 1:1000 in Tris buffered saline containing 0.1%
Tween 20 (TBST) and 0.1% BSA. After washing in TBST, membranes were
incubated with anti-mouse antibodies conjugated to alkaline
phosphatase (Southern Biotech) diluted in blocking buffer (1:1000)
for 1 hour at room temperature. Following washing in TBST,
immunoblots were developed in buffer containing
5-bromo-4-chloro-3-indolyl phosphate and 4-nitro blue tetrazolium
chloride. To quantitate the percentage (%) of total infected cell
PARP that was cleaved, densitometry of immune-reactive PARP was
performed. NIH IMAGE version 1.63 was used to measure the
integrated density (ID) of the 116 kDa uncleaved and 85 kDa cleaved
PARP bands. These values were used to calculate the percentage (%)
of PARP cleavage for each lane using the following formula:
Percentage (%) cleavage equals [(cleaved PARP ID) divided by
(cleaved PARP ID plus uncleaved PARP ID)] times 100.
[0093] PREPARATION OF HSV CONTAINING LAC Z REPORTER CONSTRUCT: The
lacZ coding sequence was isolated from plasmid pCH110 (Pharmacia,
Piscataway, N.J.). A DNA restriction fragment containing the lacZ
coding sequences was inserted into a plasmid such that lacZ was
under the control of an HSV immediate early (IE) promoter. This
reporter construct was introduced into the genome of the AICP27
virus by homologous recombination (as described above).
Analysis of LacZ Rreporter of HSV Infected Tumor Cells:
[0094] Approximately 1.times.10.sup.6 cells infected with AICP27
virus containing the lacZ reporter construct are washed three times
with phospahte buffered saline (PBS). The cells are fixed for 5 min
at 25.degree. C. by the addition of 2% formaldehyde and 0.2%
glutaraldehyde in PBS. The cells are washed again three times with
PBS. LacZ activity is measured by adding 0.1 mg/ml of of
5-bromo-4-chloro-3-indolyl-b-D-galactoside (X-gal) in PBS
containing 5 nM potassium ferrocyanide and 5 mM ferricyanide
following incubation at 37.degree. C. for 30 min. Number of blue
cells (indicative of lacZ activity) is counted using a phase
contrast microscope.
[0095] All patents, publications, gene accession numbers, and
patent application described supra in the present application are
hereby incorporated by reference in their entirety.
[0096] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
REFERENCES
[0097] 1. Aubert, M., and J. A. Blaho. 2001. Modulation of
apoptosis during herpes simplex virus infection in human cells.
Microbes Infect 3:859-66. [0098] 2. Aubert, M., and J. A. Blaho.
1999. The herpes simplex virus type 1 regulatory protein ICP27 is
required for the prevention of apoptosis in infected human cells. J
Virol 73:2803-13. [0099] 3. Aubert, M., and J. A. Blaho. 2003.
Viral oncoapoptosis of human tumor cells. Gene Ther 10:1437-45.
[0100] 4. Aubert, M., J. O'Toole, and J. A. Blaho. 1999. Induction
and prevention of apoptosis in human HEp-2 cells by herpes simplex
virus type 1. J Virol 73:10359-70. [0101] 5. Aubert, M., L. E.
Pomeranz, and J. A. Blaho. 2007. Herpes simplex virus blocks
apoptosis by precluding mitochondrial cytochrome c release
independent of caspase activation in infected human epithelial
cells. Apoptosis 12:19-35. [0102] 6. Goodkin, M. L., E. R. Morton,
and J. A. Blaho. 2004. Herpes simplex virus infection and
apoptosis. Intl Rev Immunol 23:141-72. [0103] 7. Jerome, K. R., Z.
Chen, R. Lang, M. R. Torres, J. Hofmeister, S. Smith, R. Fox, C. J.
Froelich, and L. Corey. 2001. HSV and glycoprotein J inhibit
caspase activation and apoptosis induced by granzyme B or Fas. J
Immunol 167:3928-35. [0104] 8. Jerome, K. R., R. Fox, Z. Chen, A.
E. Sears, H. Lee, and L. Corey. 1999. Herpes simplex virus inhibits
apoptosis through the action of two genes, Us5 and Us3. J Virol
73:8950-7. [0105] 9. Koyama, A. H., and A. Adachi. 1997. Induction
of apoptosis by herpes simplex virus type 1. J Gen Virol
78:2909-12. [0106] 10. Koyama, A. H., and Y. Miwa. 1997.
Suppression of apoptotic DNA fragmentation in herpes simplex virus
type 1-infected cells. J Virol 71:2567-71. [0107] 11. Kraft, R. M.,
M. L. Nguyen, X. H. Yang, A. D. Thor, and J. A. Blaho. 2006.
Caspase 3 activation during herpes simplex virus 1 infection. Virus
Res 120:163-75. [0108] 12. Leopardi, R., and B. Roizman. 1996. The
herpes simplex virus major regulatory protein ICP4 blocks apoptosis
induced by the virus or by hyperthermia. Proc Natl Acad Sci USA
93:9583-7. [0109] 13. Leopardi, R., C. Van Sant, and B. Roizman.
1997. The herpes simplex virus 1 protein kinase US3 is required for
protection from apoptosis induced by the virus. Proc Natl Acad Sci
USA 94:7891-6. [0110] 14. Nguyen, M. L., and J. A. Blaho. 2007.
Apoptosis during herpes simplex virus infection. Adv Virus Res
69:67-97. [0111] 15. Nguyen, M. L., R. M. Kraft, M. Aubert, E.
Goodwin, D. DiMaio, and J. A. Blaho. 2007. p53 and hTERT determine
sensitivity to viral apoptosis. J Virol 81:12985-95. [0112] 16.
Nguyen, M. L., R. M. Kraft, and J. A. Blaho. 2005. African green
monkey kidney Vero cells require de novo protein synthesis form
efficient herpes simplex virus 1 dependent apoptosis. Virology
336:274-290. [0113] 17. Nguyen, M. L., R. M. Kraft, and J. A.
Blaho. 2007. Susceptibility of cancer cells to herpes simplex virus
dependent apoptosis. J. Gen. Virol. 88:1866-1875. [0114] 18.
Sanfilippo, C. M., F. N. Chirimuuta, and J. A. Blaho. 2004. Herpes
simplex virus type 1 immediate-early gene expression is required
for the induction of apoptosis in human epithelial HEp-2 cells. J
Virol 78:224-39. [0115] 19. Soliman, T. M., R. M. Sandri-Goldin,
and S. J. Silverstein. 1997. Shuttling of the herpes simplex virus
type 1 regulatory protein ICP27 between the nucleus and cytoplasm
mediates the expression of late proteins. J Virol 71:9188-97.
[0116] 20. Yedowitz, J. C., and J. A. Blaho. 2005. Herpes simplex
virus 2 modulates apoptosis and stimulates NF-kappaB nuclear
translocation during infection in human epithelial HEp-2 cells.
Virology 342:297-310.
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