Download Differential chemosensitivity of breast cancer cells to

Differential chemosensitivity of breast cancer cells to
ganciclovir treatment following adenovirus-mediated herpes
simplex virus thymidine kinase gene transfer
Pei-Xiang Li, Duc Ngo, Anthony M. Brade, and Henry J. Klamut
Division of Experimental Therapeutics, Ontario Cancer Institute, Princess Margaret Hospital, and
Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
The development of resistance to radiation and chemotherapeutic agents that cause DNA damage is a major problem for the treatment
of breast and other cancers. The p53 tumor suppressor gene plays a direct role in the signaling of cell cycle arrest and apoptosis in
response to DNA damage, and p53 gene mutations have been correlated with increased resistance to DNA-damaging agents. Herpes
simplex virus thymidine kinase (HSV-tk) gene transfer followed by ganciclovir (GCV) treatment is a novel tumor ablation strategy that has
shown good success in a variety of experimental tumor models. However, GCV cytotoxicity is believed to be mediated by DNA
damage-induced apoptosis, and the relationship between p53 gene status, p53-mediated apoptosis, and the sensitivity of human tumors
to HSV-tk/GCV treatment has not been firmly established. To address this issue, we compared the therapeutic efficacy of adenovirusmediated HSV-tk gene transfer and GCV treatment in two human breast cancer cell lines: MCF-7 cells, which express wild-type p53, and
MDA-MB-468 cells, which express high levels of a mutant p53 (273 Arg-His). Treating MCF-7 cells with AdHSV-tk/GCV led to the
predicted increase in endogenous p53 and p21WAF1/CIP1 protein levels, and apoptosis was observed in a significant proportion of the
target cell population. However, treating MDA-MB-468 cells under the same conditions resulted in a much stronger apoptotic response
in the absence of induction in p21WAF1/CIP1 protein levels. This latter result suggested that HSV-tk/GCV treatment can activate a strong
p53-independent apoptotic response in tumor cells that lack functional p53. To confirm this observation, four additional human breast
cancer cell lines expressing mutant p53 were examined. Although a significant degree of variability in GCV chemosensitivity was
observed in these cell lines, all displayed a greater reduction in cell viability than MCF-7 or normal mammary cells treated under the same
conditions. These results suggest that endogenous p53 status does not correlate with chemosensitivity to HSV-tk/GCV treatment.
Furthermore, evidence for a p53-independent apoptotic response serves to extend the potential of this therapeutic strategy to tumors that
express mutant p53 and that may have developed resistance to conventional genotoxic agents.
Key words: Herpes simplex virus thymidine kinase; ganciclovir treatment; breast cancer; apoptosis; p53 tumor suppressor gene;
bystander effect; gene therapy.
H
erpes simplex virus thymidine kinase (HSV-tk) gene
transfer followed by ganciclovir (GCV) (9[(1,3-dihydroxy-2-propoxy)methyl]guanine) treatment has
shown good success as a tumor ablation strategy1,2 in a
variety of experimental models, including brain,3 head
and neck,4 skin,5 lung,6 liver,7 pancreatic,8 colon,9 prostate,10 ovarian,11 and breast cancers.12–15 Much of the
interest in this treatment strategy comes from the observation of cytotoxicity in nontransduced tumor cells that
lie in close proximity to HSV-tk-transduced cells. This
“bystander effect” eliminates the need to transduce all
cells in a target tumor cell population to achieve effecReceived November 26, 1997; accepted June 27, 1998.
Address correspondence and reprint requests to Henry J. Klamut, Room
10-721, Ontario Cancer Institute, Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9. E-mail address:
[email protected]
© 1999 Stockton Press 0929-1903/99/$12.00/10
Cancer Gene Therapy, Vol 6, No 2, 1999: pp 179 –190
tive treatment.16 –21 The cytotoxic effects of HSV-tk/
GCV treatment have been related to the induction of
apoptosis (programmed cell death) in a number of
tumor cell types.22–24 Programmed cell death is a normal
biological process used in situations in which cell deletion is required to alter tissue structure and function or
to remove genetically damaged cells.25,26 It is becoming
apparent that every cell has an intrinsic cell death
program, and that apoptosis can be triggered by a variety
of stimuli.27,28 The execution phase of apoptosis involves
activation of a proteolytic cascade of cysteine proteases
and results in distinctive morphological and biochemical
changes that include cell volume shrinkage, plasma
membrane blebbing, chromatin condensation, and DNA
fragmentation.25,26 The steps that lead to activation of
the execution phase of apoptosis are poorly understood.
However, evidence to date suggests that death-promoting signals can be transmitted by any one of several
distinct signaling pathways.29 –34 Mutations in genes
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within these signaling pathways are thought to increase
the activation threshold for apoptosis in cancer cells,
leading to genetic instability and the development of
resistance to radiation and chemotherapeutic agents.35–38
GCV is an acyclic nucleoside that is converted to
GCV triphosphate by viral thymidine kinase and endogenous mammalian kinases. This purine analog competes
with normal nucleotides during DNA synthesis and
terminates DNA replication.39,40 The DNA damage that
results is thought to signal apoptosis in HSV-tk/GCVtreated cell populations.22–24 The response of mammalian cells to DNA damage is complex and involves a
number of genes that modulate cell cycle progression,
DNA repair, and apoptosis.41– 44 The p53 tumor suppressor gene45 encodes a nuclear phosphoprotein that
can activate both G1 cell cycle arrest46,47 and apoptosis35,36,48 in response to DNA damage. Mutations in the
p53 gene are the most frequent genetic abnormality
found in human cancer,49,50 and a loss of p53 function
has been correlated with increased resistance to a wide
variety of DNA-damaging agents.51–56
Previous studies have demonstrated variability in the
susceptibility of individual tumor cell lines to HSV-tk/
GCV treatment.57–59 As HSV-tk/GCV cytotoxicity is
mediated through DNA damage-induced apoptosis, we
sought to establish whether any relationship exists between p53 gene status, p53-mediated apoptosis, and the
sensitivity of human breast tumors to this treatment
modality. To examine this issue, GCV cytotoxicity following adenovirus (Ad)-mediated gene transfer of recombinant HSV-tk was assessed in terms of cell viability,
bystander effects, and the appearance of characteristic
features of apoptotic cell death in two human breast
cancer cell lines: MCF-7 cells, which express wild-type
(wt) p53,60 and MDA-MB-468 cells, which express high
levels of a mutant p53 (273 Arg-His).61 Our analysis of
MCF-7 cells confirmed that HSV-tk/GCV treatment can
lead to a DNA damage-mediated induction of p53,
p21WAF1/CIP1, and apoptosis in breast tumor cells that
express wt p53. However, MDA-MB-468 cells exhibited
a strong apoptotic response without an increase in
p21WAF1/CIP1 protein levels, indicating that HSV-tk/GCV
treatment can induce apoptosis in the absence of functional p53. This finding raised the possibility that this
treatment strategy could be effective against breast
tumors that express mutant p53 and that may have
developed resistance to chemotherapeutic agents that
require wt p53 function. Analysis of four additional
human breast cancer cell lines expressing mutant p53
confirmed this result and demonstrated that chemosensitivity to HSV-tk/GCV treatment does not correlate
with endogenous p53 gene status.
MATERIALS AND METHODS
Cells and cell culture
The MDA-MB-468, MCF-7, MCF-7/Adr, BT-474, and T47D
human breast cancer cell lines were kindly provided by the
laboratories of Drs. R. Buick, S. Benchimol, M. Moore, and M.
Rauth of the Ontario Cancer Institute, Princess Margaret
Hospital. BT-20 human breast cancer cells and HS-574 cells,
which were established from normal tissue from a patient with
infiltrating ductal carcinoma, were obtained from the American Type Culture Collection (Manassas, Va). MCF-7, MCF-7/
Adr, MDA-MB-468, BT-20, and BT-474 breast cancer cell
lines were grown in a-minimal essential medium (a-MEM).
T47D cells were grown in RPMI 1640 medium, and HS-574
cells were grown in Dulbecco’s MEM. All media contained
10% fetal bovine sera and 100 U/mL penicillin/100 mg/mL
streptomycin (Life Technologies, Gaithersburg, Md); all cells
were incubated in the presence of 5% CO2 at 37°C. Cell lines
were tested free of mycoplasma contamination by Hoechest
33258 fluorescent detection.62
Preparation of recombinant adenoviral stocks
AdHSV-tk (recombinant Ad (rAd) containing a Rous sarcoma
virus (RSV) promoter-HSV-tk minigene) was kindly provided
by Dr. S. Woo (Baylor Medical College, Houston, Tex).
AdCMVp53 (rAd containing a cytomegalovirus (CMV) wt p53
minigene), AdCMVb-gal (rAd containing a CMV-b-galactosidase (b-gal) minigene), and the 293 cell line were kindly
provided by Dr. F. Graham (McMaster University, Hamilton,
Ontario, Canada). Large-scale rAd stocks were prepared from
single purified plaques, and all were tested free of replicationcompetent Ad essentially as described previously.63
Ad infection efficiencies
Ad infection efficiencies were determined essentially as described previously.64 Briefly, cells were seeded in six-well
dishes at a density of 100 cells/mm2; after 24 hours, cells were
exposed to different dilutions of AdCMVb-gal in 200 mL of
phosphate-buffered saline (PBS) to provide multiplicities of
infection (MOIs) in the range of 0.1–100 plaque-forming units
(PFU)/cell. After a 30-minute incubation at 37°C with gentle
shaking, 3 mL of fresh media was added to each well, and the
cells were incubated for an additional 48 hours. For b-gal
histochemistry, cells were washed in cold PBS-Ca,Mg and fixed
with a solution of 2% formaldehyde and 0.2% glutaraldehyde
in PBS-Ca,Mg for 5 minutes at 4°C. Fixed cells were washed
three times with PBS-Ca,Mg containing 2 mM MgCl2 and 0.02%
Nonidet P-40 and then overlayed with b-gal staining solution
(1 mg/mL 5-bromo-4-chloro-3-indolyl b-D-galactoside, 5 mM
potassium ferricyanide, 5 mM potassium ferrocyanide, and 2
mM MgCl2 in PBS-Ca,Mg) for 1–3 hours at 37°C. Stained
cultures were rinsed with PBS-Ca,Mg followed by dH2O and
air-dried. Transduction efficiencies, which were expressed as
the percent of the total cell population expressing b-gal, were
quantified by counting the number of blue-stained cells and
total cells in 10 –20 randomly chosen microscopic fields for a
minimum of three independent experiments.
HSV-tk expression by Northern blot analysis
Total cellular RNA was isolated using the RNeasy Kit (Qiagen, Chatsworth, Calif) according to the manufacturer’s instructions. Total RNA (10 mg) was separated by electrophoresis in 1.2% agarose-formaldehyde gels,65 transferred to zetaprobe nitrocellulose membranes (Bio-Rad, Hercules, Calif),
and hybridized to the following 32P-labeled cDNA probes
prepared by random priming:66 (a) a 2556-base pair HSV-tk
cDNA fragment, and (b) a 1.3-kilobase glyceraldehyde-3phosphate dehydrogenase (GAPDH) cDNA fragment. Hybridizations were performed overnight at 42°C in 40% formamide, 0.12 M Na2PO4 (pH 7.2), 0.25 M NaCl, 0.5% sodium
dodecyl sulfate (SDS), and 1 mM ethylenediaminetetraacetic
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LI, NGO, BRADE, ET AL: AdHSV-TK/GCV TREATMENT OF BREAST CANCER CELLS
acid (EDTA). Filters were washed in 23 standard saline
citrate (SSC)/0.1% SDS at 42°C for 20 minutes followed by
washes in 0.53 SSC/0.1% SDS and 0.13 SSC/0.1% SDS at
room temperature for 20 minutes each. Filters were exposed to
Kodak autoradiography film overnight at 270°C. Autoradiographs were analyzed densitometrically (Molecular Dynamics,
Sunnyvale, Calif) using ImageQuant software.
Cell viability assays
Cells were seeded in either 6- or 24-well plates at a density of
100 cells/mm2; after 24 hours, each well was infected with
either AdHSV-tk at 0.1, 1, 10, 25, 50, or 100 PFU/cell or
AdCMVb-gal at 100 PFU/cell followed by GCV (Hoffman-La
Roche, Nutley, NJ) at concentrations ranging from 3 to 18
mg/mL. Cell viability was determined at days 5 and 7 after
treatment using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma, St. Louis, Mo).
Briefly, MTT (in a-MEM minus phenol red) solution was
added to each well to give a final concentration of 1 mg/mL.
After a 4-hour incubation at 37°C, the formazan product was
solubilized by overlaying cells with 0.01 N HCl in isopropanol
for 4 hours at 37°C. Under our experimental conditions, there
was a direct correlation between formazan production as
detected by measuring the absorbance of the overlay solution
at 570 nm and total viable cell numbers per well. The results
are expressed as the percentage of cell viability relative to
uninfected control cultures.
Bystander effects were measured by mixing cells infected
with AdHSV-tk (100 PFU/cell) with uninfected cells at ratios
(infected to uninfected) of 1:0, 3:1, 2:1, 1:1, 1:3, 1:5, 1:10, 1:100,
and 0:1 and plating the cells onto 24-well plates at 105
cells/well. Control infected (AdCMVb-gal) and uninfected
cells plated at the same ratios and densities were included in
each study. Cells were exposed to GCV at 10 mg/mL at 24
hours after plating, and cell viability was measured at day 7
after GCV treatment using the MTT assay. The results are
expressed as the percentage of cell viability relative to corresponding uninfected control cultures.
Acridine orange and ethidium bromide (EB) staining
Cells treated with either control virus (AdCMVb-gal) or
AdHSV-tk/GCV were harvested at several timepoints posttreatment, washed with PBS, and resuspended in 1 mL of PBS
containing 5 mg/mL acridine orange (Sigma) and 5 mg/mL EB
(Sigma). After a 5-minute incubation at room temperature,
cells were pelleted by centrifugation and resuspended in 25 mL
of PBS containing 10% glycerol. Stained cell samples were
examined and photographed by fluorescence microscopy.
DNA fragmentation assays
Control and treated cultures were harvested at several timepoints post-treatment and tested for DNA fragmentation
essentially as described previously.67 Briefly, cells were
scraped, harvested, and pelleted in ice-cold PBS; next, 5 3 106
cells were resuspended in 200 mL of guanidine thiocyanate
containing 100 mM 2-mercaptoethanol and stored overnight at
270°C. DNA was precipitated overnight at 220°C after the
addition of 100 mL of 7.5 M ammonium acetate and 600 mL of
absolute ethanol and pelleted by centrifugation at 14,000 3 g
for 20 minutes at 4°C. DNA was resuspended in TE (10 mM
tris(hydroxymethyl)aminomethane (Tris)-HCl, 1 mM EDTA;
pH 8.0) containing 20 mg/mL ribonuclease A, electrophoresed
on 2% agarose gels, and visualized by EB staining. Controls
included cells infected with AdHSV-tk alone (minus GCV
Cancer Gene Therapy, Vol 6, No 2, 1999
181
treatment) and uninfected cells exposed to GCV at a concentration of 10 mg/mL.
Western blot analysis of p53 and
p21WAF1/CIP1 expression
Cells were infected with AdHSV-tk or AdCMVb-gal (control)
at 100 PFU/cell and exposed to GCV at a concentration of 10
mg/mL. Cells were harvested at various timepoints post-treatment by washing and scraping into ice-cold PBS-Ca,-Mg. Cells
were pelleted by centrifugation and solubilized (25 mL per 106
cells) in protein solubilization buffer (0.1 M Tris/HCl (pH 8.0),
1% SDS, 10 mM EDTA, and 2 mM dithiothreitol). Protein
concentrations were measured using the bicinchoninic acid
assay (Pierce, Rockford, Ill) according to the manufacturer’s
instructions. Cell lysates were loaded onto 10% SDS-polyacrylamide gels at a protein concentration of 50 mg/lane, separated
electrophoretically, and transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH) using a semidry
gel transfer apparatus (Bio-Rad).68 Transfer efficiency was
monitored by staining nitrocellulose membranes with Ponceau
red (Sigma) and post-transferred gels with Coomassie blue
R250. Membranes were blocked with a solution of 5% skim
milk powder and 1% heat-inactivated sera in Tris-buffered
saline (TBS) (0.01 M Tris/HCl (pH 7.5) and 0.15 M NaCl) for
2 hours at 4°C, washed three times with TBS containing 0.05%
Tween 20, and incubated with antibody (Ab) binding solution
(1% skim milk powder and 1% heat-inactivated sera in TBS)
containing either a 1/100 dilution of monoclonal p53 Ab (Ab-6,
Oncogene Science, Uniondale, NY) or a 1/100 dilution of a
monoclonal p21WAF1/CIP1 Ab (Ab-1, Oncogene Science) for 60
minutes at room temperature. Filters were washed with three
changes in TBS containing 0.05% Tween 20 for 10 minutes
each at room temperature and incubated with horseradish
peroxidase-conjugated donkey anti-mouse Ab (1:10,000, Jackson Laboratories, West Grove, Penn) in Ab binding solution
for 60 minutes at room temperature. Membranes were washed
again as described previously, and bands were visualized using
enhanced chemiluminescence detection reagents (Amersham,
Arlington Heights, Ill) and autoradiography according to the
manufacturer’s instructions.
RESULTS
Ad-mediated gene transfer results in high levels of
HSV-tk mRNA expression
To provide a basis for comparison of the cytotoxic effects
of AdHSV-tk/GCV treatment of MDA-MB-468 and
MCF-7 cells, we first examined Ad infection efficiencies
and HSV-tk mRNA expression levels in these two cell
lines. Ad infection efficiencies were evaluated by infecting MDA-MB-468 and MCF-7 cells with AdCMVb-gal
at MOIs ranging from 0.1 to 100 infectious particles
(PFU)/cell and determining the percentage of the total
target cell population staining positive for b-gal. The
results are summarized in Figure 1. Ad infection efficiencies were ;2-fold higher in MDA-MB-468 cells at
MOIs of 1 and 10 PFU/cell. However, in cells infected at
100 PFU/cell, .95% of both MDA-MB-468 and MCF-7
cells tested positive for b-gal expression. Northern blot
analysis indicated that HSV-tk mRNA is expressed as
early as 24 hours and peaks by 48 –72 hours postinfection
in both MDA-MB-468 and MCF-7 cells infected with
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LI, NGO, BRADE, ET AL: AdHSV-TK/GCV TREATMENT OF BREAST CANCER CELLS
Figure 1. Recombinant adenoviral vectors transduce MDA-MB-468
and MCF-7 cells at efficiencies approaching 100%. Cells were
transduced with a rAd expressing either b-gal (AdCMVb-gal) or
luciferase (AdCMVluc) at an MOI ranging from 0.1 to 100 infectious
particles (PFU)/cell. At 48 hours after infection, cells were stained for
b-gal activity. Transduction efficiency was determined as the percentage of cells staining blue in the treated cell population. Values
represent the mean 6 SD for $10 random fields, each representing
2
an area of 0.39 mm and containing a minimum of 100
cells.
AdHSV-tk at 100 PFU/cell (Fig 2a). Densitometric
analysis using endogenous GAPDH gene expression as
an internal reference (HSV-tk/GAPDH ratio; Fig 2b)
indicated that HSV-tk mRNA levels in MDA-MB-468
cells were 1.5-fold higher than in MCF-7 cells at 24 and
48 hours postinfection but had reached equivalent levels
in the two cell lines by 72 hours. These results indicated
that the transduction of MDA-MB-468 and MCF-7 cells
with AdHSV-tk at an MOI that provides for an infection
of .95% of the target cell population results in the
expression of HSV-tk mRNA at levels that differed by no
more than 1.5-fold over a period of 72 hours after
infection.
AdHSV-tk/GCV treatment results in induction of p53
and p21WAF1/CIP1 in MCF-7 cells
HSV-tk/GCV cytotoxicity is thought to be mediated by
the induction of programmed cell death in response to
DNA damage, which leads to the induction of endogenous p53 protein levels and p21WAF1/CIP1-mediated G1
cell cycle arrest.47,69,70 To establish that HSV-tk/GCV
treatment leads to the induction of p53 and p21WAF1/CIP1
protein levels in MCF-7 cells, and that mutant p53 gene
expression precludes the induction of p21WAF1/CIP1
in MDA-MB-468 cells, total cell extracts isolated at 12,
24, and 36 hours after treatment with AdHSV-tk/GCV
were examined for increases in endogenous p53 and
Figure 2. HSV-tk MRNA levels in MDA-MB-468 and MCF-7 cells
transduced with AdHSV-tk at 100 PFU/cell. a: Northern blot analysis
of total RNA isolated from MDA-MB-468 and MCF-7 cells at 0 –72
hours after infection with AdHSV-tk at 100 PFU/cell. Blots were
hybridized sequentially with 32P-labeled HSV-tk and GAPDH cDNA
probes and autoradiographed for 24 (MDA-MB-468) to 72 (MCF-7)
hours at 270°C. b: Densitometric analysis of HSV-tk and GAPDH
band intensities. Normalization of HSV-tk band intensities to
GAPDH (HSV-tk/GAPDH ratio) demonstrated that recombinant
HSV-tk mRNA levels did not vary by .1.4-fold (48-hour timepoint)
between MDA-MB-468 and MCF-7 cells over the time period
studied.
p21WAF1/CIP1 protein levels by Western blot analysis.
Results are shown in Figure 3. As expected, high levels
of mutant p53 expression were evident in cell extracts
derived from AdHSV-tk (100 PFU/cell)-infected MDAMB-468 cells in both the absence (time 0) and presence
of GCV (9 mg/mL) for #36 hours postinfection. However, no increase in endogenous p21WAF1/CIP1 protein
was observed at any of the timepoints studied. A cell
extract prepared from MDA-MB-468 cells transduced
with a rAd expressing wt p53 (AdCMVp53) was included as a positive control for the induction of endogenous p21WAF1/CIP1 protein expression in this cell line.
Endogenous p53 and p21WAF1/CIP1 protein levels were
low to undetectable in AdHSV-tk (100 PFU/cell)-infected MCF-7 cell cultures at time 0. Treatment with
GCV (9 mg/mL) resulted in a significant increase in
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LI, NGO, BRADE, ET AL: AdHSV-TK/GCV TREATMENT OF BREAST CANCER CELLS
Figure 3. AdHSV-tk/GCV treatment induces endogenous p53 and
p21WAF1/CIP1 protein levels in MCF-7 cells. Western blot analysis of
cell lysates prepared from MDA-MB-468 and MCF-7 cells at 0 –36
hours after treatment with AdHSV-tk (100 PFU/cell) and GCV (9
mg/mL). Blots were incubated with Abs to either p53 (ap53)
or p21WAF1/CIP1 (ap21); reactive bands were visualized using horseradish peroxidase-conjugated secondary Abs, enhanced chemiluminescence reagents, and autoradiography as described in Materials and Methods. Included as positive controls for p53 and
p21WAF1/CIP1 were cell lysates prepared from MDA-MB-468 and
MCF-7 cells infected with a rAd expressing wt p53 (Adp53; 100
PFU/cell). Untreated (0 hours) MDA-MB-468 cells express high
levels of endogenous mutant p53 and undetectable levels of endogenous p21WAF1/CIP1. Endogenous p21WAF1/CIP1 protein is induced in response to recombinant wt p53 overexpression (Adp53)
but not in response to AdHSV-tk/GCV treatment (12–36 hours).
Endogenous p53 (wt) and p21WAF1/CIP1 protein is undetectable in
untreated MCF-7 cells. Both are increased in cells transduced with
an adenoviral vector that expresses recombinant p53 (Adp53).
Treatment with AdHSV-tk/GCV also results in the induction of
endogenous p53 (12–36 hours) and p21WAF1/CIP1 (36 hours) protein
levels.
p53 protein levels by 12 hours afer infection and in
p21WAF1/CIP1 protein levels by 36 hours after infection. A
cell extract prepared from MCF-7 cells transduced with
AdCMVp53 was included as a positive control for both
wt p53 and the induction of endogenous p21WAF1/CIP1
protein expression. These results substantiate the notion
that HSV-tk/GCV treatment leads to an induction of
p53 and p21WAF1/CIP1 protein levels in tumor cells expressing wt p53, and establish a clear difference in the
p53 response of these two cell lines to this treatment
strategy.
MDA-MB-468 and MCF-7 cell lines display differential
sensitivities to AdHSV-tk/GCV treatment
These experiments were designed to examine the relationship between HSV-tk transgene dosage, GCV concentration, and cytotoxicity in MDA-MB-468 and
MCF-7 cell cultures. Target cell populations were infected with AdHSV-tk over a range of 1.0 –100 PFU/cell
and exposed to GCV concentrations of 3, 9 (clinically
achievable dose),3 and 18 mg/mL. Cell viability was
determined at 7 days after treatment relative to control
cultures using the MTT assay, which measures the
number of viable cells in each group at the end of the
treatment period. Controls included mock-infected and
AdCMVb-gal (100 MOI)-infected cultures treated with
Cancer Gene Therapy, Vol 6, No 2, 1999
183
Figure 4. MDA-MB-468 and MCF-7 cells display differential sensitivities to AdHSV-tk/GCV treatment. Cells were infected with
AdHSV-tk at MOIs ranging from 1 to 100 PFU/cell and treated with
GCV at concentrations of 3, 9 (clinically achievable dose), and 18
mg/mL. Cell viability was determined using the MTT assay as
described in Materials and Methods. Solid symbols, MDA-MB-468
cells; shaded symbols, MCF-7 cells. GCV concentrations are represented by: triangles, 3 ug/mL; circles, 9 ug/mL; and squares, 18
mg/mL. Results represent the mean 6 SD of a minimum of three
independent determinations and are expressed as the percentage
of viability relative to mock-infected controls.
GCV at 9 mg/mL. GCV in the range of 3–18 mg/mL of
culture medium was determined to have no significant
effect on the viability of uninfected controls. As shown in
Figure 4, treating AdHSV-tk-infected MDA-MB-468
cells with GCV at 9 mg/mL resulted in a decrease in cell
viability to 75% of controls at 1 PFU/cell, to 25% of
controls at 10 PFU/cell, and to ,5% of controls at
25–100 PFU/cell. Differences in MDA-MB-468 cell viability in AdHSV-tk-infected cells treated with GCV at 3
and 18 mg/mL ranged from 10 –20% at MOIs of 1–25
PFU/cell and decreased to ,10% at an MOI of $50.
Treatment of AdHSV-tk-infected MCF-7 cells with
GCV at 9 mg/mL decreased cell viability to 85% of
controls at 1 PFU/cell and to 70% of controls at 100
PFU/cell. Differences in cell viability in cultures treated
with GCV at 3 and 18 mg/mL averaged 22% over the
range of MOIs tested. These results demonstrate a clear
difference in the sensitivity of these two breast cancer
cell lines to AdHSV-tk/GCV treatment. At the highest
doses of AdHSV-tk and GCV used in this study, MDAMB-468 cell viability was reduced to ,1% of controls. In
comparison, MCF-7 cell viability declined to only 60%
of controls under conditions that provide for similar
levels of Ad infection and HSV-tk mRNA expression in
the two cell lines.
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portion of cells infected with AdHSV-tk and total cell
viability demonstrated that this treatment strategy results in a significant bystander effect in the MDA-MB468 cell line. In contrast, identical mixing experiments
with MCF-7 cells resulted in only a 20 –30% decline in
cell viability over the range of infected to uninfected cell
ratios studied (Fig 5).
AdHSV-tk/GCV treatment leads to apoptosis of both
MDA-MB-468 and MCF-7 cells
Figure 5. Bystander effects in MDA-MB-468 and MCF-7 cells
treated with AdHSV-tk/GCV. Bystander effects were examined by
mixing cells infected with AdHSV-tk at 100 PFU/cell with uninfected
cells at ratios (infected to uninfected) ranging from 1:10 to 1:0. Cells
were seeded onto 24-well plates at a density of 105 cells/well and
exposed to GCV at a concentration of 9 mg/mL for 7 days. Cell
viability was determined by MTT assay, and results are expressed
as the percentage of viability relative to untreated controls. Filled
bars, MDA-MB-468 cells; shaded bars, MCF-7 cells. Values represent the mean 6 SD of a minimum of three independent observations.
Bystander effects are more pronounced in AdHSV-tktreated MDA-MB-468 cells
Infecting MDA-MB-468 cells with AdCMVb-gal at 1
and 10 PFU/cell resulted in b-gal staining of 4.9% and
49% of cells, respectively (Fig 1). However, MDA-MB468 cells infected with AdHSV-tk at 1 and 10 PFU/cell
displayed 28% and 90% declines, respectively, in cell
viability at a GCV concentration of 18 mg/mL (Fig 4).
These results suggested that a bystander effect occurs
under these treatment conditions. To examine this phenomenon in more detail, a series of experiments were
performed in which cells infected with AdHSV-tk at 100
PFU/cell were mixed at different ratios with uninfected
cells and treated with GCV at a concentration of 9
mg/mL. Cell viability was determined at 7 days after
treatment using the MTT assay, and results were expressed relative to controls containing equivalent mockinfected to uninfected cell numbers. As shown in Figure
5, no significant decrease in cell viability was observed in
either MDA-MB-468 or MCF-7 cell cultures seeded at
ratios of infected to uninfected cells of 1:10 or lower. At
an infected to uninfected cell ratio of 1:5, MDA-MB-468
cell viability decreased to 30% of controls. At ratios of
1:5–1:1, viability decreased further to 10 –15% of controls; at ratios of $2:1, a decrease was observed to ,5%
of control cultures. The discrepancies between the pro-
To examine the mechanism of AdHSV-tk/GCV cytotoxicity in MDA-MB-468 and MCF-7 cells, treated cells
were examined for morphological (chromatin condensation, nuclear fragmentation) and biochemical (DNA
fragmentation) features associated with apoptotic cell
death. As shown in Figure 6, no cytotoxic effects were
observed in either MDA-MB-468 or MCF-7 cells treated
with AdHSV-tk alone (Fig 6, B and E) compared with
mock-infected controls (Fig 6, A and D). Treating
MDA-MB-468 cells with AdHSV-tk at 100 PFU/cell and
GCV at 9 mg/mL (Fig 6C) led to detachment of the
majority of cells from the culture dish by 36 hours after
treatment. Acridine orange and EB staining indicated
that this treated cell population contained large numbers
of condensed, fragmented nuclei and apoptotic bodies
characteristic of apoptosis. At 36 hours after treatment,
no significant morphological changes were observed in
MCF-7 cells treated under the same conditions (data not
shown). However, at 72 hours after treatment, morphological changes characteristic of apoptosis began to
appear in the treated MCF-7 cell population (Fig 6F)
and steadily increased to include 30 – 40% of the total
cell population by 7 days after treatment. Agarose gel
electrophoretic analysis of genomic DNA laddering (Fig
7) confirmed that HSV-tk/GCV treatment leads to the
induction of apoptosis in both MDA-MB-468 and
MCF-7 cells. However, the apoptotic response was
significantly delayed in MCF-7 cells (72 hours after
treatment) compared with MDA-MB-468 cells (36 hours
after treatment). It should be noted that in these experiments, the extent of DNA laddering is not representative of the proportion of the total cell population
undergoing apoptosis. DNA fragmentation was not observed in mock-infected controls or in cells treated with
AdHSV-tk or GCV alone. Taken together, these results
demonstrate that AdHSV-tk/GCV treatment results in
the induction of apoptosis in both MDA-MB-468 and
MCF-7 cells. However, the apoptotic response of
MCF-7 cells was significantly delayed and involved a
significantly lower percentage of the total cell population
relative to MDA-MB-468 cells treated under the same
conditions.
Breast cancer cell lines display differential
chemosensitivities to GCV treatment following
HSV-tk gene transfer
Our studies of MDA-MB-468 cells established that
AdHSV-tk/GCV treatment can lead to the activation of
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LI, NGO, BRADE, ET AL: AdHSV-TK/GCV TREATMENT OF BREAST CANCER CELLS
185
Figure 6. MDA-MB-468 and MCF-7 cells display morphological features consistent with the induction of apoptosis in response to
AdHSV-tk/GCV treatment. Phase contrast and fluorescence microscopy of MDA-MB-468 (A–C) and MCF-7 cells (D–F) stained with acridine
orange/EB revealed morphological features characteristic of apoptosis in AdHSV-tk/GCV-treated cells. A and D: mock-infected controls. B and
E: AdHSV-tk (100 PFU/cell) alone. C and F: AdHSV-tk (100 PFU/cell) and GCV (9 mg/mL). The apoptotic bodies (arrow, panel C) and chromatin
condensation (arrow, panel F) observed in AdHSV-tk/GCV-treated cells are characteristic features of apoptosis. Phase contrast: Bar, 50 mm.
Acridine orange/EB staining: Bar, 18 mm.
a strong, p53-independent apoptotic response in human
breast cancer cells that lack functional p53. As this
observation has important implications for the treatment of breast cancer cells that have developed resistance to genotoxic therapeutic agents, we examined
AdHSV-tk/GCV cytotoxicity in four additional human
breast cancer cell lines that have been reported to
express mutant p53 (MCF-7/Adr, BT-20, BT-474, and
T47D).61,71–74 The results, shown in Figure 8, are expressed as the percentage of viability relative to untreated controls. The percentages of cell viability measured for MCF-7 and MDA-MB-468 cells, as well as a
normal human control (HS-574), were also included for
comparative purposes. Whereas a significant variation in
chemosensitivity to AdHSV-tk/GCV treatment could be
seen among the five breast cancer cell lines expressing
mutant p53, all displayed a greater sensitivity to this
therapeutic modality than either MCF-7 cells or normal
HS-574 control cells. Interestingly, the multidrug-resistant subline of MCF-7 cells (MCF-7/Adr)71 showed at
least a 50-fold greater chemosensitivity to AdHSV-tk/
GCV treatment than the parental line. Although the
mechanism(s) responsible for the differential sensitivities of these cell lines to AdHSV-tk/GCV treatment are
not clear, the results suggest that endogenous p53 status
is not predictive of chemosensitivity to this treatment
modality, and point to a potential application of this
Cancer Gene Therapy, Vol 6, No 2, 1999
treatment strategy to tumor cells that express mutant
p53 and have developed resistance to conventional
genotoxic therapeutic agents.
DISCUSSION
Prodrug activation strategies involving HSV-tk gene
transfer followed by GCV treatment have shown considerable potential for cancer therapy.1,2 The bystander
effect, which refers to cytotoxic effects seen in neighboring cells that have not been transduced with HSV-tk,
makes this approach particularly attractive, as it obviates
the need to deliver a therapeutic gene to all cells in the
target tumor.16 –21 GCV cytotoxicity is thought to be
mediated through the induction of apoptosis (programmed cell death) in response to DNA damage.22–24
The p53 tumor suppressor gene product plays a major
role in regulating the response of mammalian cells to
DNA-damaging agents,51–53,55,56,75 and abnormalities in
p53-dependent apoptosis arising from mutations in the
p53 gene have been shown to contribute to the development of chemotherapeutic drug resistance.51–56,75 The
relationship between HSV-tk/GCV cytotoxicity and p53dependent apoptosis has not been firmly established in
human tumor models. In view of the high incidence of
p53 gene mutations in breast cancer49 and their associ-
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LI, NGO, BRADE, ET AL: AdHSV-TK/GCV TREATMENT OF BREAST CANCER CELLS
Figure 7. DNA laddering in MDA-MB-468 and MCF-7 cells treated
with AdHSV-tk/GCV. Agarose gel electrophoresis and EB staining of
genomic DNA extracted from MDA-MB-468 and MCF-7 cells infected with AdHSV-tk at 100 PFU/cell and treated with GCV at a
concentration of 9 mg/mL for 24 – 48 hours (MDA-MB-468) or 48 –96
hours (MCF-7). Controls include untreated cells and cells treated
with either AdHSV-tk (100 PFU/cell) alone or GCV (9 mg/mL) alone.
The DNA laddering that is characteristic of apoptotic cell death was
observed in both MDA-MB-468 and MCF-7 cells treated with
AdHSV-tk/GCV.
ation with increased resistance to drug and radiation
treatments,51–56,75 we sought to establish whether HSVtk/GCV treatment results in the predicted increase in
p53 protein levels and in the induction of apoptotic cell
death, as well as whether mutations in the p53 gene
correlate with increased resistance to this treatment
modality.
Our observation in MCF-7 cells of increases in endogenous p53 and p21WAF1/CIP1 protein levels, along with
the appearance of morphological and biochemical features characteristic of apoptotic cell death, is consistent
with the notion that DNA damage arising from AdHSVtk/GCV treatment triggers a p53-dependent apoptotic
cell death pathway.22,24,35 The relative resistance of
MCF-7 cells to this treatment strategy is also consistent
with previous studies showing that this cell line does not
readily undergo apoptosis following DNA damage.76
However, the robust apoptotic response observed in
MDA-MB-468 cells, which overexpress mutant p53,61
also points to a significant role for p53-independent
apoptosis in the cytotoxic response of tumor cells to
AdHSV-tk/GCV treatment. Furthermore, cell viability
studies demonstrated that breast tumor cells expressing
mutant p53 can be highly sensitive to the cytotoxic
Figure 8. Comparisons of the relative chemosensitivities of different
human breast cancer cell lines with AdHSV-tk/GCV treatment. Cell
viability was determined in a series of six human breast cancer cell
lines expressing either wt (MCF-7) or mutant (MCF-7/Adr, MDAMB-468, BT-20, BT-474, and T47D) p53 as well as in one normal
control (HS-574). Measurements were taken at 5 days after treatment with AdHSV-tk at an MOI of 100 PFU/cell and 10 mg/mL GCV.
A significant degree of variation in the chemosensitivities of the
different cell lines to GCV treatment was observed. However, cell
lines expressing mutant p53 consistently showed greater chemosensitivity than cells expressing wt p53, and no direct correlation
could be made between chemosensitivity and endogenous p53
gene status. Values represent the mean 6 SD of a minimum of three
independent observations.
effects of AdHSV-tk/GCV treatment. These results are
consistent with a recent study of HSV-tk/GCV cytotoxicity in mouse thyrocytes,23 and serve to expand the
predicted effective range of this treatment strategy to
breast tumors that express mutant p53 and that may
have developed resistance to other genotoxic therapeutic modalities. This latter point is illustrated by our
observation of a 50-fold increase in the sensitivity of
MCF-7/Adr cells to HSV-tk/GCV treatment. MCF-7/
Adr cells display cross-resistance to a number of chemotherapeutic agents, including vinca alkaloids, anthracyclines, colchicine, taxol, and actinomycin D71,77,78 as well
as g radiation.79 Although the mechanism by which the
development of multidrug resistance increases the sensitivity of MCF-7 cells to HSV-tk/GCV treatment is not
clear, similar results have been observed with multidrug
(vincristine)-resistant mouse leukemia P388 cells.80
The apparent lack of a correlation between cytotoxicity and endogenous p53 gene status points to the existence of alternative molecular and biochemical factors
responsible for the range of chemosensitivities to
AdHSV-tk/GCV treatment seen among the six breast
cancer cell lines studied. Possibilities include differences
in HSV-tk gene dosage, the rate of GCV uptake, the
strength of the bystander effect, and defects in one or
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LI, NGO, BRADE, ET AL: AdHSV-TK/GCV TREATMENT OF BREAST CANCER CELLS
more downstream regulators of apoptosis. In this study,
we undertook to examine some of these issues in MCF-7
and MDA-MB-468 cells, two human breast cancer cell
lines that were observed to exhibit widely different
chemosensitivities to AdHSV-tk/GCV treatment.
HSV-tk gene dosage
Variable sensitivities to GCV treatment following gene
transfer of a recombinant HSV-tk gene to mesothelioma,
non-small cell carcinoma, and ovarian carcinoma cell
lines have been attributed to differences in adenoviral
infection efficiencies as demonstrated by AdCMVb-gal
histochemistry.81 However, our observation that both
MDA-MB-468 and MCF-7 cells are infected at efficiencies approaching 100% when exposed to AdCMVb-gal
at 100 PFU/cell, and that comparable levels of HSV-tk
mRNA are expressed in each of these cell lines under
these conditions, suggests that the differential chemosensitivities of MCF-7 and MDA-MB-468 cells to GCV
are not related to differential HSV-tk gene dosage
effects.
GCV uptake
Because most nucleosides and nucleoside analogs are
highly hydrophillic, they do not readily cross the lipid
bilayer of biological membranes in the absence of functional nucleoside transport processes.82 The permeation
of nucleosides across the plasma membrane of mammalian cells is complex and is mediated by at least five
distinct transporters that differ in their sensitivity to
inhibitors and in their specificity for individual nucleosides.83 Differences in the distribution of these transporters could account for the differential GCV cytotoxicities seen in MCF-7 and MDA-MB-468 cells. However,
our observation of similar decreases in cell viability
(15–20%) as GCV concentrations were increased from 3
to 18 mg/mL suggests that GCV transport is not significantly different in these two cell lines.
Bystander effects
The absence of a significant bystander effect could
contribute to resistance to AdHSV-tk/GCV treatment at
MOIs that provide for transgene expression in ,100%
of the target cell population. Gap junctions have been
shown to mediate the cell-to-cell transfer of phosphorylated nucleotides,84 and gap junction-mediated transfer
of phosphorylated GCV molecules from HSV-tk-expressing cells to neighboring nontransduced cells has
been proposed as a mechanism for the bystander effect.18 –20 In this study, MDA-MB-468 cells were seen to
exhibit a significant bystander effect, but no bystander
effect was seen in MCF-7 cells treated under the same
conditions. The absence of a bystander effect in MCF-7
cells has been independently confirmed and has been
attributed to a deficiency in gap-junctional intercellular
communication.59 Interestingly, significant bystander effects have been observed in MCF-7 cells treated with
either a human thymidine phosphorylase gene and 59deoxy-5-fluorouridine59,85 or a liver cytochrome p450
Cancer Gene Therapy, Vol 6, No 2, 1999
187
gene in combination with cyclophosphamide.86 These
results are consistent with the view that tumors can
exhibit differential chemosensitivities to individual prodrug activation strategies.59 However, a deficiency in the
HSV-tk/GCV-mediated bystander effect does not account for the relative resistance of MCF-7 cells treated
under conditions used in this study that provide for the
transduction of .95% of the target cell population.
Bcl-2 family proteins and apoptotic thresholds
The molecular mechanisms that establish the relative
sensitivity of tumor cells to different apoptotic agents
remain to be fully elucidated. However, results to date
indicate that members of the Bcl-2 family of proteins
play a prominent role in regulating intracellular thresholds to apoptotic cell death-promoting stimuli.87 Bcl-2
functions as a negative regulator of apoptosis and has
been reported to be overexpressed in #70% of breast
cancers.88 Furthermore, high levels of bcl-2 gene expression have been correlated with an increased resistance to
therapeutic agents that activate both p53-dependent and
-independent apoptotic signaling pathways.89,90 Bax
functions as a dominant repressor of Bcl-2 activity and
promotes apoptosis, and the ratio of Bcl-2 to Bax is
considered to be an important determinant of the
threshold of a cell to death-promoting stimuli.91 Other
related genes implicated in the regulation of apoptotic
thresholds include Bcl-xL92 and BAG-1,93 both of which
are genetic homologs of bcl-2 that also function as
negative regulators of apoptosis. Like Bax, Bad and
Bcl-xS function as dominant repressors of Bcl-2 and
Bcl-xL to promote apoptosis,94,95 and the MCL1 gene
has been implicated as a mediator of p53-independent
apoptosis in human cell lines defective in p53 function.96,97 Recently, monospecific Abs, immunoblotting,
and immunohistochemical methods were used to analyze the relative levels of the Bcl-2, Bcl-xL, MCL1, Bax,
Bak, and BAG-1 proteins in a series of human breast
cancer cell lines that included each of the breast cancer
cell lines used in this study.74 MCF-7 cells were shown to
express Bcl-2 at levels that were 8-fold higher than in
MDA-MB-468 cells, and MCF-7/Adr cells have been
reported to express decreased levels of Bcl-2 relative to
the parental cell line.98 Thus, Bcl-2 levels do seem to
correlate with the relative sensitivities of MCF-7, MDAMB-468, and MCF-7/Adr cells to AdHSV-tk/GCV treatment. However, high levels of Bcl-2 expression in BT474 cells and undetectable levels of Bcl-2 in BT-20 cells
are inconsistent with the relative sensitivities of these
cell lines to AdHSV-tk/GCV treatment. In fact, no
direct correlation could be made between the levels of
expression of any one of the Bcl-2 family of proteins and
the sensitivity of individual breast cancer cell lines to
AdHSV-tk/GCV treatment. This finding raises the possibility that multiple genetic and biochemical factors
contribute to the sensitivity of individual tumors to this
treatment strategy, and the relative contribution of each
of these factors to HSV-tk/GCV cytotoxicity will need to
be established if prognostic assays for the responsiveness
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LI, NGO, BRADE, ET AL: AdHSV-TK/GCV TREATMENT OF BREAST CANCER CELLS
of individual tumors to this treatment modality are to be
developed.
In summary, we have demonstrated that AdHSV-tk/
GCV treatment of breast cancer cells can lead to
increases in endogenous p53 levels and to the induction
of apoptotic cell death. However, this cytotoxic response
does not seem to depend upon the expression of a
functional p53 gene, and tumor cell lines expressing
mutant p53 exhibit a high degree of variability in their
sensitivity to this treatment modality. Evidence for a
p53-independent apoptotic response to AdHSV-tk/GCV
treatment serves to extend the potential of this treatment strategy to breast tumors that express mutant p53.
However, further studies will be needed to elucidate the
precise molecular and biochemical mechanisms responsible for HSV-tk/GCV cytotoxicity and the development
of predictive assays for the responsiveness of individual
tumors to this treatment strategy.
10.
11.
12.
13.
14.
ACKNOWLEDGMENTS
We thank S. Woo for the AdHSV-tk construct and F. Graham
for the AdCMVb-gal and AdCMVp53 constructs. This work
was supported in part by the Medical Research Council of
Canada, the Foundation for Gene and Cell Therapy, and an
Amgen predoctoral fellowship (to P.-X.L).
15.
16.
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