Yonago Acta medica 2005;48:63–74

Cell Death Induction of Thymidine Kinase Gene Transfer
Followed by Ganciclovir Treatment in Oral Squamous Cell
Carcinoma Cell Lines
Itaru Kuratate*†, Mitsuhiko Osaki* and Kazuo Ryoke†*Division of Organ Pathology, Department of Microbiology and Pathology, †Divisionof Oral and Maxillofacial Biopathological Surgery, Department of Medicine of Sensory
and Motor Organs, School of Medicine, Tottori University Faculty of Medicine, Yonago
683-8503 JapanThe tumoricidal effect of herpes simplex virus thymidine kinase (HSV-tk) gene transfer followed by ganciclovir (GCV) treatment has been demonstrated in relation to the
bystander effect. We examined the mode of cell death after GCV treatment in threehuman oral squamous cell carcinoma (SCC) cell lines that had been infected with adenovirus possessing HSV-tk gene. Oral SCC cell lines displayed high susceptibility to
HSV-tk/GCV treatment despite low transduction. Evidence suggests that apoptosis was
not found by internucleosomal DNA fragmentation, cell cycle kinetics or gene expressions indicative of apoptosis. However, we observed different levels of expression ofannexin V-positive/propidium iodide-negative cells in all the cell lines and occasional
TUNEL-positive cells in one cell line. To address these controversial findings, we further
confirmed the morphological phenotypes of cell death with semi-thin and ultra-thinsections, which revealed that the cells undergoing death were consistent with necrosis,
i.e. swelling of cytoplasm and intracellular organs with membrane disruption. Furthermore, no apoptotic bodies were detected within cytoplasm of apparently intact cells. A
nonapoptotic mechanism may play a central role in the HSV-tk/GCV-induced cell deathin oral SCC cell lines.
Key words: apoptosis; ganciclovir; necrosis; thymidine kinase

Squamous cell carcinoma (SCC) is the most frequent malignancy in the oral cavity. The prognosisof advanced oral SCC has remained poor over thelast few decades despite progress in aggressive approaches using combination therapy. Novel strategies should be explored in the oral cancer field.
One of the potential improvements in current therapeutic strategies is herpes simplex virus
thymidine kinase (HSV-tk) gene transfer followed by ganciclovir (GCV) treatment based on

the enzyme/prodrug system concept (Borrelli etal., 1988). HSV-tk-transduced cells selectively
convert prodrug GCV into monophosphate GCV.
Triphosphate GCV is the final activated form with
an ability to kill cells by interfering in DNA synthesis through chain termination and single-strand
breaks (Elion, 1982). Another promising aspect of
this strategy is the existence of the so-called “bystander effect” (Freeman et al., 1993; Colombo etal., 1995), which mediates cytotoxicity to the adja-

Abbreviations: Ad, adenovirus; FITC, fluorescein isothiocyanate; GCV, ganciclovir; HSV-tk, herpes simplex virus
thymidine kinase; IC50, median inhibitory concentration; MOI, multiplicity of infection; PBS, phosphate-bufferedsaline; pfu, plaque forming unit; PI, propidium iodide; SCC, squamous cell carcinoma; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling63

I. Kuratate et al.
Table 1. Oral squamous cell carcinoma cell lines and p53 status
Cell line

Histology*

p53 status

Mutation

Site mutation codon

HSC-3
HSC-4
SCCKN

por
well
mod

mutantmutantmutant

exon8
exon7
exon5

TAAG insersion (305 and 306)
248CGC to CAG
178CAC to -AC (frame shift)*well, well differentiated type; mod, moderately differentiated type; por, poorly differentiated type.

phological changes that occurred at ultra-structurallevels (Kerr et al., 1972).
The purpose of the present study was to assess the feasibility of adenovirus-mediated HSVtk gene transfer followed by GCV to oral SCC celllines, in addition to verifying whether apoptotic responses are associated with HSV-tk/GCV-induced
cell death. In addition, we examined the ultrastructural level alterations to identify molecularbiological changes.

cent untransduced cells. There have been two hypothesis proposed to explain the bystander effect:(i) transfer of cytotoxic molecules via gap junctions
because phosphorylated GCV is unable to diffuse
freely across the cytoplasmic membrane (Bi et al.,1993) and (ii) phagocytosis of apoptotic bodies containing GCV-metabolites from HSV-tk-transduced
cells by untransduced intact cells (Freeman et al.,
1993; Colombo et al., 1995).
HSV-tk/GCV treatment has been successfully
applied in both human and animal tumor cell linesof different origins (Moolten et al., 1990; Glaser
et al., 2001; Lanuti et al., 1999; Li et al., 1999;
Boucher et al., 1998). There have been, however,few studies on oral SCC cell lines (O’Malley et al.,
1996; Fukui et al., 2001). While the validity of thisapproach has been confirmed, the molecular mechanism of HSV-tk/GCV-induced cell death remains
to be elucidated. The most commonly describedcell death mechanism was related to the inductionof apoptosis, not only for the execution pathway
but also the bystander effect (Colombo et al., 1995;
Samejima and Maruelo, 1995). On the other hand,a few studies have suggested nonapoptotic induction, i.e. necrosis (Kaneko and Tsukamoto, 1995;
Vile et al., 1997; Katabi et al., 2002). Recently,
activation of caspase-independent apoptotic pathways was also suggested because of the incomplete
inhibition of cell death by caspase inhibitors suchas z-VAD-fmk (Beltinger et al., 1999; Rivas etal., 2001). Both molecular biological analysis andmorphological examinations are indispensable for
apoptotic evaluation. Most studies exposing the
HSV-tk/GCV-induced cell death mechanism havebeen performed with apoptosis-related molecularbiological analysis, while a few studies using ultrathin examinations have been performed where
apoptosis was originally evaluated from the mor-

Materials and Methods
Cell lines
Three cell lines derived from human tongue SCCs
were used (Table 1). We obtained HSC-3 andHSC-4 from the Health Science Research Resources Bank (Osaka, Japan), and SCCKN from Riken
Gene Bank (Tsukuba, Japan). Cell line 293 derived
from human embryonic kidney was used for bothvirus purification and titer determination. All celllines were grown in Dulbecco’s MEM (COSMO
BIO, Tokyo, Japan) complemented with 1/100 diluted Gibco solution (29.2 mg/mL L -glutamine
10,000 unit/mL penicillin G, 10,000 µg/mL streptomycin) and 10% inactivated fetal bovine serum
and maintained at 37˚C in an incubator under 5%
CO2 /95% air atmosphere.
Adenovirus vectors
Two replication-deficient recombinant adenoviruses containing LacZ marker gene (Ad.β -gal) as
a control and luciferase vector, and adenoviruses
containing HSV-tk (Ad.tk) as the therapeutic vector under the control of cytomegalovirus promoter

64

Cell death of oral SCC by ganciclovir

were kindly provided from Professor Kenzo Sato
(Division of Molecular Biology, Department of Molecular and Cellular Biology, School of Medicine,
Tottori University Faculty of Medicine, Yonago,
Japan). The Ad.β -gal and Ad.tk were propagatedin 293 cells, which include the Ad5 E1 region inchromosomal DNA. These viruses were purified
by sequential centrifugation in CsCl step gradients.
Virus titers were determined as plaque forming
units (pfu) using a modified end-point cytopathic
effect assay. The titers of stock in 5% glycerol and
95% culture medium of Ad.β -gal and Ad.tk were2.27 × 1010 pfu/mL and 1.35 × 1011 pfu/mL, respectively.

tion at 37˚C, cells were trypsinized and replated in
96-well culture plates at a density of 3 × 103/well.
After overnight incubation, the medium was replaced with selective 100 µL medium containing
different concentrations of GCV (0–1,000 µg/mL).
Uninfected cells without GCV treatment were also
incubated as controls. Cell viability was assessed
at 96 h after GCV treatment using an MTT assay(CellTiter 96 Nonradioactive Cell Proliferation
Assay, Promega, Madison, WI). According to the
manufacturer’s instruction, 15 µL of the dye solution was added to each well and the plates wereincubated for 4 h. Subsequently, 100 µL of thesolubilizing solution was added. After overnight incubation, the absorbance at 570 nm wavelength wasmeasured using a microplate reader. Cell viability
was expressed as the relative percentage to control.
The result represents the mean ± SE of triplicates.

Adenovirus infection efficiency
Cells were seeded in 24-well culture plates at adensity of 2 × 10 4 /well. After overnight incubation, stock Ad.β -gal was diluted in 100 µL of
medium and added at varying multiplicities of infection (MOIs) of 0, 6.25, 12.5, 25, 50 or 100. The
cells were exposed for 1 h with constant agitation,and then the fresh medium was added up to 500µL. Plates were incubated for an additional 24, 48
and 72 h at 37˚C. After rinsing with phosphatebuffered saline (PBS) twice, cells were fixed with asolution of 2% formaldehyde and 0.2% glutaraldehyde in PBS for 10 min at 4˚C. Cells were rinsed
with PBS twice and incubated in a solution containing the X-gal (0.2% 5-bromo-4-chloro-3-indolylβ - D -galactopyranoside) for 4 h at 37˚C. The number of blue cells expressing transgene was counted.
Adenovirus infection efficiency was expressed as amean of the percentage of stained cells to the total
cells from three randomly selected microscopic
fields at ×200 magnification.

DNA fragmentation
Both floating and attached cells were harvested
from wells at 0, 12, 24, 48 and 72 h incubation
times after HSV-tk/GCV treatment. Cells wereincubated for 10 min at 4˚C in 100 µL lysis buffer containing 10 mM Tris-HCl (pH 7.4), 10 mM
EDTA and 0.5% Triton X-100. After centrifugation for 5 min at 16,000 rpm, the supernatants were
decanted and digested with RNase A (100 µg/mL,
final) at 37˚C for 1 h and then with proteinase K(200 µg/mL, final) for 1 h at 37˚C. After thesetreatments, DNA was extracted by phenol, andisopropanol precipitation. These preparations were
subjected to electrophoresis in 2% agarose gels andstained with 0.5 µg/mL ethidium bromide and electrophoretic patterns of the gel under UV light wasphotographed.

Cell viability assay

Cell cycle kinetics

Cells were seeded in 60 mm dishes at a densityof 3 × 105/dish. After overnight incubation, bothstock Ad.β -gal and Ad.tk were diluted in 100 µL of
medium and adjusted at MOIs of 1.25, 5, 10 and 20.
Each dish was exposed for 1 h with viral diluted
medium or condition medium, and then fresh medium was added up to 300 µL. After 16 h incuba-

Cells were infected with Ad.tk at MOIs of 5 for
HSC-3 or 2.5 for HSC-4 and SCCKN, respectively.
After 16 h incubation, the medium was exchanged
with GCV medium at a concentration of 1 µg/mL.
Both floating and attached cells were harvested
from wells at 0, 12, 24, 48 and 72 h incubation times
after HSV-tk/GCV treatment. Then, 1 × 10 6 /mL

65

I. Kuratate et al.
suspensions were prepared for flow cytometry assay.
To analyze cell cycle kinetics, 2 × 105 cells
were fixed in 70% ethanol and stored at 4˚C until
ready for use. Cells were centrifuged and resuspended in 1 mL PBS containing RNaseA (DNase
free, 0.1 mg/mL), and stained with 50 µg/mL propidium iodide (PI). Samples were analyzed within 1
h after the staining procedure. Fluorescence intensity was determined by an EPICS XL flow cytometer (Coulter, FL).

sulfonyl fluoride, 1 ng/mL leupeptin, 10 ng/mL
soy bean trypsin inhibitor, 1 ng/mL aprotinin, 10
ng/mL N-tosyl-L -phenylalanyl chloromethyl ketone
for 1 h on ice. Lysates were centrifuged at 15,000rpm for 10 min and then supernatants were decanted. Protein concentrations were determined using
the Bradford protein assay (Bio-Rad Lab., Richmond, CA). Equal amounts (25 µg) of total protein
were loaded onto each lane of 10% SDS polyacrylamide gels, electrotransferred to a polyvinylidene
difluoride membrane (Millipore, Bedford, MA),and then probed with the respective antibodies.
Blots were developed with peroxidase-labeled antimouse or anti-rabbit antibodies (1:2000; MBL, Nagoya, Japan) using enhanced chemiluminescence(ECL detection system; Amersham, Bucks, United
Kingdom).

Annexin V assay
To detect the early stage of apoptosis, annexin
V-fluorescine isothiocyanate (FITC) and PI double
staining were performed according to the manufacturer’s instructions for the apoptosis detection kit(Trevigen, MD). Briefly, 1 × 105 cells were exposedwith 100 µL of labeling reagent containinig 1 µLof annexin V-FITC conjugate and 10 µL of PI (50µg/mL). After 15 min incubation at room temperature in the dark, an additional 400 µL of 1 ×binding buffer was added. Samples were analyzed
within 1 h after the staining procedure. Sample
analysis was also performed using an EPICS XL
flow cytometer.

TUNEL method

The following antibodies were used for Western
blotting; anti-P53 (BP53-12, diluted 1:500; Novocastra Lab., Claremont Place, United Kingdom),anti-Bax (P-19, 1:250; Santa Cruz Biotechnology,
Santa Cruz, CA), anti-Bcl-2 (100, 1:250; Santa
Cruz Biotechnology, Santa Cruz, CA), anti-caspase-3 (CCP32, 1:250; Transduction Laboratories,
Lexington, KY) and anti- β -actin (Ab-1,1:1500;
SIGMA, St. Louis, MO).

Cells cultured on cover slips were transduced andincubated for 24, 48 and 72 h with and without
GCV (1 µg/mL). After incubation, attached cells
were washed with PBS twice. Terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin
nick- end labeling (TUNEL) staining was performed according to kit protocol with minor modification using an Apop Tag Plus peroxidase In Situ
Apoptosis Detection Kit (Intergen, Purchase, NY).
Briefly, after fixation with 95% ethanol, cells were
exposed to terminal deoxynucleotidyl transferasewith digoxigenin-11-dUTP and dATP in a moist
chamber for 90 min at 37˚C. Antidigoxigenin
antibody-peroxidase was used for detection ofdigoxigenin-11-dUTP labeling for 30 min at room
temperature, followed by color development with
3,3'-diaminobenzidine containing H2O2 solution.
Methyl green was used for counterstaining.

Expression of apoptosis related protein

Morphological examinations

Both floating and attached cells were harvested
from wells at 0, 12, 24, 48 and 72 h incubation
times after HSV-tk/GCV treatment, and solubilized in lysis buffer with 50 mM Tris-HCl (pH 7.4),
125 mM NaCl, 0.1% NP-40, 1 mM phenyl methyl

Both floating and attached cells were harvested
from wells at 72 h with and without GCV (1µg/mL) in all cell lines. After centrifugation andwashing with PBS twice, cell pellets were fixed in
Kalnovsky solution for 4 h at 4˚C. After overnight

Antibodies

66

Cell death of oral SCC by ganciclovir

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Fig. 1. Adenovirus infection efficiency in oral SCC
cell lines. Cells were infected with Ad.β -gal at varying
MOIs of 0, 6.25, 12.5, 25, 50 or 100 pfu/cell, respectively. At 48 h after the infection, cells were fixed andstained by X-gal. The percentages of cells expressing
the transgene were counted. Infection efficiency was
expressed by the percent average of blue-staining cells
in three randomly selected × 200 microscopic fields. ,HSC-3, , HSC-4 and , SCCKN.

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washing with 0.1 M phosphate solution bufferedat pH 7.4 at 4˚C, cell pellets were postfixed in 1%
osmium tetroxide solution for 2 h. After washingwith 0.1 M phosphate solution buffered at pH 7.4,
cell pellets were dehydrated in a graded ethanol
series and embedded in Epon 812. After polymerization, 1 µm semi-thin sections were served
for light microscopic examinations after toluidine
blue staning. Then 60 nm ultra-thin sections wereserved for electron microscopic examination after
uranyl acetate and Reynold’s lead citrate staining.
Specimens were examined with an H-800 electron
microscope (Hitachi, Tokyo).

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Fig. 2. Cell viabilities after HSV-tk/GCV treatment.
Infected cells with either Ad.tk or Ad.β -gal were treatedwith GCV at concentrations ranging from 0 to 1,000µg/mL. Uninfected cells without GCV treatment wereused as control. Cell viabilities were measured at 96
h after GCV treatment using the MTT assay. Results
were calculated as the percentage of viability relative tocontrol. The result represents the mean ± SE of triplicates. The IC50 were determined. (a) HSC-3, (b) HSC-4
and (c) SCCKN cell lines. , GCV alone; , Ad.β -gal
+ GCV; , Ad.tk 1.25 MOI + GCV; , Ad.tk5 MOI +GCV; , Ad.tk 10 MOI + GCV and , Ad.tk 20 MOI +GCV.

Results
Adenovirus infection efficiency
Adenovirus infecction efficiencies in human oral
SCC cell lines were quantified using direct cellcounting with X-gal staining. We assessed the infection efficiency at the 48 h time point, when the
β -gal expression level was sufficient. For 100%
infection, an MOI 50 was needed for HSC-3 andSCCKN cell lines, and impossible for the HSC-4
cell line within an MOI 100 (Fig. 1). However, in-fection of Ad.tk at these high titers failed to be cytotoxic to oral SCC cell lines. Then we performed
an MOI escalation study to determine the sublethal

67

Cell death of oral SCC by ganciclovir

MOI value of Ad.tk infection (data not shown).
The results showed MOIs of 20 and less for the
HSC-3 cell line and 2.5 and less for HSC-4 and
SCCKN cell lines, respectively.

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Cell viability
Cell viability was determined from the colorimetricquantity of viable cells. HSV-tk gene transfer decreased the median inhibitory concentration (IC50)
at least 2 logs lower in all cell lines (Fig. 2). Cell
viability was dependent on GCV concentration.
As the HSC-3 cell line only had resistance to Adtk infection, we could examine cell viabilities atseveral ranges of MOI (Fig. 2a). Though the IC50
decreased in proportion to the MOI escalation, theresults were not different between MOIs of 20 and
10. This fact suggested the upper limit bounds of
MOI escalation of this strategy.

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Fig. 3. DNA fragmentation after HSV-tk/GCV treatment. Cells were infected with Ad.tk at MOI 5 for
HSC-3, 2.5 for HSC-4 and SCCKN, and followed by 1
µg/mL GCV. Cells were harvested in a time-dependent
manner. Uninfected cells without GCV treatment wereused as control. DNA extracts were loaded on 2%
agarose gel by electrophoresis and visualized by staining with 0.5 µg/mL ethidium bromide. (a) HSC-3, (b)HSC-4 and (c) SCCKN cell lines. M, marker; C, control.

DNA fragmentation

Cell cycle kinetics

Internucleosomal DNA fragmentation was examined for standard apoptotic hallmarks. They all
showed smearing, whose peaks were 72 h, 72 h and
24 h for cell lines HSC-3, HSC-4 and SCCKN, respectively (Fig. 3).

Figure 4 shows the results of cell cycle kinetic
analysis. The cells broke down severely showing
a time-dependent decrease of G1 and G2/M peaks
without cell cycle arrest. Sub-G1 fraction, whichmeans hypodiploid cells correspond to dead cells,
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Fig. 4. Cell cycle kinetics analysis by flow cytometry. Cells were infected with Ad.tk at MOI 5 for HSC-3, 2.5 for
HSC-4 and SCCKN, and followed by 1 µg/mL GCV. Cells were harvested in a time-dependent manner. Uninfected
cells without GCV treatment were used as control. The number within each panel shows the percentage of Sub-G1
fractions. C, control.

68

Cell death of oral SCC by ganciclovir����Fig. 5. Annexin V-FITC and PI double stainning by flow cytometry. Cells were infected with Ad.tk at MOI 5 for
HSC-3, 2.5 for HSC-4 and SCCKN, and followed by 1 µg/mL GCV. Cells were harvested in a time-dependent
manner. Uninfected cells without GCV treatment were used as control.
a: Representative results of HSC-4. The number within each panel shows the percentage of each quadrant. C, control.
b: Increases in percentage in the lower right quadrant relative to control. , HSC-3, , HSC-4 and , SCCKN.
c: Increases in percentage in the upper right quadrant relative to control. , HSC-3, , HSC-4 and , SCCKN.

increased in a time-dependent manner in all thecell lines.

Expression of apoptosis related protein
Protein expression levels of P53, P21, Bax, Bcl-2
and caspase-3 activation were examined by Western blot analysis (Fig. 6). P53 was stably expressedin HSC-3 and HSC-4 cell lines, and undetectable
at all time points in the SCCKN cell line. P21 andBax was consistently expressed at any time points
in all cell lines. On the other hand, Bcl-2 slightly
but steadily increased within 24 h after treatment
in HSC-3 and SCCKN cell lines. No caspase-3
cleavage or activation was observed throughout theexperiment in any cell lines.

Annexin V assay
Annexin V has an affinity for the phosphatidylserine that is translocated from the inner leaflet to theouter leaflet of the plasma membrane in the earlystage of apoptosis (Martin et al., 1995). During thelate stage, the plasma membrane showed increased
permeability and PI entered the cytoplasm andstained chromatin indicating necrosis. Figure 5ashows representative kinetics of HSC-4 in the celllines. The proportion of annexin V-positive/PInegative cells in the lower right quadrant shows theearly stage of apoptosis increased in a time-dependent manner (Fig. 5b). The proportion of annexin
V-positive/PI-positive in the upper right quadrantshows necrosis also increased in a time-dependent
manner (Fig. 5c).

TUNEL method
In situ DNA strand breaks were examined by the
TUNEL method. TUNEL -positive cells withbrown-labeled nuclei were occasionally detected69