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Fig. 6. Western blot analysis of P53, P21, Bax, Bcl-2 andcaspase-3 of each cell lines. 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) HSC-3, (b)HSC-4 and (c) SCCKN cell lines. C, control.

Fig. 7. TUNEL staining of treated SCCKN 24 h after
GCV treatment. Cells were infected with Ad.tk at MOI
2.5, and followed by 1 µg/mL GCV. TUNEL-positive
cells with brown-labeled nuclei are occasionally detected in only SCCKN cell line (arrows).

A few cells showing chromatin aggregation to the
nuclear membranes, also displayed cytoplasmic
membranes disruption. Treated cells presented
structural changes of intracellular organelles involving enlargement of mitochondria with the disappearance of cristae, vesiculation and vacuolization of endoplasmic reticulum. Apoptotic bodies
were not observed in any of the treated cells.

in only the SCCKN cell line infected with Ad.tk
at MOI 2.5 followed by 1 µg/mL GCV after 24 h(Fig. 7). However, we could not evaluate correctly
at 48 h and 72 h because most treated SCCKN
cells had easily come off in the staining procedure.
However, the untreated SCCKN cells showed no
TUNEL signal.
Morphological examinations
There were no significant differences in morphological findings in both semi-thin and ultra-thinsections after HSV-tk/GCV treatment among thecell lines. Semi-thin sections of untreated cells
showed variations in size and shape and occasionally small vesicles in the cytoplasm with a few mitoses (Fig. 8a). In contrast, a large number of treated cells became swollen with or without membrane
disruption, sometimes involving nuclei disruption(Fig. 8b). Cytoplasmic vesicles were markedly
increased. There were few cells with chromatin aggregation in HSC-4 and SCCKN cell lines.
Ultra-thin sections revealed intact nuclei,
intracellular organelles and their membranes in untreated cells (Fig. 9a). Otherwise, a large numberof treated cells contained enlarged and rounded
nuclei in which heterochromatin was sparse. Cytoplasmic membranes were also disrupted (Fig. 9b).

abFig. 8. Semi-thin section micrographs of HSC-4 cells.
(a) Untreated cells. Some cells occasionally have small
vesicles, and some display mitoses. (b) Treated cells 72
h after GCV treatment. Cells were infected with Ad.tk
at MOI 2.5, and followed by 1 µg/mL GCV. Cells become swollen and intracytoplasmic vesicles remarkably
increase. Few cells show chromatin aggregation (arrows).

70

Cell death of oral SCC by ganciclovirabFig. 9. Ultra-thin section micrograph of HSC-4 cells. (a) Untreated cells. (b) Treated cells 72 h after GCV treatment. Cells were infected with Ad.tk at MOI 2.5, and followed by 1 µg/mL GCV. A large number of cells containenlarged and rounded nuclei of which hetero chromatin are sparse. Some of treated cells show chromatin aggregation. But their cytoplasmic membranes are still disruptured. Inset of (b) displays enlargement of mitochondria withdisappearance of cristae, vesiculation and vacuolization of endoplasmic reticula. Bars in (a) and (b) = 1 µm.

opposite (Kaneko and Tsukamoto, 1995). It is possible that the DNA fragmentation assay might notdetect low levels of apoptosis. Confirmation of thephenotype of dying cells is needed by reference tomorphology using electron microscopy (Vile et al.,1997). In fact, some cases failed to demonstrate
DNA laddering even when the presence of apoptotic cells was further confirmed using electron
microscopy (Freeman et al., 1993; Craperi et al.,1999). In the present results, DNA fragmentation showed unspecific DNA degradation, that is,smearing (Fig. 3). Accordingly, both semi- andultra-thin examinations revealed the phenotype ofdying cells was necrotic in all cell lines.
Previous studies of flow cytometry demonstrated that HSV-tk/GCV treatment caused either
S- and/or G2/M-phase cell cycle arrest before
undergoing cell death (Kaneko et al., 1995; Weiet al., 1998; Craperi et al., 1999). The present results showed cell death progressing with no cell
cycle arrest (Fig. 4). The DNA content of sub-G1
increased in a time-dependent manner. A rapid
increase was observed in the HSC-4 cell line at 72
h (42.7%), in which DNA fragmentation definitely
showed smearing suggesting no occurrence of regular DNA double-strand breaks. Annexin V has
been used as another biological marker to detect
the early stage of apoptosis before the occurrence

Discussion
This study clearly demonstrated that HSV-tk/GCV
treatment showed diverse but obvious sensitivity tooral SCC cell lines independent of their histological status (Table 1 and Fig. 2). Oral SCC cell lines
displayed high susceptibility to HSV-tk/GCV treatment despite low transduction, suggesting possible
tumoricidal effects even in the case of inability of
100% gene delivery (Fig. 1). In the HSC-3 cellline, however, HSV-tk/GCV treatment showed theupper limit bounds at MOI 10. As reported previously, a higher expression of HSV-tk gene may not
be able to further enhance the tumoricidal effect,
though a threshold level of HSV-tk gene expression
is necessary for maximum cell killing (Chen et al.,1995).
The tumoricidal mechanism of HSV-tk/GCV
treatment has focused on exploring the molecular biological events associated with apoptosis(Samejima and Maruelo, 1995; Wei et al., 1998; Rivas et al., 2001). To date, the most reliable method
for identifying apoptotic cell death is the detectionof the occurrence of internucleosomal DNA strand
breaks (Wyllie, 1980). Concerning past HSVtk/GCV treatment studies, several reported the existence of DNA laddering (Samejima and Maruelo,
1995; Wei et al., 1998), but several reported the71

I. Kuratate et al.
of morphological changes in single cells (Martin
et al., 1995). The present results showed that annexin V-positive/PI-negative cells corresponding
to apoptotic cells, increased in a time-dependent
manner in all cell lines (Fig. 5b), although further
semi- and ultra-thin examinations revealed necrotic morphological features. While the externalization of phosphatidylserine during apoptosis has
been presented, intracellular events have not beenadequately elucidated. Although the meaning of
the annexin V-positive reaction induced by HSVtk/GCV treatment is not contested, this phenomenon implies the feasibility of inspection and exemplification by phagocytic cells (Depraetere, 2000;
Hanayama et al., 2002). Changes in the propertiesof their surface membrane would improve the combination with a targeting approach such as immunotherapy.
Since the tumoricidal effect of HSV-tk/GCV
treatment is conceivable via DNA damage, the lossof function of p53 can develop resistance to apoptosis. Li et al. reported, however, the HSV-tk/GCV
cytotoxic response did not depend on the expression of a functional p53 (Li et al., 1999; Craperi etal., 1999). Similarly, we demonstrated an obvious
tumoricidal effect without either p53 or p21 expression (Figs. 4 and 6). These results were consistentin all cell lines and suggested that p53-dependent
cell cycle regulation or apoptosis induction may notcorrelate with the tumoricidal effect of HSV-tk/
GCV treatment to oral SCC. Alternatively, HSVtk/GCV treatment might have considerable potential to oral SCC that highly expresses mutated p53
in vivo (Naglar et al., 2002) and in vitro (Sakai andTsuchida, 1992). A previous exogenous p53 transduction study suggested co-expression of p53 did
not enhance the cytotoxicity of HSV-tk/GCV treatment, although p53 was able to increase amount of
apoptosis which was markedly less than the total
cell death in vitro (Xie et al., 1999).
Dimerization between Bax and Bcl-2 is an
important factor to direct either death promotion ordeath inhibition. Bax accumulation was reported
after GCV exposure in glioma cell lines (Craperi
et al., 1999). However, they could not rule out
the possible occurrence of necrotic cell death. In

contrast, over-expression of Bcl-2 inhibited HSVtk/GCV-induced activation of caspase and apoptosis (Beltinger et al., 2000). However, no synchronous correlation could be found between theexpression levels of Bcl-2 family and the sensitivity
to the oral SCC cell lines.
No cleavage or activation of caspase-3 wasdetected in oral SCC cell lines. This suggested
the existence of an alternative molecular pathway
responsible for the tumoricidal effect of HSV-tk/
GCV treatment to oral SCC cell lines even though
the route of cell death is unknown.
One exception in the present study was an occasional positive TUNEL signal in SCCKN cells(Fig. 7). However, no other evidence of apoptosis
was detected with other biological assays. Cautionary notes suggested that a positive TUNEL signal
should not be considered as a specific marker of
apoptosis because the assay would also suggest
necrotic cell death (Charriaut-Marlangue and BenAri, 1995; Grask-kraupp et al., 1995).
Apoptosis was originally described on thebasis of the morphological features by electron
microscopy (Kerr et al., 1972) even though somefeatures may also be detectable by light microscopy. Apoptotic bodies including cellular remnants
should be observed either in the intercellular spaceor within the cytoplasm of intact cells. A large
number of treated cells showed disintegration of thecell structure involving irregular scattered heterochromatin, swelling of cytoplasm and intracellularorganelles, and cytoplasmic membrane disruption
in all cell lines. Furthermore, intact cells containing apoptotic bodies that deposit the fragmented
components from neighboring dead cells were notfound. Based on the annexin V/PI double staining
at 72 h, the HSC-4 cell line should include the cellsof early stage apoptosis of 19.1% (Fig. 5). However, we could not find firm evidence of apoptosis
in semi- and ultra-thin section examinations. Consequently, apoptosis may not play a central role in
the tumoricidal mechanism. The hypothesis thatthe bystander effect is mediated by phagocytosis is
unacceptable for oral SCC cells in vitro.
A previous study using Fas ligand demonstrated an artificial deficiency of caspase-8 resulted

72

Cell death of oral SCC by ganciclovir

in a switch of cell death from apoptosis to necrosis
in Jurket cell lines (Kawahara et al., 1998). Thisimplies that caspase activation itself was dispensable for determining cell death. Kitanaka andKuchino (1999) suggested the existence of caspaseindependent programmed cell death with necroticlike morphology, that can be activated either alone
or in concert with the caspase-dependent apoptotic
program. Differential sensitivity and the type ofcell death induced by HSV-tk/GCV treatment appear to be dependent upon cell-type difference, in
addition to the status of the apoptosis-related gene.
In summary, we demonstrated that oral SCC
cell lines exhibited an obvious sensitivity to HSVtk/GCV treatment suggesting the presence of ap53- and caspase-3-independent death signaling
pathway. However, the mechanism and the molecular pathway responsible for HSV-tk/GCV-induced
cell death are still unknown. A better understanding of the cell death mechanism is essential toestablish an appropriate and effective modality infuture studies.

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Debatin KM. Mitochondrial amplification of deathsignals determines thymidine kinase/ganciclovirtriggered activation of apoptosis. Cancer Res 2000;
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9 Craperi D, Vicat JM, Nissou MF, Mathieu J, Baudier
J, Benabid AL, et al. Increased bax expression isassociated with cell death induced by ganciclovir ina herpes thymidine kinase gene-expressing glioma
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12 Freeman SM, Abboud CN, Whartenby KA, Packman
CH, Koeplin DS, Moolten FL, et al. The “bystander
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13 Fukui T, Hayashi Y, Kagami H, Yamamoto N,
Fukuhara H, Tohnai I, et al. Suicide gene therapy
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14 Glaser T, Castro MG, Lowenstein PR, Weller M.
Death receptor-independent cytochrome c releaseAcknowledgments: All authors thank Professor Kenzo
Sato (Division of Molecular Biology, Department of
Molecular and Cellular Biology, School of Medicine,
Tottori University Faculty of Medicine) for his appropriate advice and kindly providing two replication-deficient
recombinant adenovirus vectors, Ad.β -gal and Ad.tk.
We also thank Professor Kazuo Yamada (Division of
Medical Biochemistry, Department of Pathophysiological and Therapeutic Science, School of Medicine, Tottori
University Faculty of Medicine) for his invaluable advice, and Mr. Norihisa Itaki (Division of Organ Pathology, Department of Microbiology and Pathology, Schoolof Medicine, Tottori University Faculty of Medicine) for
his skilful technical assistance. Finally, we thank all themembers of Division of Organ Pathology, Departmentof Microbiology and Pathology, School of Medicine,
Tottori University Faculty of Medicine for their helpful
discussions.

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Received June 20, 2005; accepted August 29, 2005
Corresponding author: Mitsuhiko Osaki, PhD

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