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Enhancement of Cytarabine Sensitivity in Squamous Cell Carcinoma Cell Line Transfected With Deoxycytidine Kinase
Hiromi Kojima, MD;
Minoru Iida, MD;
Hidemi Miyazaki, MD;
Tomohiko Koga, MT;
Hiroshi Moriyama, MD;
Yoshinobu Manome, MD
Arch Otolaryngol Head Neck Surg. 2002;128:708-713.
ABSTRACT
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Background Cytarabine is the most effective agent known for the treatment of acute
myeloid leukemia. Its antitumor effect is expressed by combining with DNA
during replication and then destroying the DNA chain. However, cytarabine
has only limited activity against most solid tumors, including squamous cell
carcinoma of the head and neck. The reason for this is thought to be that
in cell lines of solid tumors the expression of cytidine deaminase, an enzyme
that degrades cytarabine, is high, whereas the expression of deoxycytidine
kinase (dCK), which phosphorylates cytarabine (a prodrug), is weak.
Objective To determine whether head and neck squamous cell carcinomas can be made
more sensitive to the cytotoxic effects of cytarabine by shifting the balance
from the degradative to the activation pathway.
Methods Human SCC-25 squamous carcinoma cells were transfected by either retroviral
vector or adenoviral vector containing DCK gene and
were identified for dCK expression by Northern blot analysis. In vitro cytotoxic
assay after cytarabine exposure was performed using these cells.
Results Both retroviral and adenoviral vector-mediated transduction of the dCK
complementary DNA resulted in marked sensitization of tongue squamous carcinoma
cell lines to the cytotoxic effects of cytarabine in vitro.
Conclusion The dCK-cytarabine system may be a useful approach for gene therapy
of squamous cell carcinomas of the head and neck.
INTRODUCTION
SQUAMOUS CELL carcinomas of the head and neck (HNSCCs) have been increasing
as major health concerns around the world. Although considerable advances
in surgical techniques, radiation therapies, and chemotherapy have been made
during the past several decades, the long-term survival of patients with advanced
HNSCC has not been significantly improved. In addition, no effective salvage
therapy exists for patients in whom standard treatment fails.
Gene transfer offers the possibility of new approaches to cancer treatment.
Head and neck cancers are particularly well suited to gene transduction strategies.1 Multiple gene transfer strategies that involve the
introduction of foreign genes that directly kill tumor cells, that restore
a defective tumor-suppressor gene, and induce apoptosis, or that enhance the
immune response, are presently under investigation for HNSCC.2-5
One gene transduction strategy for the treatment of cancer involves the introduction
of a drug sensitivity gene, which encodes an enzyme that can intratumorally
activate a prodrug. An example of this strategy is the transfer of the herpes
simplex virus thymidine kinase (HSVtk) gene, which
encodes the protein that activates ganciclovir, a nucleoside analogue.6-8
Cytarabine is the most effective agent in the treatment of acute myeloid
leukemia.9-10 It is incorporated
into replicating DNA and terminates DNA chain elongation.11-13
However, unfortunately, cytarabine has limited activity against most solid
tumors, including HNSCC. Resistance mechanisms include relatively rapid deamination
of cytarabine by cytidine deaminase as compared with phosphorylation to cytidine
monophosphate by deoxycytidine kinase (dCK).14
We hypothesized that HNSCC could be made more sensitive to the cytotoxic effects
of cytarabine if the balance is shifted from the degradative to the activation
pathway.
The present work demonstrates that the adenovirus-mediated transfer
of the DCK gene followed by administration of cytarabine
increases the sensitivity of HNSCC cells to the cytotoxic effects of cytarabine
in vitro.
MATERIALS AND METHODS
TUMOR CELL LINE
Human SCC-25 squamous carcinoma cells (American Type Culture Collection
[ATCC], Rockville, Md) were grown as monolayers in Dulbecco modified Eagle
minimum essential medium supplemented with 10% heat-inactivated fetal bovine
serum; penicillin, 100 U/mL; streptomycin sulfate, 100 µg/mL; 2mM L-glutamine;
and hydrocortisone, 0.4 µg/mL. The amphotropic PA317 retrovirus packing
cell line (ATCC) was grown in Dulbecco modified Eagle minimum essential medium
supplemented with 10% heat-inactivated fetal bovine serum; penicillin, 100
U/mL; streptomycin sulfate, 100 µg/mL; and 2mM L-glutamine.
CONSTRUCTION OF dCK-EXPRESSING VECTORS AND CELL LINES
A 0.8-kilobase (kb) fragment of the human dCK complementary DNA was
cloned into the EcoRI site of the pMV7 retroviral
vector (pMV7-dCK).15-17
The neo gene is transcribed from a thymidine kinase
promoter (tk).
Supernatant from PA317/pMV7-dCK retroviral producer cells was used to
transduce SCC-25 target cells. The cells were incubated for 24 hours and then
selected for 2 weeks in the presence of geneticin sulfate 400 µg/mL
(Gibco BRL, Gaithersburg, Md). Polyclonal populations of SCC-25/Neo (transduced
by pMV7) and SCC-25/dCK (transduced by pMV7-dCK) cells were identified for
dCK expression by Northern blot analysis.
NORTHERN BLOT ANALYSIS
Total cellular RNA was isolated as described by others.18
The RNA (20 µg per lane) was separated in agarose-formaldehyde gels,
transferred to nitrocellulose filters, and hybridized to the following phosphorus
32–labeled DNA probes: (1) a 0.8-kb NcoI and BamHI fragment of dCK from the pET3d-dCK plasmid17; (2) a 1.3-kb HindIII fragment
from the p1Aneo plasmid containing the neomycin 3'-phosphotransferase
complementary DNA sequence17; (3) a 3.3-kb HindIII/EcoRI lacZ fragment from the pSV-ß-galactosidase vector (Promega Corp,
Madison, Wis); and (4) a 1.5-kb EcoRI insert of a
human ß-actin gene purified from the HFBCC49 plasmid (ATCC). Hybridizations
were performed as described by others.19
IN VITRO CYTOTOXIC ASSAY
Cells (2 x 103/200 µL) were seeded into individual
wells of a 96-well microtiter plate (Linbro Division, Flow Laboratories Inc,
Hamden, Conn). Twelve hours later, the cells were treated with cytarabine
for 96 hours. The cells were fixed after cytarabine exposure and stained with
0.05% methylene blue.20 The dye was eluted
with 0.33M hydrochloric acid for 15 minutes with agitation. Absorbance was
measured with a microplate reader (Model 550, Bio-Rad Laboratories, Hercules,
Calif) at 595 nm. Values were determined within the linear range and standardized
to a control curve.21 Statistical analysis
was performed with an unpaired, 2-tailed t test.
RECOMBINANT ADENOVIRUS
The dCK-cDNA was cloned into the NotI site
of a shuttle plasmid, Ad.CMV-ßgal, as previously described.17
The resulting shuttle plasmid, pCMV-dCK, was cotransfected into 293 cells
with the pJM17 plasmid containing the adenoviral type 5 genome as described
by others.22-23 The calcium phosphate
precipitation method was used for DNA transfection. Recombinant adenovirus
was isolated from a single plaque, expanded in the 293 cells, and purified
by double cesium gradient ultracentrifugation as described by others.24 The titer of purified adenovirus was determined by
means of a spectrophotometer at 260 nm and by plaque assays.
RESULTS
EXPRESSION OF dCK IN SCC-25 CELLS
Cells from the SCC-25 squamous cell carcinoma cell line were transduced
by the pMV-dCK or control pMV-7 with the use of viral supernatant from the
respective PA317 producer cell lines. Polyclonal selection for stable vector
integration was performed by adding geneticin sulfate (Gibco BRL) to the culture
medium. Northern analysis confirmed that the parental SCC-25 cells (SCC-25/WT)
and SCC-25/Neo cells (cells transduced by the control pMV7 vector) did not
express dCK mRNA (Figure 1). In
contrast, SCC-25 cells transduced by pMV7-dCK (SCC-25/dCK) expressed high
levels of the dCK transcript. The neomycin 3'-phosphotransferase gene
was expressed in both the SCC-25/Neo and SCC-25/dCK clones (Figure 1). Since transduction of dCK did not affect the cell growth,
dCK itself was considered not toxic to the cells.
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Figure 1. Northern blot analysis demonstrating
the expression of deoxycytidine kinase (dCK) messenger RNA in SCC-25 cells
after retrovirally mediated gene transfer. The full-length dCK was used to
probe for expression of dCK messenger RNA (top). The neomycin 3'-phosphotransferase
(neoR) complementary DNA was used as a control to show expression
of neoR messenger RNA in both SCC/Neo and SCC-25/dCK cells (middle).
Hybridization to the ß-actin probe demonstrates equal loading of the
lanes (bottom). The neo gene is transcribed as a
full-length genomic length (5'-LTR to 3'-LTR) message in the pMV7
vectors, which explains why the transcript in the SCC-25/dCK cells is larger
than the transcript in the SCC-25/Neo cells.
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CYTARABINE SENSITIVITY OF TRANSFECTED CELLS
To determine the sensitivity of SCC-25/WT, SCC-25/Neo, and SSC-25/dCK
cells to cytarabine, cytotoxicity curves were generated after 96 hours of
drug exposure (Figure 2). After
a 96-hour exposure, the concentration of cytarabine that resulted in 50% cytotoxicity
(IC50) for the SCC-25/dCK, SCC-25/WT, and SCC-25/Neo cells was
5.0nM, 100nM, and 120nM, respectively (P<.001)
(Figure 2). These findings indicate
that transduction of dCK increases the sensitivity of cells to cytarabine.
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Figure 2. Cytotoxic assay showing the sensitivity
of SCC-25 cells to cytarabine after retrovirally mediated transduction of
the deoxycytidine kinase (DCK) gene. Cells were exposed
to the indicated concentrations of cytarabine for 96 hours. Cytotoxicity was
determined by fixation, staining with methylene blue, and measurement of the
absorbance at 595 nm. The symbols used in this figure are parental SCC-25
(SCC-25/WT) (squares), SCC-25/Neo (triangles), and SCC-25/dCK (circles) cells,
respectively. The results are expressed as the mean of 8 experiments; bars
show the SDs. The SCC-25/dCK cells were significantly more sensitive to cytarabine
than were either the SCC-25/WT or SCC-25/Neo cells (P<.001;t test). ß-Gal indicates ß-galactosidase; MOI,
multiplicity of infection.
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GENE TRANSFER EXPERIMENTS USING AN ADENOVIRAL VECTOR SYSTEM
On the basis of our data demonstrating that DCK
gene expression enhanced cytarabine sensitivity to SCC-25 cells in vitro,
we hypothesized that DCK might be an effective chemosensitization
gene for gene therapy. Accordingly, we constructed a replication-deficient
recombinant adenovirus carrying the CMV promoter–CK gene minicassette
(Ad.CMV-dCK). To test the activity of this vector, SCC-25/WT cells were transduced
with either Ad.CMV-ßgal or Ad.CMV-dCK in vitro. Forty-eight hours later,
the total RNA was harvested and analyzed for expression of the transgene.
The SCC-25 cells transduced with Ad.CMV-dCK expressed high levels of dCK messenger
RNA. In contrast, dCK expression was undetectable in Ad.CMV-ßgal–infected
cells (Figure 3A). The level of
dCK expression was dependent on the multiplicity of infection (MOI) (Figure 3B).
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Figure 3. Northern blot analysis of deoxycytidine
kinase (dCK) expression in SCC-25 cells transduced with Ad.CMV-dCK. A, Forty-eight
hours after transduction (multiplicity of infection [MOI] = 20), cells were
harvested, and the total RNA (20 µg) was analyzed for the expression
of transgenes. B, SCC-25 cells were transduced at the indicated MOIs to assess
the effect of the viral titer on the level of gene expression. Hybridization
to the ß-actin probe demonstrated equal loading of the lanes.
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Cytotoxic assays were performed to determine whether SCC-25 transduction
by Ad.CMV-dCK confers cytarabine sensitivity. Twenty-four hours after transduction,
SCC-25 cells were exposed to various concentrations of cytarabine for 96 hours.
The SCC-25 cells transduced by Ad.CMV-ßgal at different MOIs (MOI = 0,
20) showed no difference in sensitivity to cytarabine (Figure 4). In contrast, SCC-25 cells transduced by Ad.CMV-dCK exhibited
a huge increase in sensitivity to cytarabine (MOI = 20). The IC50
values for these cell lines were 100nM (Ad.CMV-ßgal, MOI = 0), 130nM
(Ad.CMV-ßgal, MOI = 20), and 0.23nM (Ad.CMV-dCK, MOI = 20) (Figure 4). We obtained similar results with
2 different tongue squamous cell carcinoma cell lines (SCC-4, KOSC-2 Cl3-43;
Human Science Research Resources Bank, Osaka, Japan). The IC50
values for these cell lines were 120nM (SCC4: Ad.CMV-ßgal, MOI = 0),
100nM (SCC-4: Ad.CMV-ßgal, MOI = 20), and 2nM (SCC-4: Ad.CMV-dCK, MOI
= 20) and 200nM (KOSC-2 Cl3-43: Ad.CMV-ßgal, MOI = 0), 240nM (KOSC-2
Cl3-43: Ad.CMV-ßgal, MOI = 20), and 3nM (KOSC-2 Cl3-43: Ad.CMV-dCK, MOI
= 20).
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Figure 4. Cytotoxic assay showing the sensitivity
of SCC-25 cells to cytarabine after adenoviral vector transduction. SCC-25
cells were infected at various multiplicities of infection (MOIs) of recombinant
adenovirus (MOI = 0, viral vehicle only [squares]; Ad.CMV-ßgal, MOI =
20 [triangles], or Ad.CMV-dCK, MOI = 20 [circles]). After 24 hours, cells
were exposed to the indicated concentrations of cytarabine for 96 hours. Cytotoxicity
was determined by staining with methylene blue. The results are expressed
as the mean of 8 experiments; bars show the SDs. The difference in cell killing
was highly statistically significant for the cells treated with Ad.CMV-dCK
at MOI of 20 (P<.001). ß-Gal indicates ß-galactosidase.
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COMMENT
The standard therapy for HNSCC is a combination of radiotherapy and
surgery, while chemotherapy is selected for recurrent or inoperable cases.
A representative chemotherapy is a combination of cisplatin and fluorouracil.
However, unlike hematologic malignancies, HNSCC is not very sensitive to these
chemotherapies, and the development of new therapeutic agents has been hoped
for. Accordingly, it would be very advantageous for the treatment of HNSCC
if some innovation made it possible to effectively use antineoplastic agents
whose safety and antitumor efficacy have already been confirmed in relation
to hematologic malignancies.
Transduction of genes that sensitize tumor cells to prodrugs represents
a promising strategy for cancer therapy. The following systems are known to
increase the chemical sensitivity: HSVtk in combination with ganciclovir as
a prodrug, the combination of cytosine deaminase-flucytosine, human platelet-derived
endothelial cell growth factor (PD-ECGF)–floxuridine, PD-ECGF–tegafur,
and PD-ECGF/fluorouracil.25-27
Although there are a few reports about HSVtk-ganciclovir therapy of head and
neck cancers, no other systems have been studied.1
It is known that cytarabine manifests its antitumor effect by combining
with DNA during replication and then destroying the DNA chain. However, cytarabine
does not have any effect on most solid tumors, although it shows a powerful
effect against hematologic malignancies. Cytarabine penetrates cells by a
carrier-mediated process using a nucleoside transporter that binds nitrobenzylthioinosine.28 Cytarabine is converted to its active form via phosphorylation
by 3 successive enzymes, consisting of dCK, deoxycytidylate kinase, and nucleoside
diphosphate kinase.12, 29 The cytotoxic
effects of cytarabine can be abrogated by blocking this incorporation of cytidine
triphosphate into DNA. Alternately, cytarabine can be metabolized to inactive
intermediates directly through the inactivation of cytidine monophosphate
by deoxycytidylate deaminase. Thus, cytarabine activation depends on a relative
balance between activating and degradative enzymes. The phosphorylation of
cytarabine by dCK seems to be a rate-limiting step of the activation of cytarabine.30 Unlike hematologic malignancies, many solid cancers
show little intracellular expression of dCK, and their sensitivity to cytarabine
seems to be low for this reason. Moreover, as the reason that cytarabine does
not show activity against solid tumors, it has been suggested that these tumors
differ from hematologic malignancies in terms of their cell cycle or hemodynamics.31-32
We therefore hypothesized that cytarabine metabolism might be shifted
from intracellular deamination toward phosphorylation and activation after
overexpression of dCK. That is, we surmised that the sensitivity to cytarabine
might be improved to the level of that shown by hematologic malignancies by
introducing the DCK gene into the HNSCC cells. Our
previous study had shown that transduction of brain tumor (glioma) cells by
retroviral and adenoviral vectors expressing the DCK
gene increased the sensitivity of these cells to the cytotoxic effects of
cytarabine in vitro and in vivo.17
Cancer cells of the central nervous system show low activity of cytidine
deaminase, an enzyme that degrades cytarabine, resulting in a longer half-life
of cytarabine in those cells. Moreover, cytarabine can be administered into
the medullary cavity. For these and other reasons, cytarabine has proved to
be comparatively easy to use in the treatment of brain tumors. However, cell
lines of other types of solid tumors show strong expression of cytidine deaminase,
reported to be 100 to 1000 times greater than that in the cells of hematologic
malignancies.33 The results of the present
study clearly demonstrated that it is possible to intensify the antitumor
effect of cytarabine even in relation to HNSCC by transducing the DCK gene into HNSCC cells.
The dCK-cytarabine system has potential advantages for cancer gene therapy
for 4 reasons. First, cytarabine has been used for many years, as a result
of which its pharmacokinetics have been extensively studied and a high-dose
regimen has been established. Moreover, the adverse reactions caused by cytarabine,
of which bone marrow suppression is representative, have been thoroughly investigated,
and thus the level of safety of this drug is also very high.
Second, an immune response is induced when foreign genes are introduced
in vivo by means of an adenovirus, and it is said that the targets of cytotoxic
T lymphocytes are the proteins that are produced by the transduced foreign
genes.34 However, unlike HSVtk and other currently
used chemosensitization genes such as cytosine deaminase, DCK is a human gene and thus limits the likelihood of a significant
anti-dCK immunologic response in humans.
Third, in HNSCC the cause of death is almost always local recurrence,
while cases said to be due to distant metastasis are rare. Accordingly, for
unresectable cases and cases with local recurrence, administration of an adenoviral
vector directly into the tumor can be expected to result in efficacy of the
transduced genes. In addition, such direct administration of an adenoviral
vector into a tumor is easy to accomplish, and the level of expression of
the adenovirus in the tumor is thought to be high. It is also thought that
an immune response against the adenovirus is less likely to occur in intratumoral
administration of the adenoviral vector than in intravenous administration.
Fourth, the usual clinical dosage of cytarabine is approximately 20
mg/kg per day, which results in a plasma drug concentration in the range of
101nM to 102nM. In the present study, the cytarabine
IC50 for the SCC25 cells transfected with the DCK gene by means of adenoviral infection was 2.3 x 10-1nM, which is much lower than the stated plasma drug concentration range
achieved in actual clinical therapy.
For these reasons, although further studies including in vivo experiments
will be required, we believe that the dCK-cytarabine system may be a useful
approach for gene therapy of HNSCC, as well as possibly other solid tumors.
AUTHOR INFORMATION
Accepted for publication October 26, 2001.
Corresponding author and reprints: Hiromi Kojima, MD, Department
of Otorhinolaryngology, Jikei University School of Medicine, 3-25-8 Nishishinbashi
Minato-ku, 105-8461 Tokyo, Japan.
From the Departments of Otorhinolaryngology (Drs Kojima, Iida, Miyazaki,
and Moriyama) and Microbiology (Dr Manome), Jikei University School of Medicine,
Tokyo, Japan, and the Division of Clinical Laboratory, Chiba Social Insurance
Hospital, Chiba, Japan (Dr Koga).
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