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Experimental Therapeutics, Preclinical Pharmacology |
Is Associated with Up-Regulation of p27Kip1 in Human Hepatocellular Carcinoma Cells1
Department of Surgery II, Osaka University Medical School, Osaka 565-0871, Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in colon cancer,
hepatocellular carcinoma (HCC), and other malignancies. In our
preliminary clinical studies, we have observed outstanding effects with
this combination therapy in patients with advanced HCC. However, the
underlying mechanism by which IFN- modulates the effects of 5-FU is
unknown. We, therefore, conducted a mechanistic study using two HCC
cell lines, PLC/PRF/5 and HuH7. IFN- significantly enhanced the
growth inhibitory effect of 5-FU in PLC/PRF/5 cells but not in HuH7
cells, and the isobolographic analysis indicated that this effect was
synergistic. Flow cytometric analysis showed a delay in the progression
of G0-G1 to S phase in PLC/PRF/5, and a
sustained induction of the cyclin-dependent kinase inhibitor
p27Kip1 and down-regulation of cyclin D1 was observed.
Moreover, increased expression of p27Kip1 was associated
with reduced CDK-2-associated kinase activity. Another difference in
the two cell types was that PLC/PRF/5 expressed abundant IFN receptors,
but HuH7 did not. Apoptosis assays were not helpful in explaining the
mechanism. Our results suggest that the synergistic effects of 5-FU and
IFN- may in part be attributable to alterations in cell cycle
progression via up-regulation of p27Kip1. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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30 per 100,000 men per year (1)
.
Despite various forms of therapies, including hepatectomy,
transarterial embolization, and percutaneous ethanol injection therapy,
many patients show disease recurrence and finally progress to the
advanced stages with vascular invasion and multiple intrahepatic
metastases. The prognosis for HCC is generally poor, and the 5-year
survival rate is limited to 25–58% after surgery (2
, 3)
.
Chemotherapy is commonly used for the treatment of human malignancies.
Although a single chemotherapeutic agent is usually ineffective in
suppressing HCC, previous clinical trials suggested that combination of
several chemotherapeutic agents may be effective against advanced HCC.
For example, combination therapies such as
5-FU/IFN-/cisplatin/methotrexate or
5-FU/IFN-/cisplatin/doxorubicin were useful for patients with
advanced, unresectable HCC (4
, 5)
. It has also been
demonstrated that the regimen of 5-FU and IFN- induces an enduring
partial response in 30% of patients with HCC who had low levels of
serum -fetoprotein (6)
.
Combination therapy with 5-FU and IFN- was initially proposed by
Wadler et al. (7)
in 1988, using colon cancer
cell lines. Subsequently, this combination therapy was applied to
various types of human malignancies. In patients with colorectal
cancer, esophageal cancer, gastric cancer, or HCC, satisfactory results
were obtained (6
, 8, 9, 10, 11, 12)
, but contrary data were also
reported in the studies for colorectal cancer, gastric cancer,
pancreatic cancer, and HCC (13, 14, 15, 16)
. In our department,
prospective clinical trials have been in progress using 5-FU and
IFN- in patients with inoperable and extremely advanced HCC.
Although these studies should continue for several more years,
we have thus far obtained satisfactory results with this protocol.
Several in vitro studies have provided some explanations
about the cooperative effects obtained with combination of 5-FU and
IFN-. The evidence includes reduced 5-FU clearance and alteration of
5-FU metabolism by IFN-; increase in the amount of FdUMP that can
bind to thymidylate synthetase, resulting in inhibition of conversion
from dUMP to dTMP during normal DNA synthesis (17
, 18)
. It
appears that IFN- reduces the uptake of thymidine and the activity
of thymidine kinase in conjunction with the action of 5-FU. Moreover,
it is suggested that cooperative effects may be exerted through
immunomodulatory actions (19)
. However, there are only a
few studies that have examined the effects of 5-FU and IFN- on
fundamental cell biology, such as the cell cycle and apoptosis
(20
, 21)
.
Several clinical trials have demonstrated the considerable
effectiveness of 5-FU combined with IFN- in patients with advanced
HCC (4, 5, 6
, 22)
, but it has not yet been clarified how
IFN- modulates antitumor activity of 5-FU in HCC cells. In the
present study, we examined several aspects of cell biology, including
cell proliferation, apoptosis, and changes in the cell cycle, in HCC
cell lines PLC/PRF/5 and HuH7, during treatment with 5-FU and IFN-.
In addition, expression of the IFN receptor was examined. Because
IFN- clearly enhanced the growth-inhibitory effect of 5-FU in
PLC/PRF/5 cells but not in HuH7 cells, comparison of several
forementioned features may provide evidence associated with the
underlying mechanism of this combination therapy.
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MATERIALS AND METHODS |
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was obtained from Otsuka Pharmaceutical Co.
(Tokyo, Japan), and 5-FU was obtained from Kyowa Hakko Co. (Tokyo,
Japan). The two human HCC cell lines, PLC/PRF/5 and HuH7, were
purchased from the Japanese Cancer Research Resources Bank. They were
maintained in DMEM supplemented with 10% FBS at 37°C in a humidified
incubator with 5% CO2 in air.
Growth Inhibitory Assays with 5-FU and IFN-.
Cells (3 x 103) per well were added in
triplicate to a 96-well microplate, and 24 h later, the medium was
replaced by 0.1 ml of fresh medium containing various concentrations of
5-FU and/or IFN-. Concentrations of 5-FU tested were 0.05, 0.5 and 5
µg/ml, and those of IFN- were 50 and 500 units/ml. Tumor cells
suspended in complete medium were used as a control for cell viability.
The medium was changed every 48 h, and 4 days after the addition
of 5-FU and/or IFN-, the numbers of viable cells were assessed by
MTT (Sigma Co, St. Louis, MO) assay. Briefly, 10 µl (50 µg) of MTT
were added to each well. The plate was incubated for 4 h at
37°C. Unreacted MTT was then removed, leaving the resultant formazan
crystals at the bottom of the well. Then, 0.1 ml of 2-propanol was
added to each well to dissolve the crystal. The absorbance of the plate
was measured in a microplate reader at a wavelength of 570 nm. These
assays were repeated, and similar results were obtained. Also in other
parts of the present study, experiments were repeated at least twice,
and no discrepant results were obtained.
Growth Curves.
Cells were uniformly seeded (5 x 103/well
for PLC/PRF/5 and 2 x 103/well for HuH7) in
triplicates into 24-well dishes. Twenty-four h later (day 0), the
culture medium was removed and replaced by 1 ml of fresh medium with or
without 0.5 µg/ml 5-FU and 500 units/ml IFN-. Medium was changed
every 48 h, and on days 2, 4, 6, 8, and 10, viable cells were
counted using a hemocytometer by trypan blue dye exclusion. The
doubling time was calculated using growth curves between days 4 and 8,
as described previously (23)
.
Cell Cycle Analysis.
Flow cytometric analysis was performed, as described previously
(23)
. Briefly, cells were washed twice with PBS and then
fixed in 70% cold ethanol for 4 h before being washed and
resuspended in 1 ml of PBS. Propidium iodide (Sigma; 50 µl of 1 mg/ml
solution in PBS) and RNase (Sigma; 1 µl of 10 mg/ml solution) were
added for 30 min at 37°C. Samples were filtered through 44 µm nylon
mesh, and data were acquired with a FACSort (Becton Dickinson
Immunocytometry Systems, San Jose, CA). Analysis of the cell cycle was
carried out using ModFIT software (Becton Dickinson Immunocytometry
Systems).
Western Blot Analysis.
Cells were washed twice with ice-cold PBS and collected with a rubber
scraper. After centrifugation, the cell pellets were resuspended in
lysis buffer [50 mM HEPES, 150 mM NaCl, 2.5
mM EGTA, 1.0 mM EDTA, 1.0 mM DTT,
0.1% Tween 20, 10% glycerol, and protease inhibitors (10 µg/ml
leupeptin, 10 µg/ml aprotinin, 1.0 mM
phenylmethylsulfonyl fluoride; pH 7.5)]. After sonication, the
extracts were clarified at 15,000 x g for 5 min at
4°C, and the supernatant fraction was collected. Western blot
analysis was performed, as described previously (23)
.
Antibodies.
The following rabbit polyclonal Abs were used at appropriate
concentrations as recommended by the manufacturer:
anti-p27Kip1 Ab (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA); anti-p21waf1/cip1 Ab (Santa
Cruz); anti-human IFN receptor /ß Ab (Santa Cruz Biotechnology;
Refs. 24
and 25
); anti-cyclin D1 Ab (Upstate
Biotechnology Inc., Lake Placid, NY); anti-CDK2 Ab (UBI); and
anti-human cyclin E Ab (Upstate Biotechnology Inc.).
In Vitro Assay for CDK2-associated Activity.
The in vitro CDK2-associated kinase assay was performed as
described previously (26)
. Cells were collected and
sonicated in kinase buffer (lysis buffer plus 10
mM ß-glycerophosphate, 1
mM NaF, and 0.1 mM sodium
orthovanadate). After centrifugation, the supernatant fraction was
collected. Immunoprecipitation with 1 µg of CDK2 polyclonal Ab
(Upstate Biotechnology Inc.) was performed using protein A-Sepharose
beads (Sigma), followed by washing of the beads four times with kinase
buffer and twice with reaction buffer [50 mM
HEPES (pH 7.5), 10 mM
MgCl2, 1 mM DTT, 2.5
mM EGTA, 10 mM
ß-glycerophosphate, 1 mM NaF, and 0.1
mM sodium orthovanadate]. The final pellets were
resuspended in 45 µl of reaction buffer containing 2 µg of histone
H1 (Sigma) and 5 µCi of [
-32P]ATP and
incubated for 30 min at 30°C. The reaction mixture was then subjected
to SDS-PAGE, and the intensity of phosphorylation of the histone H1
substrate was determined by autoradiography.
Detection of Apoptosis.
To detect in situ apoptosis, we used the TUNEL
(3)
method with the Apop Tag kit (Oncor S7100,
Gaithersburg, MD) as described previously (26)
. This
method can detect fragmented DNA ends of apoptotic cells. Cells (5 x 105/well) were uniformly seeded into 10-cm
diameter dishes and cultured for 24 h. The medium was removed and
replaced by 10 ml of fresh medium with or without 0.5 µg/ml 5-FU and
500 units/ml IFN-. Cells free in the medium and attached to the dish
were harvested 72 h after the addition of 5-FU or IFN- and then
fixed with 10% buffered formalin for detection of apoptotic cells.
Terminal deoxynucleotidyl transferase was omitted from the nucleotide
mixture for the negative control. As a positive control, we used
paraffin-embedded sections of 10% buffered, formalin-fixed rodent
mammary glands. For quantification of apoptotic cells, 10 microscopic
fields were randomly selected at xx25, >700 total cells were counted
in each sample, and the percentage of apoptotic cells was calculated.
Statistical Analysis.
Data are expressed as mean ± SE. Differences between groups were
examined for statistical significance using Mann-Whitney’s
U test or 
2 test.
Evaluation of Cooperative Effects.
Calculations of synergistic cytotoxicity were determined by
isobolographic analysis as described by Berenbaum (27
, 28)
. The nature of the effect of a particular dose combination
was determined by isobolographic analysis, as follows. The point
representing that combination would lie on, below, or above the
straight line joining the doses of the two drugs that, when given
alone, produced the same effect as that combination, representing
additive, synergistic, or antagonistic effects, respectively.
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RESULTS |
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/ß receptor Ab, PLC/PRF/5 displayed prominent
bands at Mr 40,000 (soluble
form), Mr 51,000 (short form), and
Mr 102,000 (long form), which were
about 7.7-, 2.8-, and 2.4-fold more intense, respectively, than the
corresponding bands expressed in HuH7 (Fig. 1)
. Nonspecific, cross-reactive bands
(Mr 140,000 and
Mr 80,000) appeared at similar
densities between the two cell lines and served as loading controls.
Coomassie blue staining of the gel also confirmed equal loading of the
total protein (data not shown).
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have cooperative effects on
inhibition growth of HCC cells, growth-inhibitory assays were
performed. Cells were exposed to 5-FU and/or IFN- for 4 days at
various concentrations. 5-FU alone (Fig. 2A
), 0.5 µg/ml 5-FU
inhibited cell growth by 23.2 ± 0.06% in PLC/PRF/5 (left
panel) and 24.0 ± 0.05% in HuH7 (right panel).
At 5 µg/ml, 5-FU reduced cell growth to 49.2 ± 0.03% and
49.2 ± 0.08% of control cells, in PLC/PRF/5 and HuH7,
respectively. No growth-inhibitory effect was observed in cells with
IFN- alone at 50 or 500 units/ml. When 5-FU and IFN- were
administered simultaneously at various concentrations, significant
cooperative effects were observed at 0.5 µg/ml 5-FU plus 50 or 500
units/ml IFN- in the PLC/PRF/5 cell line (P = 0.010
and 0.046, respectively), and the isobolographic analysis indicated
that these cooperative effects were synergistic (Fig. 2B
).
However, these effects were not observed with 5 µg/ml 5-FU plus
IFN-. In HuH7 cells, the growth-inhibitory effects induced by 0.5 or
5 µg/ml 5-FU were not affected by the addition of IFN-.
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. 5-FU (0.5 µg/ml) and IFN- (500
units/ml) were selected for treatment, because under these conditions
IFN- enhanced the growth-inhibitory effects of 5-FU in the PLC/PRF/5
cell line. In PLC/PRF/5 cells, the doubling time was 41.8 ± 2.5,
54.3 ± 5.9, 72.2 ± 14.2, and 144.8 ± 34.6 h for
cultures of control, IFN-, 5-FU, and 5-FU plus IFN-,
respectively. The difference between the latter two groups was
significant (P = 0.021). In HuH7 cells, the doubling
time was 29.4 ± 4.0, 35.6 ± 3.7, 107.2 ± 6.7, and
145.5 ± 8.9 h for each treatment, and no significant
difference was found between 5-FU alone and 5-FU plus IFN-. A
significant difference was also observed in cell numbers on day 10
between the 5-FU group and combination group in PLC/PRF/5 cells
(P = 0.020) but not in HuH7 cells.
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. These studies were performed in
cultures that were grown previously in serum-free medium for 72 h
to arrest the cells in
G0-G1, because after
re-addition of 10% FBS, the cells would synchronously progress through
the cell cycle. Cells were then collected 6, 12, 24, 48, 72, and
96 h later. Flow cytometric data confirmed that after serum
starvation, the majority of cells (PLC/PRF/5, 69.0%; HuH7, 74.0%)
were in G0-G1. After
refeeding the serum-starved cultures of PLC/PRF/5 with 10% FBS minus
drugs (control cultures), cells remained in
G0-G1 until 12 h and
then progressed into the S (34%) and G2-M (34%)
phases within 24 h (Fig. 4B
),
and one full cell cycle was completed at 48 h (Fig. 4A
). In cultures refed with serum plus 5-FU, the
distribution of the cell cycle was similar until 12 h, compared
with control cultures. At 24 h, the cells progressed into S phase,
but there was an appreciable number of cells with S-phase DNA content
(48%; Fig. 4B
) with a right-sided downward slope (Fig. 4A
), perhaps because of interruption of the normal process
of DNA synthesis. In turn, this caused a delay in progression to
G2-M phase (15%). The combination of 5-FU and
IFN- apparently delayed the cell cycle. At 24 h, there was
still a marked accumulation of cells in
G0-G1 phase (63%),
although at the same time point the percentage in
G0-G1 phase decreased to
approximately one-half in the other groups (Fig. 4B
). There
was a gradual increase in the S-phase fraction from 48 to 72 h
(Fig. 4B
), but even by 72 h the cell cycle had not
returned to the initial distribution pattern and remained significantly
different from control or 5-FU-treated cultures (Fig. 4A
).
In cultures refed with serum plus IFN-, the percentages of cells in
each phase were not largely different from those of the control
cultures (Fig. 4B
).
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on cell cycle progression was similar to that observed in PLC/PRF/5
(Fig. 4C
). In cultures subjected to combination therapy,
when compared with cultures of 5-FU alone, there was no difference in
cell cycle distribution at 24 h, although there was a slight
increase in S-phase fraction (42%) at 48 h. At 72 h, unlike
PLC/PRF/5, the distribution of the cell cycle was not different from
that of other groups.
Detection of Cell Cycle Regulatory Proteins.
The above flow cytometric analyses indicated that PLC/PRF/5 cells
displayed an apparent delay in G1-S transition
between 5-FU and combination treatments. Therefore, we examined the
expression of several cell cycle regulatory proteins involved in
G1-S transition, including G1 cyclins (D and E
type) and the CDK inhibitors p21waf1/cip1 and
p27Kip1. Among these molecules examined, the
levels of p27Kip1 and cyclin D1 changed as the
cell cycle progressed. In control cultures of PLC/PRF/5, expression of
p27Kip1 diminished with time and was lowest at
24 h (Fig. 5A
, left
panel). In the 5-FU or IFN- group,
p27Kip1 decreased at 24 h and remained
relatively low at 48 h. By contrast, p27Kip1
expression in the combination therapy group remained high until 24 h, and an appreciable amount of p27Kip1 was still
detectable at 48 h. Densitometric analysis indicated that the
densities of the band of combination group was almost 3.6-fold higher
than that of the 5-FU group. In HuH7 cells,
p27Kip1 expression was high at 6 h, and the
level did not vary much with time in the control cultures, nor was any
difference induced by the drugs (Fig. 5A
, right panel).
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, left
panel). When drugs were combined, cyclin D1 expression
markedly decreased at 48 h. In HuH7 cells, cyclin D1 level was
relatively low at 6 h, and at subsequent time points it was
slightly increased in the control and IFN- groups. By contrast, it
decreased at 24 and 48 h in the 5-FU and combination groups (Fig. 5B
, right panel). The expression of cyclin E and
p21waf1/cip1 in either cell line was uniform,
irrespective of the drug used and time course (data not shown).
In Vitro Assay for CDK2-associated Activity.
To investigate whether high expression of p27Kip1
seen in PLC/PRF/5 was functional, an in vitro kinase assay
was performed, using cell extracts that were harvested 24 h after
the addition of the drug. Because the major function of
p27Kip1 is to bind the cyclin E/CDK2 complex and
inhibit its kinase activity, we assayed the CDK2 immunoprecipitates for
in vitro CDK2-associated kinase activity using histone H1 as
the substrate. Cell extracts prepared from control, 5-FU-, and
IFN--treated cultures yielded strong phosphorylation of histone H1
(Fig. 6
, Lanes 2–4). By
contrast, extracts from cultures treated with 5-FU plus IFN-
displayed an 3.2-fold decrease in kinase activity when compared with
5-FU alone (Fig. 6
, Lane 5). When the extract from the
control culture was immunoprecipitated with nonimmune rabbit IgG, no
kinase activity was detected (Fig. 6
, Lane 1).
|
. The percentages of apoptosis in
PLC/PRF/5 were 1.3 ± 0.1, 4.3 ± 0.3, 6.1 ± 0.5, and
6.6 ± 0.7%, in the control, 5-FU, IFN-, and drug combination
group, respectively. On the other hand, the frequency of apoptosis was
low in HuH7 at 0.6 ± 0.2, 0.7 ± 0.1, 0.9 ± 0.1, and
0.9 ± 0.1%, for each respective group.
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, and combination groups,
respectively. For HuH7, sub-G1 proportions for
each group after 72 h were 8.6, 8.7, 8.4, and 9.5%. |
DISCUSSION |
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may enhance the
tumor-inhibitory effect of 5-FU. In this study, we used two hepatoma
cell lines that were both sensitive to 5-FU but insensitive to IFN-
alone. Differences between the cell lines emerged in their responses to
the 5-FU and IFN- combination. When combined with 0.5 µg/ml 5-FU,
IFN- induced growth-inhibitory effects in a dose-dependent manner in
PLC/PRF/5. Thus, within the range, 5-FU and IFN- had synergistic
effects on cell growth, as confirmed with the isobolographic analysis.
Importantly, the concentration of 5-FU used in our study (0.5 µg/ml)
was almost the same level as that in plasma of patients when the drug
is clinically administrated by continuous infusion (29)
.
Conversely, no cooperative effects were seen in HuH7 cells. The
synergistic effects in PLC/PRF/5 were apparent, although the difference
was not so dramatic (Fig. 2A
). This may be because 4 days of
culture was too short of a time period. Indeed, the difference in
proliferation between the two cell types was more obvious when the cell
culture was extended up to 10 days (Fig. 3)
. It is also possible that
the difference in sensitivity to 5-FU might play some role in the
cooperative effects; the doubling times in the 5-FU group were 1.7-fold
and 3.7-fold longer than the control group in PLC/PRF/5 and in HuH7
cells, respectively. Therefore, 5-FU might be enough to suppress HuH7,
which cannot be exceeded by the addition of IFN-.
Analysis of cell cycle progression provided a clue to the underlying
mechanism of the synergistic effects seen in PLC/PRF/5. A marked delay
in cell cycle progression was found at 24 h, with
G0-G1 accumulation in the
combination therapy group in PLC/PRF/5 (Fig. 4B
). However,
in the HuH7 cell line, the DNA content pattern at the same time point
was similar between combination group and 5-FU groups. The difference
between the two cell lines could be explained by induction of
p27Kip1 (Fig. 5A
). PLC/PRF/5 cells
exhibited an 3.5-fold rise in p27Kip1
expression in the presence of both 5-FU and IFN- at 24 h
compared with treatment with 5-FU alone, whereas in HuH7 cells, no
significant difference in p27Kip1 expression was
observed between the two treatments. Furthermore, a CDK2-associated
kinase assay showed considerably lower kinase activity in
p27Kip1-overexpressing extracts of PLC/PRF/5. It
is possible that the levels of CDK2 or cyclin E and another CDK
inhibitor, p21waf1/cip1, may affect the extent of
activity of CDK2-associated kinase (23)
, but the levels of
these proteins did not change by drug treatment in either cell line
(data not shown). These findings suggest that
p27Kip1 induced by 5-FU and IFN- in PLC/PRF/5
is functional and may contribute to the delay in progression of the
cell cycle from G1 to S phase.
It is of interest that p27Kip1 may play a crucial role in chemotherapy against HCC, because recent studies have highlighted the relevance of p27Kip1 in the progression of various types of human malignancy (30 , 31) . We showed previously that expression of p27Kip1 was reduced in subsets of colon cancers (32) , and we have found recently that loss of p27Kip1 is associated with shorter disease-free survival in HCC (33) . The present data are consistent with those of the latter report. Thus, it seems that low expression of p27Kip1 favors sound growth and expansion of HCC cells, and conversely, up-regulated p27Kip1 may effectively function to suppress HCC.
A linkage between IFN- and inhibition of the cell cycle has been
examined. IFN- induced
G0-G1 phase accumulation in
NIH-3T3 murine fibroblasts, human Burkitt’s lymphoma Daudi, and the
lymphoid cell line U-266 (34
, 35)
. It was also reported
that treatment of Daudi cells with IFN- inhibited the activity of
CDK2 but not CDC2 (36)
, suggesting that a decrease in CDK2
kinase may be responsible for the
G0-G1 arrest. By contrast,
in the present study, we did not observe a significant increase in the
G0-G1 population nor
p27Kip1 induction with treatment of IFN-
alone. However, p27Kip1 was strongly induced by
simultaneous administration of IFN- and basal treatment with 5-FU in
PLC/PRF/5. Therefore, it is likely that the susceptibility of the cells
to IFN- in inducing p27Kip1 may differ among
cell types, and that 5-FU may aid IFN--mediated
p27Kip1 induction, which leads to reduction in
CDK2 kinase activity in at least certain HCC cell lines.
We also demonstrated the possible involvement of cyclin D1 in cell
cycle control (37)
. Decreased cyclin D1 expression was
observed in the late cell cycle (at 48 h) in the combination group
of PLC/PRF/5 cells but not in the group treated with 5-FU alone (Fig. 5B
). This might contribute to the sustained arrest in
G0-G1 at 48 h (Fig. 4B
). On the other hand, although cyclin D1 expression
decreased in the combination group in HuH7 cells at 24 and 48 h,
similar down-regulation of cyclin D1 was observed also in the group
treated with 5-FU alone (Fig. 5B
), consistent with a lack of
cooperative effect in this cell line.
Recently, it has been reported that expression of the IFN- receptor
varies among hepatoma cell lines and among patients with hepatitis C
virus-associated hepatitis (38
, 39)
. HuH7, which was
resistant to combination therapy, exhibited lower IFN receptor
expression than PLC/PRF/5. This suggests a possible relationship
between resistance to IFN- and the extent of expression of IFN
receptors. Introduction of IFN receptor cDNA into HCC cell lines may
clarify the relevance of signal transduction from the receptor to the
cytoplasmic and nuclear events involved in cell cycle control. We have
performed recently a retrospective study of IFN receptor expression in
primary HCC tumors and found that a wide range of expression exists in
these tumors (data not shown). A clinical investigation is currently
under way in our laboratory in an effort to examine the possible
correlation between the extent of expression of the IFN receptor and
the effects of combination therapy.
Apoptosis assays did not explain the synergistic effects of 5-FU and
IFN- in PLC/PRF/5 cells. The TUNEL assay indicated that although
there was an 4–5-fold increase in frequency of apoptosis in the
combination group compared with control (6.6 versus 1.3%),
IFN- alone also induced similar levels of apoptosis (6.1%). In HuH7
cells, TUNEL-positive cells were minimal (<1%), irrespective of
treatment. Our findings obtained by the TUNEL method matched those
measured by flow cytometry in PLC/PRF/5 cells. However, in HuH7, there
was a discrepancy between data obtained by the TUNEL method and flow
cytometric analysis. It is probable that the increased
sub-G1 population seen in HuH7 cells may be
attributable to debris of fragmented but not apoptotic cells, because
we observed many necrotic cells under the microscope (data not shown).
In addition to the phenomena examined in the present study, several
aspects remain to be clarified. As mentioned in the "Introduction,"
changes in the 5-FU metabolic pathway could possibly underlie the
mechanism of IFN- synergism. Indeed, we found that the activity of
thymidylate synthetase was slightly decreased without an increase in
the amount of FdUMP after treatment with 5-FU and IFN- in PLC/PRF/5
cells (data not shown). It is also possible that the antitumor effect
might be exerted via host immunomodification and changes in vascularity
in vivo (40, 41, 42)
. These aspects should be
addressed in the future. Regardless of the other possibilities, it is
apparent from the present study that IFN- modulated the progression
of the cell cycle, possibly via induction of
p27Kip1 in 5-FU-treated PLC/PRF/5 cells. Among
the complex and diverse effects of combination therapy (5-FU and
IFN-), our data provide a novel explanation with regard to the
intracellular events associated with cell cycle regulation.
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FOOTNOTES |
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1 This work was supported by a Grant-in Aid for
Cancer Research from the Ministry of Health and Welfare of Japan. 
2 To whom requests for reprints should be
addressed, at Department of Surgery II, Osaka University Medical
School, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone:
81-6-6879-3251; Fax: 81-6-6879-3259; E-mail: msakon{at}surg2.med.osaka-u.ac.jp
3 The abbreviations used are: HCC, hepatocellular
carcinoma; 5-FU, 5-fluorouracil; FBS, fetal bovine serum; MTT,
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Ab,
antibody; CDK, cyclindependent kinase; TUNEL, terminal
deoxynucleotidyl transferasemediated dUTP-biotin nick end
labeling.
Received 11/29/99; revised 4/12/00; accepted 4/12/00.
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