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Experimental Therapeutics, Preclinical Pharmacology |
Department of Oral Pathology, Hiroshima University, Faculty of Dentistry [Y. K., T. T., T. K., S. S., T. T., M. Z., M. M., H. N.], and Clinical Laboratory, Hiroshima University Dental Hospital [I. O.], Hiroshima 734-8553, Japan
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ubiquitin-mediated proteolysis is involved in the turnover of various
cell cycle-regulatory proteins, including the tumor suppressor protein
p53 and various cyclins as well as the CDK inhibitor
p27Kip1 (12, 13, 14, 15, 16)
. Accumulating
evidence suggests that the ubiquitin-proteasome pathway is a major
pathway of proteolysis in eukaryotic cells and plays important roles in
many physiological functions by the degradation of various oncoproteins
or transcriptional regulators (c-Myc, c-Fos, c-Jun, and nuclear
factor-
B/IB) and various cell cycle-regulatory proteins
(12, 13, 14, 15, 16)
. Some of these proteins are related to the
regulation of apoptosis, and it is known that ubiquitin-mediated
proteolysis is involved in the regulation of apoptosis in mammalian
cells (12
, 14)
. In proteolytic events during apoptosis,
activation of the proteolytic enzyme is a characteristic feature of
apoptosis. In particular, cystein proteases of the caspase
family are known to be as central components of proteolytic machinery
(17)
. Apoptosis induced by proteasome inhibitor is
attributable to activation of CPP32, a member of the caspase family of
cystein proteases, and appears to occur independently from
interleukin-1ß converting enzyme activity (18)
.
The proteasome inhibitors are capable of inducing apoptosis in proliferating cells but not in quiescent, differentiated cells (18, 19, 20, 21) . p53-dependent apoptosis was induced by proteasome inhibitors in mammalian cells (22 , 23) . Besides, in human leukemic HL60 cells, apoptosis induced by inhibition of proteasome-mediated proteolysis is revealed to be accompanied by an increase in the concentration of p27Kip1 (18) .
In the present study, we treated OSCC cells with LLnV or lactacystin, which are specific proteasome inhibitors, to examine the induction of apoptosis by inhibition of the proteasomal function. We also investigated the expression of various cell cycle-regulatory and apoptosis-related proteins after the treatment of proteasome inhibitors and discussed possible mechanisms of apoptosis induced by the proteasome inhibitors.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Treatment with Proteasome Inhibitor.
We used LLnV and lactacystin, which are specific proteasome inhibitors.
LLnV inhibits the chymotrypsin-like activity of the proteasome, and
lactacystin targets the catalytic ß-subunit of the proteasome. LLnV
was obtained from the Peptide Institute (Osaka, Japan). Lactacystin was
obtained from the Kyowa Medix Co. (Tokyo, Japan). The compounds were
dissolved in an amount of DMSO. LLnV and lactacystin were added
to the OSCC cells at the final concentration of 50 and 10
µM, respectively.
Electron Microscopy.
Cells were fixed in 2.5% glutaraldehyde, postfixed in 1% osmium
tetroxide, and embedded in epoxy resin. Thin sections were stained in
uranyl acetate and lead citrate and examined under a Hitachi H500H
transmission electron microscope.
Determination of DNA Fragmentation.
For qualitative analysis of DNA fragmentation, cells were harvested at
the indicated times by centrifugation and lysed by the addition of 100
µl of lysis buffer consisting of 10 mM Tris-HCl (pH 7.4),
10 mM EDTA, and 0.1% of Triton X-100. They were incubated
by addition of RNase A and proteinase K (37°C for 60 min). After
centrifugation, the soluble DNA fragments released into the supernatant
were precipitated by the addition of 0.5 volume of 7.5 M
ammonium acetate and 2.5 volumes of ethanol. DNA pellets were dissolved
in TE and loaded onto a 2.0% agarose gel and separated at 100 V
for 45 min. DNA fragments were visualized after staining with ethidium
bromide by transillumination with UV light.
Cell Growth.
Cells were plated onto a 24-well plate (Sumilon; Sumitomo Bakelite Co.,
Tokyo, Japan) and allowed to attach for 24 h. The culture medium
was then replaced with medium that contained 50 µM LLnV
or 10 µM lactacystin. For a control, cells were incubated
in the medium containing the vehicle, 50 µM DMSO.
Trypsinized cells were counted by cell counter (Coulter Z1, Coulter,
FL) at 6, 12, and 24 h after treatment. Viable cells, as detected
by the trypan blue dye exclusion test, were also counted at 6, 12, and
24 h after treatment.
Flow Cytometric Analysis.
Cell cycle distribution was determined by DNA content analysis after
propidium iodide staining using Cycle Test Plus DNA reagent kit (Becton
Dickinson, San Jose, CA). Cells were cultured as described above and
fixed in 70% ethanol and stored at 4°C before analysis. Flow
cytometric determination of DNA content was analyzed by a FACScalibur
(Becton Dickinson) flow cytometer. For each sample, 20,000 events were
stored. The fractions of the cells in
G0-G1, S, and
G2-M phases were analyzed using CELLQuest, a cell
cycle analysis software.
Western Blot Analysis.
We examined the expression of cell cycle-regulatory and
apoptosis-related proteins in OSCC cell lines treated with LLnV by
Western blot analysis. The cells were lysed in a buffer containing 50
mM Tris-HCl (pH 7.4), 125 mM NaCl, 0.1% (v/v)
NP40 (Sigma Chemical Co., St. Louis, MO), 5 mM EDTA, 0.1
M NaF, 10 µg/ml leupeptin (Sigma), 0.1 µg/ml trypsin
inhibitor (Sigma), 0.1 µg/ml aprotinin (Sigma), and 50 µg/ml
phenylmethylsulfonyl fluoride (Wako, Osaka, Japan). The protein
concentration was determined by Bradford protein assay (Bio-Rad,
Richmond, CA) using BSA (Sigma) as a standard. Fifty µg of protein
were solubilized in Laemmli’s sample buffer by boiling and subjected
to 10% SDS-PAGE, followed by electroblotting onto a nitrocellulose
filter (Schleicher & Schuell, Dasse, Germany). The filter was blocked
1 h at 4°C with PBS buffer (137 mM NaCl, 8.1
mM
Na2HPO4·12H2O,
2.68 mM KCl, and 1.47 mM
KH2PO4) containing 5%
nonfat dry milk powder. Western blot analysis was performed using an
antihuman p27Kip1, cdk4, Rb, and CPP32 mouse
monoclonal antibody (Transduction Laboratories, Lexington, Kentucky),
antihuman p53 and Bcl-2 mouse monoclonal antibody (Dako, Copenhagen,
Denmark), antihuman cyclin D and p21 mouse monoclonal antibody,
antihuman cdk2, and cyclin E rabbit polyclonal antibody (Santa Cruz
Biotechnology, Santa Cruz, CA), and anti-phospho Rb (Ser-780) rabbit
polyclonal antibody (MBL, Nagoya, Japan). Primary antibodies
were added to PBS containing 5% nonfat dry milk powder and incubated
for 60 min at room temperature. Incubation with a secondary
peroxidase-coupled goat antimouse antibody was performed under the same
conditions. For detection of the immunocomplex, the ECL Western
blotting detection system (Amersham, Aylesbury, United Kingdom) was
used. Anti-actin mouse monoclonal antibody (C4; Boehringer
Mannheim, Australia) was used for normalization of Western blot
analysis.
Protein Kinase Assay.
For cdk2/cyclin E-associated kinase activity, 100 µg of the lysate
were precleared in lysis buffer with 40 µl of 1:1 slurry of protein
A-agarose (Oncogene Research Products, Cambridge, MA) for 30 min at
4°C. Samples were incubated with 1 µg of anti-cdk2 or cyclin E
rabbit polyclonal antibody (Santa Cruz Biotechnology) for 3 h at
4°C and subsequently centrifuged. The precipitates were washed three
times with lysis buffer and three times with kinase buffer [50
mM Tris-HCl (pH 7.4), 10 mM
MgCl2, and 1 mM DTT]. The
precipitates were then suspended in 35 µl of kinase buffer containing
6 µg of histone H1 (Sigma; type III-S), followed by a 5-min
preincubation at 30°C. Subsequently, 5 µl of a 60 µM
[
-32P]ATP solution (3 µCi) were added, and
the kinase reaction was carried out at 30°C for 10 min. The reaction
was stopped by adding 20 µl of 4x Laemmli’s sample buffer and
boiling. The samples were subjected to 10% SDS-PAGE, followed by
autoradiography.
CPP32/Caspase 3 Activity.
CPP32/caspase 3 activity was determined using CPP32/Caspase-3
Colorimetric Protease assay kit (MBL, Nagoya, Japan). The assay is
based on spectrophotometric detection of the chromophore pNA
after cleavage from the labeled substrate DEVD-pNA. The
pNA light emission can be quantified using a microtiter
plate reader at 405 nm.
Antisense Oligonucleotide and Cell Viability.
The oligonucleotides were purchased from Greiner Japan (Tokyo, Japan).
The antisense oligonucleotide sequence was
p27Kip1 antisense (5'-GACACTCTGACGTTTGACAT-3'),
which was complementary to the region around the initiation codon of
p27Kip1 (25
, 26)
. We used
p27Kip1 sense (5'-CTGTGAGAGTG CAAACTGTA-3')
oligonucleotides as a control. The oligonucleotides (100
µM) and lipofectin (Life Technologies, Inc., Rockville,
MD) were incubated at 37°C for 15 min. The oligonucleotide-lipofectin
mixture was diluted with serum-free medium and added to the cells,
giving a final concentration of 1.0 µM. Western blot
analysis was performed to investigate whether antisense effectively
inhibits the expression of p27Kip1 protein. After
4 days, we treated these cells with LLnV, and cell viability was
determined by trypan blue exclusion.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Apoptotic body
formation was observed at the ultrastructural level (Fig. 1E
). Oligonucleosomal laddering was also demonstrated in the
cells at 24 h after treatment with proteasome inhibitors (Fig. 1F
). These apoptotic changes induced by LLnV or lactacystin
occurred most prominently in HSC2 cells. There were no morphological
changes and laddering in the cells treated with DMSO. Apoptotic change
was not shown in most of normal oral epithelial cells and gingival
fibroblasts at 24 h after LLnV treatment (Fig. 1G
). At
24 h after LLnV treatment, cell viability was 76 and 92% in
normal oral epithelial cells and gingival fibroblasts, respectively, in
comparison with 18% in HSC2 cells.
|
. After
24 h of the LLnV treatment, >80% cells died and detached from
the culture dish. DMSO did not show inhibitory effects. We used LLnV in
the following analyses mainly because LLnV treatment brought about
apoptosis more remarkably than lactacystin.
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. The
ratios of the cell cycle populations of nontreated cells at
G0-G1, S, and
G2-M phases were about 53, 27, and 20%,
respectively. After 12 h treatment with LLnV, the ratios of the
cell cycle populations at
G0-G1, S, and
G2-M phases were about 78, 12, and 10%,
respectively. The decrease of G1 cells and
increase of apoptotic cells became prominent at 24 h after LLnV
treatment.
Accumulation of p27Kip1 Protein by Treatment with
Proteasome Inhibitor.
Proteasome inhibitors have been reported to induce apoptosis in the
p53-dependent pathway (22
, 23)
. Another report showed that
the inhibition of proteasomal activity is accompanied by accumulation
of p27Kip1 protein (18)
. Therefore,
we examined expression of p53 and p27Kip1 after
the LLnV treatment. No change of p53 protein level was observed, but
expression of p27Kip1 protein increased in all
OSCC cells (Fig. 3A
). We
compared the signal intensity of p27Kip1
expression before and after the LLnV treatment by densitometric
scanning. The relative expression levels of signal intensity at 12 h after the LLnV treatment were 7.9, 2.9, and 1.7 in HSC2, HSC3, and
Ho-1-U-1 cells, respectively. The highest level of accumulation of
p27Kip1 protein was detected in HSC2 cells.
|
). Expressions of cyclin D1 was reduced with time.
Expression of p53 was not detected before or after incubation with
LLnV. Expressions of p21, cdk2, cyclin E, and cdk4 also did not change
with the LLnV treatment (Fig. 3B
).
Because accumulation of p27Kip1 and reduced
expression of cyclin D1 were observed with the LLnV treatment, we
examined cdk2 kinase activity, cyclin E-associated kinase activity, and
expression of phospho pRb (Ser-780) to elucidate the functional effects
of LLnV on the cell cycle (Fig. 4)
. After
cell extracts were immunoprecipitated with an antibody against cdk2 or
cyclin E, cdk2 kinase activity and cyclin E-associated kinase activity
were measured by using histone H1 as a phosphorylation substrate.
Expression of phospho pRb (Ser-780) was examined by using anti-phospho
pRb (Ser-780), which reacts with only phosphorylated pRb at the site of
Ser-780. This site is phosphorylated by cdk4/cyclin D1 but not by
cdk2/cyclin E/A (27)
. LLnV treatment inhibited cdk2 kinase
activity and cyclin E-associated kinase activity in HSC2 cells. The
level of p27Kip1 in the immunoprecipitates was
determined by Western blot analysis. The amount of cyclin E-associated
p27Kip1 increased at 12 and 24 h after LLnV
treatment (Fig. 4A
). Expression of phospho pRb also
disappeared by 12 h of LLnV treatment, but pRb did not change
expression level (Fig. 4B
).
|
). CPP32/caspase 3 activity
was also measured. CPP32/caspase 3 activity increased time dependently
(Fig. 5B
). Expression of Bcl-2 was reduced time dependently
(Fig. 5A
).
|
). Accumulation of p27Kip1 protein
was also observed in HSC2 cells with p27Kip1
sense oligonucleotide treatment but not in HSC2 cells with
p27Kip1 antisense oligonucleotide treatment after
12 h of LLnV treatment (Fig. 6A
). Treatment with antisense
p27Kip1 oligonucleotide blocked the expression of
p27Kip1. After 24 h of LLnV treatment, cell
viability was 
20% in HSC2 cells and HSC2 cells with
p27Kip1 sense oligonucleotide treatment but
50% in HSC2 cells with p27Kip1 antisense
oligonucleotide treatment (Fig. 6, B and C).
Treatment with p27Kip1 antisense oligonucleotide
inhibited the apoptosis induced by LLnV both time and dose dependently
(Fig. 6, B and C
). In Fig. 6, B and C,
an unexpected protective influence of the sense
oligonucleotides was shown by 24 h treatment with 10 or 25
µM LLnV but not by 50
µM LLnV. We suggest that sense oligonucleotides
may influence to a lesser degree in proteasome inhibitor
treatment. However, sense oligonucleotides did not influence 50
µM LLnV used in all experiments of the present
study. To examine whether p27Kip1 is upstream or
downstream of CPP32/caspase 3 or Bcl-2, we examined the expression of
CPP32/caspase 3 and Bcl-2 in antisense
p27Kip1-treated cells. CPP32 expression was
detected, but Bcl-2 expression was not detected in HSC2 cells with
p27Kip1 antisense oligonucleotide treatment after
12 h of LLnV treatment (Fig. 6A
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Reduced expression of
p27Kip1 has been shown recently in various
carcinomas and suggested to be brought about by increased
proteasome-mediated degradation rather than altered gene expression
(28, 29, 30, 31, 32, 33)
. We have also found reduced expression of
p27Kip1 in OSCC (34)
. Therefore, we
assumed that inhibition of proteasome-mediated degradation by
proteasome inhibitors might induce accumulation of
p27Kip1 protein in OSCC cells. The present
results clearly demonstrated that treatment with proteasome inhibitors
induced accumulation of p27Kip1 protein after
apoptotic changes in OSCC cells. Therefore, we suggest that
accumulation of p27Kip1 protein may play an
important part in apoptosis. This hypothesis is supported by the
findings that treatment with antisense p27Kip1
oligonucleotide inhibited apoptosis induced by proteasome inhibitors in
HSC2 cells (Fig. 6)
. These findings are consistent with recent reports that overexpression of p27Kip1 protein leads to apoptosis in various cancer cell lines (35 , 36) . However, details of the mechanism of apoptosis induced by overexpression of p27Kip1 protein are still unclear.
Both cdk2 kinase activity and cyclin E-associated kinase activity were
reduced after treatment with proteasome inhibitor (Fig. 4)
.
Accumulation of p27Kip1 protein may directly
inhibit cdk2/cyclin E-associated kinase activity, which may cause cell
cycle arrest at the G1 phase. Expressions of
cyclin D1 and phospho pRb (Ser-780) were also reduced by the treatment
with proteasome inhibitor (Fig. 3B and 4).
Cyclin D1
turnover is governed by ubiquitination and proteasomal degradation,
which are positively regulated by cyclin D1 phosphorylation on
threonine 286, but it does not depend on cdk4 catalytic activity
(37)
. The reduced expression of cyclin D1 may bring about
loss of phospho pRb (Ser-780) expression, because the site of Ser-780
in phospho pRb is only phosphorylated by cdk4/cyclin D1
(27)
. We conceive that the events after LLnV treatment may
result in inhibition of cyclin D1 transcription, and effects on
proteolysis are minor. Moreover, flow cytometric analysis shows that
cell cycle population at the G1 phase increased
at 12 h after LLnV treatment. Therefore, we suggest that the
accumulation of p27Kip1 protein and the reduction
of cyclin D1 protein by treatment with proteasome inhibitor brought
about cell cycle arrest at the G1 phase.
Human T-cell leukemia cells, proliferating fibroblastic cells, and
pheochromocytoma cells, which have a wild-type p53 protein, were
reported to show the accumulation of p53 protein after the proteasome
inhibitors treatment (22
, 23)
. All OSCC cells used in the
present study have a p53 mutation (38
, 39)
, and they did
not express p53 protein (Fig. 3A
). Because mutations in the
p53 gene increase the stability of the p53 protein,
p53 overexpression has been accepted as an indicator of possible p53
mutations. In our previous study, other OSCC cells with a p53 mutation
in codon 248 overexpressed p53 protein by Western blot analysis
(40)
. In HSC2 cells, point mutation appeared at the
boundary between exon 6 and intron 6, and this point mutation would
lead to the synthesis of aberrant p53 protein, terminating at the stop
codon of nucleotides 65–67 of exon 7 (38)
. In HSC3, a
4-bp insertion was noted between codon 305 and 306 and contained a stop
codon in the reading frame (38)
. We conceive that point
mutations in these cells could not increase the stability of the p53
protein. In the present study, proteasome inhibitor treatment did not
bring about p53 accumulation in these cells (Fig. 3A
). This
result is supported by the report that mutant type p53 proteins were
not affected by proteasome inhibition (41)
. We supposed
that apoptosis of OSCC cells was induced by a p53-independent pathway.
A precursor of CPP32 protein, a member of the caspase family, was found
to be processed by a granzyme and became an active form
(42)
. We observed the activation of CPP32/caspase 3 in
HSC2 cells treated with proteasome inhibitor (Fig. 5)
. This result is
the same as reported findings that proteasome inhibitors enhanced
CPP32-like activity (18
, 43
, 44)
. Drexler
(18)
suggests that inhibition of CPP32 activation may also
be accomplished by the proteasome through constant degradation or
processing of a protein, such as blocking the cell cycle progression
located upstream of CPP32, which is critical for CPP32. Activation of
CPP32 is thought to be necessary for proteasome inhibitor-induced
apoptosis and plays an important role in this apoptosis signaling
pathway. Besides, it is also known that Bcl-2 protein blocks the
activation of CPP32 in mammalian cells (45
, 46)
. It has
been reported recently that apoptosis induced by the defects in the
ubiquitin-activating enzyme E1 was blocked by overexpression of Bcl-2
(47)
. The reduction of Bcl-2 protein induced by the
treatment with proteasome inhibitor is considered to act favorably for
CPP32-related apoptosis of HSC2. Moreover, CPP32 expression was
detected and Bcl-2 was not detected in HSC2 cells with
p27Kip1 antisense oligonucleotide treatment after
12 h of LLnV treatment (Fig. 6A
). Treatment with
p27Kip1 antisense oligonucleotide inhibited CPP32
activity slightly. This finding suggests that p27 may function upstream
of CPP32 protein but not of Bcl-2.
We examined whether proteasome inhibitors induced apoptosis in the
normal oral epithelial cells and gingival fibroblasts (Fig. 1G
). Apoptosis was not induced in the most of these cells by
the LLnV treatment, which is quite different from the findings in OSCC
cells. It is interesting that the proteasome inhibitor was capable of
inducing apoptosis in cancer cells but not in normal cells. Although
further studies must be done to determine the difference between normal
cells and OSCC cells, treatments with proteasome inhibitor may be used
as a novel therapy for OSCC.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Grant 07457429 from the
Ministry of Education, Science, Sports and Culture, Japan, and Tsuchiya
Foundation, Hiroshima, Japan. 
2 To whom requests for reprints should be
addressed, at Department of Oral Pathology, Hiroshima University,
Faculty of Dentistry, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553,
Japan. Phone: 81-82-257-5634; Fax: 81-82-257-5619.
3 The abbreviations used are: CDK,
cyclin-dependent kinase; Rb, retinoblastoma; pRb, Rb protein; OSCC,
oral squamous cell carcinoma; LLnV,
carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal;
pNA, p-nitroanilide.
Received 9/ 9/99; revised 11/29/99; accepted 11/29/99.
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) or 10 µM lactacystin (
).
DMSO-treated HSC2 cells were used as control (•). Trypsinized cells
were counted by cell counter at 6, 12, and 24 h after treatment.
Data are expressed as a percentage of cell number at 0 h.
Bars, SD. B, effect of proteasome
inhibitor on cell cycle of HSC2 cells. HSC2 cells were incubated with
50 µM LLnV for 0, 12, and 24 h, and the cell cycle
population was analyzed by flow cytometry.
