(Received for publication, October 11, 1995, and in revised form, September 9, 1996)
From the Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Nagoya City University, Mizuho, Nagoya 467, Japan
Interleukin (IL)-1 is a multi-functional cytokine
and regulates cell growth either positively or negatively. Previous
studies have shown that IL-1-induced ornithine decarboxylase (ODC)
activity down-regulation is involved in the anti-proliferative effect
of IL-1 on human A375 melanoma cells. In this study, we examined the
IL-1-induced molecular events resulting in ODC activity
down-regulation in C2-1, a A375 cell line stably transfected with human
type I IL-1 receptor. Recombinant human (rh) IL-1
inhibited the
growth and down-regulated the ODC activity of C2-1 cells in a
dose-dependent manner. Kinetics studies showed that both
the DNA synthesis and ODC activity of C2-1 cells progressively
decreased from 12 h after IL-1 addition. Northern hybridization
showed that IL-1 had no influence on ODC mRNA level. However,
rhIL-1 induced both a decrease of ODC protein and an ODC-inhibiting
activity in IL-1-treated C2-1 cells. IL-1 specifically up-modulated the
mRNA level of antizyme, a protein essential for ODC regulation, but
had little effect on its stability. IL-1-induced antizyme up-modulation
preceded IL-1-induced down-regulation of ODC protein, ODC activity, and DNA synthesis in C2-1 cells. Run-on transcription analysis confirmed that the increased antizyme mRNA expression was due to elevated antizyme gene transcription. Furthermore, the action of IL-1 to inhibit
the ODC activity and growth of C2-1 cells was blocked by expressing the
antisense RNA of human antizyme in C2-1 cells. These results suggest
that IL-1-induced antizyme expression is responsible for IL-1-induced
ODC activity down-regulation in human melanoma cells.
Interleukin-1 (IL-1)1 is one of the multi-functional cytokines produced predominantly by activated monocytes and macrophages and participates in many host reactions (1, 2). It has been not only incriminated in various disease states such as endotoxic shock (3) and autoimmune diseases (4) but also shown to play beneficial roles in immune defense, homeostasis, and immune surveillance (1, 2, 5). In vitro, IL-1 regulates cell growth in either a positive or a negative manner. With respect to its negative effect on cell growth, IL-1 has been reported to suppress the growth of a variety of normal and malignant cell types including pancreatic Langerhans cells (6), endothelial cells (7), and tumor cell lines derived from melanoma (8), breast carcinoma (9), myeloid leukemia (10), ovarian carcinoma (11), and lung adenocarcinoma (12).
Two agonist species of the IL-1 family, IL-1 and IL-1
, although
encoded by different genes and sharing only 26% amino acid sequence
homology (13), can bind the same cell surface receptors and mediate
similar biological actions (14). Two types of IL-1 receptors (IL-1R)
have until now been identified: type I IL-1R is an 80-kDa glycoprotein
preferentially expressed on fibroblasts and T cells (15, 16), whereas
type II IL-1R is a 60-kDa glycoprotein primarily present on B cells,
macrophages, and neutrophils (17). Only type I IL-1R can transduce IL-1
signal, whereas type II IL-1R works as a decoy receptor to dampen
signaling via type I IL-1R (18, 19). The binding of IL-1 to type I
IL-1R has been proposed to cause activation of different second
messenger pathways including activation of a GTP-binding protein with
no associated increase in adenyl cyclase (20), activation of adenyl
cyclase (21, 22), hydrolysis of three phospholipids by
nonphosphatidylinositol phospholipase Cs (23, 24), release of ceramide
from sphingomyelin after activation of sphingomyelinase (25), and
release of arachidonic acid from phospholipids via cytosolic
phospholipase A2 after its activation by phospholipase
A2-activating protein (26, 27). Very recent studies suggest
the involvement of either activation of mitogen-activated protein
kinases and tyrosine kinases or down-regulation of protein phosphatase
(28-32). However, the precise transmenbrane and/or intracellular
pathway(s) for different aspects of IL-1 action has not been fully
elucidated.
The first intracellular alteration probably associated with the anti-proliferative effect of IL-1 in A375 human melanoma cells was reported to be down-regulation of ornithine decarboxylase (ODC) activity (34). Subsequently, IL-1-induced IL-6 production was shown to mediate in part the anti-proliferative effect of IL-1 on A375 melanoma cells (35). IL-1 treatment also caused A375-6 cells to be arrested in G0/G1 phase of the cell cycle (36). Furthermore, studies documented by Rangnekar et al. emphasized the importance of an IL-1-induced early gene expression program (38), and they proposed that IL-1-induced gro (37) and Egr-1 (39) genes expression might be associated with IL-1-induced growth arrest in A375 cells.
Very recently, we transfected A375-5 cells, a twin subclone of A375-6 expressing no detectable IL-1R (33), with a human type I IL-1R cDNA and obtained a series of IL-1R-positive stable transfectants (40). Among these transfectants, some were IL-1-sensitive and others were IL-1-resistant. The most obvious difference between the two categories of transfectants was that upon IL-1 treatment, all the sensitive transfectants down-regulated their ODC activities, whereas all the resistant transfectants failed to do so. These results combined with the observation that putrescine, a resultant product of ODC reaction, reversed the anti-proliferative effect of IL-1 (40), and our previous studies (34, 36) led us to conclude that IL-1-induced ODC activity down-regulation plays a very essential role in the anti-proliferative effect of this cytokine on A375 melanoma cells (40). However, how ODC activity is down-regulated by IL-1 remains largely unknown. In the present study, we addressed this question by the use of C2-1, an IL-1-sensitive transfectant A375 cell line (40). Our results suggest that IL-1-induced up-modulation of AZ expression is responsible for IL-1-induced ODC activity down-regulation in C2-1 melanoma cells.
RPMI 1640 and TPA were purchased from
Sigma. Fetal bovine serum was purchased from Bocknek
(Toronto, Canada). Recombinant human IL-1 (rhIL-1
, 2 × 107 units/mg) and rh tumor necrosis factor
(107 units/mg)were provided by Dr. M. Yamada (Dainippon,
Osaka, Japan). Recombinant human IL-6 was provided by Dr. Y. Akiyama
(Ajinomoto, Yokohama, Japan). Recombinant human interferon
was
obtained from Nippon Roche (Kamakura, Japan). Oncostatin M was a
generous gift from Dr. C. Reynolds (NCI, National Institutes of Health, Frederick, MD). DL-[1-14C]Ornithine
hydrochloride (56 mCi/mmol) was purchased from Amersham Corp. Rabbit
polyclonal anti-hODC IgG antibodies and mouse monoclonal anti-hODC IgG
antibodies (41) were generous gifts from Drs. A. Kadota and K. Nakayama
(Idemitsu Kosan Co., Ltd., Sodegaura, Chiba, Japan).
The C2-1 cell line, a stable hIL-1RI
transfectant of A375-5 human melanoma cells (40), was maintained in
culture medium (RPMI 1640 supplemented with 100 units/ml of penicillin,
100 µg/ml of streptomycin, 15 mM HEPES, and 5% fetal
bovine serum) at 37 °C in a humidified atmosphere containing 5%
CO2. The C2-1 cells were detached with 0.05%
trypsin-0.02% EDTA in phosphate-buffered saline. After washing with
culture medium and being adjusted to 2 × 104
cells/ml, C2-1 cells were distributed into Falcon 3003 dishes (Becton
Dickinson) at a volume of 10 ml/dish. After preincubation for 2 days to
allow complete recovery of cells from trypsinization, the culture
supernatants were replaced by prewarmed fresh culture medium in the
presence of rhIL-1 or other reagents at different concentrations as
specified.
Cells in suspension
(4 × 104 cells/ml) were distributed into 96-well flat
bottomed plates (Falcon, Lincoln, NJ) at 0.1 ml/well and then subjected
to incubation for 24 h at 37 °C under humidified air containing
5% CO2 Thereafter, 0.1-ml aliquots of culture medium in
the absence or the presence of different concentrations of rhIL-1
were added in triplicate, and the cells were cultured for different
periods of time as specified. Cell proliferation was determined by
crystal violet stain as described previously (40). For the measurement
of DNA synthesis, the cells were pulsed with 0.5 µCi/ml of
3H-thymidine for the last 2 h. Then, the cells were
collected on glass filters, and the 3H-thymidine
incorporation was measured with a scintillation counter (LSC-1000;
Aloka, Tokyo, Japan).
The cells were collected by trypsinization, washed twice with ice-cold TED buffer (25 mM Tris, 0.15 mM EDTA, 2.5 mM dithiothreitol, pH 7.5) containing 0.15 M NaCl, and resuspended in an appropriate amount of TED buffer. Thereafter, the cells were sonicated for 30 s on ice with a handy sonicator (Tomy Seiko Co., Ltd., Tokyo, Japan) and centrifugation for 5 min at 1000 × g in a chilled microfuge. The supernatant thus obtained was used as cell lysate. The protein content of the cell lysate was determined using a protein assay kit (Bio-Rad) with bovine serum albumin (Sigma as the standard. After preparation of cell lysates, they were treated for 10 min at room temperature with or without 4 µM DFMO.
ODC AssayODC activity in the cell lysate was immediately measured by the method of Seely and Pegg (42) with minor modifications. Briefly, the enzyme reaction mixture consisting of 100 µl of TED buffer, 25 µl of 0.8 mM pyridoxal phosphate, 15 µl of 8 mM L-ornithine, and 10 µl of DL-[1-14C]ornithine was prepared in advance. After the addition of 100 µl of TED buffer (blank) or cell lysate, the mixture was incubated at 37 °C for 30 min with constant shaking. Then the reaction was stopped by the addition of 0.5 ml of 0.5 N HCl, and the mixture was shaken for another 2 h. ODC activity was determined as the release of [14C]CO2 (dpm/h/mg of protein), which was collected on a 4-cm2 filter paper soaked with 0.5 N NaOH and measured with a fluid scintillation counter (LSC-1000; Aloka, Tokyo, Japan).
ELISAAn indirect double antibody ELISA was established for
the determination of ODC protein in the cell lysates. Cell lysates was adjusted with TED buffer to a concentration of 1 mg/ml protein and
further diluted with coating buffer (0.1 M
carbonate/bicarbonate, pH 9.6) to a final concentration of 50 µg/ml
(coating solution). For coating, 0.1 ml of coating solution was
distributed into each well of a 96-well plate (Nunc-Immuno Plate,
MaxiSorpTM; Roskilde, Denmark) and incubated at 4 °C overnight. The
coated plate was blocked for 1 h at 37 °C with 0.4 ml/well of
washing buffer (0.1 mM Na2HPO4,
0.02 mM KH2PO4, 140 mM
NaCl, 0.02 mM KCl, 0.05% Tween 20, pH 7.4) containing
0.5% bovine serum albumin (Sigma, fraction V). After
blocking, monoclonal mouse anti-human ODC antibodies (5 µg/ml in
phosphate-buffered saline) were added at a volume of 0.1 ml/well, and
the plate was incubated at 37 °C for 3 h. Thereafter, 0.1 ml of
1/1000 diluted horseradish peroxidase-conjugated goat anti-mouse IgG
antibodies (purified F(ab)2, Cappel; Organon Teknika
Corp., West Chester, PA) was added into each well, and the plate was
incubated for 3 h at 37 °C. Three times of washes with washing
buffer were carried out between each step to completely remove reagents
not bound to the solid phase. Finally, 0.1 ml of
O-phenylenediamine substrate solution was added into each
well, and the plate was incubated at 37 °C for 15-20 min to allow
yellowish color to develop before terminating the enzymatic reaction by the addition of 0.1 ml/well of 2 N
H2SO4. The amount of ODC protein in each sample
was measured as absorbance at 490 nm using a ELISA plate reader
(Bio-Rad). Each sample was measured in triplicate wells.
Total RNA
was extracted from monolayer cultures of C2-1 cells according to
Chomczynski and Sacchi (43). After size fractionation on an
agarose-formaldehyde gel and transfer to a nitrocellulose (NC) filter,
the specific mRNA on the filter was detected by hybridization with
a 32P-labeled cDNA probe at 42 °C for about 17 h in hybridization buffer comprising 50% formamide, 5 × SSPE
(1 × SSPE is 0.15 M NaCl, 10 mM
NaH2PO4, 10 mM EDTA, pH 7.4),
5 × Denhardt's solution, 1% SDS, and 100 µg/ml denatured
salmon sperm DNA. The following probes were used: (i) a 611-bp cDNA
fragment corresponding to bases 2-612 of reported human ODC cDNA
(44). This cDNA fragment was amplified by reverse
transcriptase-polymerase chain reaction using A375-5 cell-derived
mRNA as template. The primers used were 5-GCCGGCGAATTCCTGGAGAGTTGCC-3
and 5
-CCTGCGAATTCTGAGCGTGGCACCG-3
. The fragment obtained was cloned into the EcoRI site of
pGEM-3Z to create pGEM-hODC, and the fidelity of the cloned fragment
was confirmed by sequencing; (ii) a 550-bp cDNA fragment of human AZ that was also amplified by reverse transcriptase-polymerase chain
reaction method using A375-5 cell-derived mRNA as template. As hAZ
gene had not been cloned at the beginning of this work, the primers,
designed according to the sequence of rat antizyme gene (45), were
5
-CCTGCAGCGGATCCTCAACAGCCACTG-3
and
5
-GGATGCCCGGGTCTCACAATCTCAAAG-3
. The amplified fragment was cloned
into the SmaI site of pGEM-3Z to create pGEM-hAZ. Sequencing
the cloned hAZ cDNA fragment by dideoxy terminator method showed
that it corresponds to bp 186-726 of the full-length human AZ cDNA
cloned from Daudi cells (49); (iii) a PstI-digested 1300-bp
fragment of human glyceraldehydephosphate dehydrogenase (GAPDH) (46).
The probes were labeled by random priming (Multi Prime DNA Labeling
Kit, Amersham Corp.). After hybridization, the filters were washed
twice at room temperature in 2 × SSC (1 × SSC is 0.15 M NaCl, 15 mM sodium citrate, pH 7.0) for 30 min followed, if necessary, by washing in 0.2 × SSC until a
reasonably low background was obtained. The filters were
autoradiographed using a Bio-Image analyzer (Fuji BAS 2000, Tokyo,
Japan).
Nuclei preparation and in
vitro transcription was performed according to the method of
Celano et al. (47) with minor modifications. Briefly, 6 × 106 C2-1 cells were suspended in 0.5 ml of ice-cold
lysis buffer (20 mM Tris-HCl, pH 7.4, 10 mM
NaCl, 3 mM MgCl2, and 0.5% Nonidet P-40, v/v)
and incubated on ice for 10 min. Nuclei were pelleted at 1000 × g for 5 min and washed once with lysis buffer. The nuclei pellet was resuspended in 0.1 ml of storage buffer (50 mM
Tris-HCl, pH 8.3, 5 mM MgCl2, 0.1 mM EDTA, and 40% glycerol, v/v), immediately snap-frozen
in liquid nitrogen, and stored at 80 °C. In vitro nuclear transcription was carried out in 0.1 ml of transcription buffer
(10 mM Tris-HCl, pH 7.5, 5 mM
MgCl2, 80 mM KCl, 0.1 mM EDTA, and
0.5 mM dithiothreitol) containing 4 mM ATP,
GTP, CTP, and 100 µCi of [
-32P]UTP at 30 °C for
30 min. Subsequently, the mixture was treated at 30 °C for 10 min
with DNase I (final concentration, 300 µg/ml) followed by treatment
at 42 °C for another 30 min with proteinase K (final concentration,
100 µg/ml) in the presence of 50 µg of carrier tRNA, 1% SDS, and
10 mM EDTA. RNA in the mixture was extracted by the acid
guanidium phenol method (43) followed by two times of ethanol
precipitation. The
-32P-labeled nuclear RNA was
dissolved in hybridization buffer to a concentration of 106
cpm/ml and hybridized, under the same conditions as those of Northern
hybridization, to NC filters onto which a number of DNA plasmids were
immobilized. The following plasmids were used: pGEM-hAZ, pUC-hMnSOD
(48), pUC-hGAPDH (46), and control plasmids including pUC118 and
pGEM-3Z. All plasmids were linearized by digestion with appropriate
restriction enzymes and denatured by alkalai treatment before blotting
on to NC filter (5 µg/slot).
The 1063-bp full-length hAZ cDNA was
cut out by EcoRI from pAZ7.1 reported recently (49) and
cloned into the EcoRI site of the expression vector pBK-RSV
by standard procedure to generate recombinant plasmid pRhAZ(AS) in
which expression of the antisense transcript of hAZ was controlled by
the RSV long terminal repeat. Pure pBK-RSV and pRhAZ(AS) were prepared
by two cycles of CsCl untracentrifugation. For transfection, 50 µg of
plasmids were electroporated into 5 × 106 C2-1 cells
in 0.5 ml of Opti-MEMI under conditions (0.4-cm cuvette, 0.31 kV, 950 microfarad) by the use of the Gene Pulser II system (Bio-Rad). Under
these conditions, electroporation efficiencies were 55-65% as
estimated by in situ -galactosidase staining of pSV-
-galactosidase-transfected C2-1 cells. After electroporation, cells were immediately suspended in RPMI 1640 supplemented with 10%
fetal bovine serum and incubated at 37 °C for 48 h in a
humidified atmosphere containing 5% CO2. Thereafter, cells
were detached with 0.05% trypsin-0.02% EDTA in phosphate-buffered
saline, washed, and used for the examination of their responses to
IL-1-induced ODC activity down-regulation and growth inhibition by
methods as described above.
Treatment with
rhIL-1 for 3 days dose-dependently inhibited the growth
of C2-1 cells (Fig. 1A) in a manner similar
to those of other IL-1-sensitive A375 melanoma cells (8, 34, 40). Fig.
1B showed that the DNA synthesis of C2-1 cells was
suppressed by rhIL-1
in a time-dependent manner, which
occurred from 12 h after IL-1 addition. As reported previously
that ODC activity down-regulation was important for IL-1-induced
inhibition of growth and DNA synthesis in human melanoma cells (34,
40), we subsequently investigated the effect of IL-1 on the ODC
activity of C2-1 cells. Culture of C2-1 cells at 37 °C for 48 h
in the presence of various concentrations of rhIL-1
down-regulated
their ODC activity in a dose-dependent manner (Fig.
2A). Time course study revealed that the ODC
activity of cultured C2-1 cells increased transiently within 6 h,
began to decrease from between 6 and 12 h, and continued to
decrease thereafter after rhIL-1
addition (Fig. 2B). At
48 h after rhIL-1
addition, the ODC activity was down-regulated to about 15% of that before rhIL-1
addition. The transient increase of ODC activity was due to medium change (refer to the legend of Fig.
2) because the similar increase was also observed in the absence of
rhIL-1
(data not shown). At 12 h after rhIL-1
addition, the
ODC activity of C2-1 cells was still as high as that before rhIL-1
addition (Fig. 2B), whereas the rate of DNA synthesis was
reduced about 25% (Fig. 1B). This discrepancy is perhaps
due to the transient increase of ODC activity, because about 25% of the ODC activity was suppressed at 12 h after rhIL-1
addition compared with highest ODC activity time point caused by medium changes.
IL-1 Had No Effect on ODC mRNA Level in C2-1 Cells
The
effect of rhIL-1 on the ODC mRNA level of C2-1 cells was
investigated by Northern hybridization (Fig. 3).
Treatment with rhIL-1
ranging from 0.1 to 1000 units/ml for 48 h showed that there were no obvious alterations of ODC mRNA level
in C2-1 cells (Fig. 3A), nor were there any
time-dependent variations of ODC mRNA expression in
C2-1 cells upon treatment with 100 units/ml rhIL-1
(Fig.
3B). In a separate experiment in which total RNA was
extracted at 30 min, 1 h, and 2 h after the addition of 100 units/ml of rhIL-1
, the ODC mRNA level of cultured C2-1 cells showed no alterations (data not shown). These results suggested that
IL-1 neither suppressed ODC gene tanscription nor destabilized ODC
mRNA in C2-1 melanoma cells.
IL-1 Induced Down-regulation of ODC Protein in C2-1 Cells
We
next investigated the influence of rhIL-1 on the ODC protein level
of C2-1 cells (Fig. 4). rhIL-1
down-regulated ODC protein level in C2-1 cells in a dose-dependent manner
(Fig. 4A). The kinetics of rhIL-1
-induced down-regulation
of ODC protein in C2-1 cells (Fig. 4B) was similar to that
of rhIL-1
-induced ODC activity down-regulation as illustrated in
Fig. 2B. These results suggested that IL-1-induced ODC
activity down-regulation was associated with and might be a result of
IL-1-induced down-regulation of ODC protein in melanoma cells.
Existence of ODC Enzyme Inhibitor(s) in IL-1-treated C2-1 Cells
A careful comparison of Fig. 4 with Fig. 2 revealed that
IL-1-induced ODC protein down-regulation could not completely explain IL-1-induced ODC activity down-regulation, because they were not completely parallel. Therefore, we examined whether there existed IL-1-induced intracellular ODC enzyme inhibitor(s), which might be
responsible for part of the IL-1-induced ODC activity down-regulation. Theoretically, if there were such inhibitor(s) in IL-1-treated C2-1
cells, mixing the lysate of IL-1-treated C2-1 cells with that of
untreated C2-1 cells would suppress at least in part the ODC activity
existing in the lysate of untreated C2-1 cells. In order to measure the
exact ODC activity, half of the cell lysates were pretreated with DFMO
at 4 µM, which was predetermined to be enough to inhibit
ODC activity. The results in Table I demonstrated that
the ODC activity of the IL-1-treated cell lysate was almost undetectable, and a mixture containing 50% untreated and 50%
rhIL-1-treated lysates was much lower than half of the sum of ODC
activities of untreated and rhIL-1
-treated lysates, indicating that
there were inhibitory activities for ODC in IL-1-treated C2-1
cells.
|
It is well
known that AZ, a small intracellular protein that functions to inhibit
ODC activity by monomerizing ODC homodimer and to down-regulate ODC
protein by accelerating its degradation in 26 S proteasome, is critical
in the regulation of ODC activity (49-53). We therefore investigated
whether IL-1 could up-modulate the AZ expression in C2-1 cells.
Treatment with various concentrations of rhIL-1 at 37 °C for
24 h showed that as little as 0.1 units/ml of rhIL-1
up-modulated AZ mRNA expression in C2-1 cells, and the action was
dose-dependent (Fig. 5A). The
time course study revealed that rhIL-1
-induced up-modulation of AZ
mRNA in C2-1 cells occurred at around 3-6 h after rhIL-1
addition (Fig. 5B). Comparison of the kinetics of
rhIL-1
-induced up-modulation of AZ mRNA (Fig. 5B)
with those of rhIL-1
-induced down-regulation of ODC protein (Fig.
4B) and ODC activity (Fig. 2B) indicated that
upon rhIL-1
treatment, AZ mRNA up-modulation preceded
down-regulation of ODC protein and activity.
IL-1 Activated the Transcription of AZ Gene in C2-1 Cells
Because rhIL-1 exhibited no obvious effect on the
stability of AZ mRNA in C2-1 cells (data not shown), the effect of
rhIL-1
on the transcription of AZ gene was investigated by nuclear
run-on experiments. Treatment with rhIL-1
at a concentration of 100 units/ml for 12 h greatly increased (approximately 6-fold) the transcription rate of AZ gene in C2-1 cells (Fig. 6).
The transcription of MnSOD gene (as a positive control) was also
increased, whereas rhIL-1
had no effect on GAPDH gene transcription.
In a separate experiment, when nuclei derived from C2-1 cells treated
with rhIL-1
for 6 and 24 h were examined, similar results were
obtained (data not shown).
Expression of Antisense RNA of hAZ Blocked IL-1-induced ODC Activity Down-regulation and Growth Inhibition
To further examine
whether IL-1-induced up-modulation of AZ mRNA expression was
responsible for IL-1-induced ODC activity down-regulation, we
constructed a recombinant plasmid (pRhAZ(AS)), which directed the
synthesis of antisense hAZ RNA, and transfected this plasmid into C2-1
cells to see if IL-1-induced ODC activity down-regulation could be
blocked (Fig. 7). Transient transfection of C2-1 cells
with pRhAZ(AS) blocked about 70% of rhIL-1-induced ODC activity
down-regulation, whereas transfection with the vector plasmid pBK-RSV
in an identical way had no effect (Fig. 7A). Furthermore, transfection of C2-1 cells with pRhAZ(AS) also blocked approximately 60-70% of rhIL-1
-induced growth inhibition of these cells (Fig. 7B). The failure of antisense hAZ RNA to completely block
the rhIL-1
-induced ODC activity down-regulation and growth
inhibition in C2-1 cells was presumably because the transfection
efficiency was not 100%. These results not only provided evidence that
AZ up-modulation was responsible for IL-1-induced ODC activity
down-regulation but also further strengthened our view that ODC
activity down-regulation was essential for IL-1-induced growth
inhibition in A375 melanoma cells (40).
TPA and Other Cytokines Failed to Increase the AZ mRNA Level of C2-1 Cells
Besides IL-1, a number of cytokines including
interferon (8, 55), tumor necrosis factor (34, 35), IL-6 (35, 56), and oncostatin M (57), as well as TPA2 can
suppress the growth of human melanoma cells. Because the growth of C2-1
cells could also be inhibited by these reagents (data not shown), we
therefore investigated whether or not these reagents could modulate the
expression of AZ mRNA in C2-1 cells. Treatment of C2-1 cells for
24 h with any of these reagents at a dose larger than their
ED50 failed to induce elevation of AZ mRNA level (Fig.
8), suggesting that up-modulation of AZ gene expression
in melanoma cells was IL-1-specific.
Treatment with rhIL-1 inhibited the growth (Fig. 1A)
and down-regulated the ODC enzyme activity (Fig. 2A) of C2-1
cells in a similar dose-dependent manner. Time course
studies showed that both ODC enzyme activity and DNA synthesis of C2-1
cells were down-regulated by rhIL-1
in similar kinetics; of
particular note is that they both occurred at 12 h after the
addition of rhIL-1
(Figs. 1B and 2B). These
results are similar to those observed in other IL-1-sensitive A375
melanoma cells (34, 40). ODC catalyzes the conversion of ornithine to
putrescine, the first step and a major site of regulation of polyamine
biosynthesis, and is involved in the regulation of cell growth,
differentiation, and cell cycle control (44, 50, 51, 57). Based on the results in Figs. 1 and 2 of the present study and previous reports, several aspects of evidences support the notion that IL-1-induced ODC
activity down-regulation is an essential step in IL-1-induced growth
inhibition in melanoma cells: (i) IL-1-induced ODC activity down-regulation precedes IL-1-induced inhibition of DNA synthesis and
proliferation in A375 cells (34); (ii) putrescine, a physiological product of ODC reaction and precursor of polyamines, can overcome most
when high doses of IL-1 are used (34, 40) and all when low doses of
IL-1 are used (40) of IL-1-induced growth inhibition; (iii) in melanoma
cells whose ODC activities cannot be down-regulated by IL-1, their
growth also cannot be suppressed by IL-1 (40); (iv) treatment of
synchronously cultured melanoma cells with IL-1 delays progression from
G0/G1 to S, retards progression through G2/M of the first cell cycle, and blocks progression from
G0/G1 to S of the second cell cycle (36). This
consequence is compatible with IL-1-induced ODC activity
down-regulation because it has been reported that ODC activity begins
to be activated in late G1, increases gradually through S
phase, and reaches its peak at the S/G2 transition of the
cell cycle (51, 58). Nevertheless, how IL-1 causes ODC activity
down-regulation is unknown.
Because the expression of ODC gene is usually stimulated (34, 59) in
cells whose growth and ODC activities are promoted by IL-1 (1-2, 34,
59), we first investigated whether IL-1 could affect ODC mRNA level
in C2-1 cells. The results that rhIL-1 did not have any obvious
influence on the ODC mRNA level of C2-1 cells (Fig. 3) indicate
that IL-1 does not act on the transcription and the stability of ODC
mRNA in C2-1 cells. On the other hand, IL-1 decreased ODC protein
level of C2-1 cells in a dose- and time-dependent manner
(Fig. 4) similar to IL-1-induced ODC activity down-regulation.
Furthermore, rhIL-1
treatment induced the production of ODC activity
inhibitor(s) in C2-1 cells (Table I). These data led us to
propose that both IL-1-induced ODC protein down-regulation and ODC
inhibitor(s) accounted for IL-1 induced ODC activity
down-regulation.
ODC protein level is regulated at two major sites, i.e.
translation and degradation (50-51, 59-62). Theoretically, either
decreased translation or increased degradation can result in the
down-regulation of ODC protein level. The fact that in IL-1-treated
C2-1 melanoma cells, down-regulation of ODC protein was accompanied by
the production of ODC inhibitor(s) allowed us to investigate the effect
of IL-1 on the expression of AZ, a small intracellular protein known to act as both a specific ODC inhibitor and a promotor for the degradation of ODC protein (50-54, 63). Treatment with rhIL-1 up-modulated AZ
mRNA level in a dose- and time-dependent manner (Fig.
5). More important is that rhIL-1
-induced AZ mRNA up-modulation
occurred at as early as 6 h after IL-1 addition (Fig.
5B), which preceded the IL-1-induced down-regulation of ODC
protein (Fig. 4B) and enzyme activity (Fig. 2B).
The transcription of the AZ gene was further shown to be activated by
IL-1 treatment (Fig. 6), whereas rhIL-1
treatment had no effect on
the stability of AZ mRNA (data not shown). These results suggest
that in C2-1 cells, IL-1 probably exerts its growth inhibitory effect
by up-modulating AZ expression, which in turn down-regulate ODC
activity by simultaneously inhibiting ODC activity and promoting ODC
protein degradation. These results also explain why IL-1-induced ODC
protein down-regulation was not parallel with IL-1-induced ODC activity
down-regulation, because ELISA perhaps measured both free ODC and
AZ-bound ODC (without enzymatic activity) molecules that had not been
degraded. The importance of IL-1-induced AZ expression in IL-1-induced
ODC activity down-regulation is further supported by the fact that
antisense AZ RNA expression blocked to a similar extent (about 70%)
the IL-1-induced ODC activity down-regulation (Fig. 7A) and
growth inhibition (Fig. 7B).
To confirm the role of AZ in IL-1-induced ODC activity down-regulation, another approach would be the direct measurement of AZ protein level in untreated and IL-1-treated cells by methodologies such as Western blotting. Unfortunately, antibodies against human AZ are not available for us, and it is reported that the amount of AZ is quite low (51, 63). How did IL-1-treated C2-1 cell lysate inhibit the ODC activity of untreated C2-1 cell lysate (Table I)? There may be excess AZ in the treated cells. This seems likely because in the treated cell lysate most of the remaining apparent ODC activity was not inhibited by treatment with DFMO, suggesting that there was no ODC activity in the cell lysate. Probably up-modulated AZ protein from the treated cell lysate inhibited ODC activity by binding ODC.
The effects of other cytokines capable of inhibiting the growth of human melanoma cells (34-35, 55-57) on the AZ mRNA levels were also examined, and they all failed to down-modulate AZ mRNA expression in C2-1 cells (Fig. 8). Tumor necrosis factor and interferon has been reported to cause growth inhibition of human melanoma cells by suppressing c-myc expression (58), whereas IL-1 does not inhibit c-myc expression in A375 melanoma cells (38); it is also reported that a number of mutant melanoma cell lines resistant to both IL-6 and oncostatin M remained sensitive to IL-1 (57), and two IL-1-resistant A375 cell lines were still sensitive to IL-6 and oncostatin M (40). These evidences combined with the results of Fig. 8 indicate that IL-1-induced AZ mRNA up-modulation is quite unique to and important for the anti-proliferative effect of IL-1 on melanoma cells. Another line of evidence to support the involvement of AZ mRNA up-modulation in IL-1-induced growth inhibition is that in two IL-1-resistant clones derived from A375-6 cells (56), IL-1 failed to induce AZ mRNA up-modulation as well as ODC activity down-regulation.2
The observation that IL-1 up-modulated AZ expression by accelerating AZ gene transcription (Fig. 6) is of interest. So far as we know, there is no report showing that any factors can influence AZ gene transcription. Polyamines are important intracellular modulator for AZ but have no influence on AZ mRNA level (51, 63). How IL-1 activates the transcription of AZ gene in C2-1 melanoma cells needs further studies. Nevertheless, the results of the present study not only confirm our previous proposition that down-regulation of ODC activity is an essential step in the IL-1-induced growth inhibition in melanoma cells but also demonstrate that IL-1-induced ODC activity down-regulation is the result of IL-1-induced AZ up-modulation. Furthermore, the present study also suggests for the first time that AZ gene expression may have a novel regulatory role in controlling cell growth.
We thank Dr. S. Itoh for technical assistance in sequencing. We also thank Drs. Y. Murakami and S. Hayashi (Jikei University, School of Medicine) for kind suggestions. We appreciate the generosity of Dr. C. Reynolds, who provided rh oncostatin M, and Drs. A. Kadota and K. Nakayama, who provided monoclonal and polyclonal anti-hODC antibodies.