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Znt. J. Cancer: 65,805-811 (1996)
0 1996 Wiley-Liss, Inc.
Publication of the international Union Against Cancer
Publication de I'Union lnternationala Contre le Cancer
INTERLEUKIN 1 (IL-1) PRODUCTION IS NOT ESSENTIAL
FOR ACQUIRED RESISTANCE OF HUMAN A375 MELANOMA
CELLS TO ANTI-PROLIFERATIVE EFFECT OF IL-1
Saotomo ITOH',Hidetoshi HAYASHI',
Naoko WATANABE~,
Yoshiro KOBAYASHI~,
Takemasa TAKII'and Kikuo 0 ~
'DepartPnent of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Nagoya City Univemity,Mimho, Nagoya 467;
2Departmentof Biomolecular Science, Faculty of Science, Toho University,Funabashi, Chiba 274, Japan.
The proliferation of human melanoma cell line A375-6 is
inhibited by interleukin I (IL- I). However, the cells acquired
resistance to IL-I after a long period of culture. We have
reported that 2 resistant subclones, A375-R8 and -R19, produced IL- la constitutively and that IL- I induced IL-6 production
in an autocrine manner. Therefore, we supposed that IL-la
production renders the cells resistant to IL- I. To investigatethe
relationship between IL- la production and IL- I resistance, we
transfected the IL- Ia expression plasmid to the IL- I-sensitive
clone, A375-6, and the anti-sense mRNA expression plasmid to
IL-I-resistant cells, A375-R8 and -R19. A375-6MS, a transfectant of mature IL-la expression plasmid, expressed IL-la
mRNA and produced IL- I activity at a level comparable to the
resistant cells. The transfectant also produced IL-6 and exhibited augmented expression of Mn-SOD mRNA. However, IL- I
sensitivity of this transfectant was not affected. With respect to
sensitivity to anti-proliferative effects of other cytokines, such
as IL-6 and TNFa, there was no difference between the
transfectant and parent cells. Although A375-R8PH I 0 and
-R19PH 10, transfectants of IL- la anti-sense mRNA expression
plasmid, exhibited a decrease in the level of IL-l production,
their IL-I sensitivity did not differ from parent cells. These
results, therefore, suggest that IL- la production is not essential
or suffkient for the acquisition of resistance to the antiproliferativeeffect of IL- I.
o 1996 Wilq-Liss, Inc.
IL-1 is a multifunctional cytokine that regulates immunoreaction, inflammation, hematopoiesis and homeostasis (Dinarello, 1994). IL-1 also regulates the proliferation of various
cell types in both positive and negative manners. Thymocytes,
B cells and fibroblasts are representatives that proliferate in
response to I G 1 (Gery et al., 1972; Freedman et al., 1988;
Schmidt et al., 1982). However, the proliferation of many cell
types, including several human mammary tumor cell lines, the
murine myeioid leukemic cell line (Ml) and human endothelial cells, is inhibited by IL-1 (Gaffney and Tsai, 1986; Onozaki
et al., 1987; Maier et al., 1990).
The human melanoma cell line A375-6 is the first found to
be inhibited of its proliferation by IL-1 (Onozaki et al., 1985).
We have noticed that A375-6 cells became resistant to IL-1
after a long period of culture. Two IL-1-resistant subclones,
A375-RB and -R19, were obtained, and their characteristics
have been studied. These 2 subclones expressed functional
IL-1 receptor regardless of their resistance to IL-1 and
produced IL-la constitutively. They also produced IL-6 constitutively, and the production was shown to result from the
autocrine effect of endogenous IL-la. The cells also became
resistant to the anti-proliferative effect of IL-6 (Araki et at.,
1994).
The progression of human melanoma has been extensively
studied. In an early stage (the radial growth phase), the growth
of melanoma cells depends on several growth factors and can
be inhibited by several cytokines, such as transforming growth
factor (TGF)-P, IL-1, IL-6 and oncostatin M. However, in an
advanced stage, melanoma cells in the vertical growth phase
and metastases often produce IL-1 and/or IL-6 and acquire
independence from growth factors and resistance to the
cytocidal or cytostatic cytokines (Kerbel, 1992). This growth
factor independence and cytokine resistance seem to be
0 ~ ~ ~ 1 1 . 3
related to malignancy of melanoma (Lu and Kerbel, 1994).
Therefore, elucidating the mechanism of acquired resistance
to IL-1 would help to clarify the mechanism of melanoma
progression.
In this study, we investigated the relationship between
acquisition of IL-1 resistance and constitutive production of
IL-la in A375 human melanoma cells by transfecting IL-la
expression plasmids to IL-1-sensitive cells and IL-la antisense plasmids to resistant cells, followed by observing alterations in the responsiveness of transfected cells to the antiproliferative effect of rhIL-la.
MATERIAL AND METHODS
Reagents
RPMI 1640 was purchased from Sigma (St. Louis, MO);
fetal bovine serum (FBS) was from Sanko Junyaku (Tokyo);
human recombinant IL-la (2 x lo7 U/mg), tumor necrosis
factor (TNF)a (1.9 x lo6 U/mg) were provided by Dr. M.
Yamada (Osaka, Japan). Human recombinant IL-6 was kindly
supplied by Dr. Y. Akiyama (Yokohama, Japan); human
recombinant IL-2 was from Shionogi (Osaka, Japan); human
recombinant IL-1 receptor antagonist (IL-1Ra) was from
Upjohn (Kalamazoo, MI). Human manganese superoxide
dismutase (Mn-SOD) cDNA was kindly provided by Dr. K.
Matsushima (Kanazawa, Japan).
Cell culture
The human melanoma cell line A375 was originally given by
Dr. R. Ruddon (NCI, Bethesda, MD). By limiting dilution,
IL-1-sensitive cells A375-6 were obtained. Two IL-1-resistant
subclones, A375-R8 and -R19, were obtained by limiting
dilution of A375-6 cells that had acquired resistance to IL-1
after routine passage for 3 months (Araki et aL, 1994). Cells
were cultured in RPMI 1640, 100 U/ml of penicillin G, 100
Fg/ml of streptomycin, 15 mM HEPES and 5% heatinactivated FBS at 37°C.
Construction of expression plasmids
Human mature IL-la cDNA was obtained from the 12-0tetradecanoylphorbol 13-acetate-stimulated human myelomonocytic cell line (THP-1) by reverse transcriptase polymerase chain reaction (RT-PCR) using primers containing BamHI
site and cloned into pGEM-3Z to create pGEM-hmlLla.
Cloned hIL-la cDNA was confirmed by sequencing to be a
3To whom correspondence and reprint requests should be sent, at
Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Nagoya City University, Mizuho, Nagoya 467, Japan. Fax: (81)
052-836-3419.
Abbreviations: IL, interleukin; IL-lR, interleukin 1 receptor; ILlRa, interleukin 1 receptor antagonist; TNF, tumor necrosis factor;
HEPES, N-2-hydroxyethyl iperazine-N'-2-ethanesulfonicacid; MTT,
3-(4,5-dimethylthiazol-2-yl~-2,5-diphenyl
tetrazoliurn bromide; FBS,
fetal bovine serum; SOD, superoxide.dismutase.
Received: July 14,1995 and in revised form November 14,1995.
806
ITOH ETAL
512-base fragment identical to the sequence from base 372 to
base 875 of reported hIL-la cDNA (Furutani et al., 1986),
which covers the mature IL-la region and the 26-base nucleotide down-stream of the stop codon TAG.
A SmaI/Hinc 11-digested fragment from pGEM-hmILla
containing the mature IL-la region was ligated with Klenow
fragment-treated HindIII-digested pRSV-X . pRSV-neo' using a DNA ligation kit (Takara, Ohtsu, Japan; Fig. 1). The
structures of pRSV-hmILla and pRSV-hmILlaAS were verified by restriction enzyme mapping.
Another IL-la anti-sense expression plasmid, pEF-PH10,
was constructed as follows: the PstI-HindIII-digested fragment
(0.9 kbp) of pHL4 (Kobayashi et aL, 1988), which contains the
coding region of pre-IL-la, was inserted into pUG131 treated
with PstI and EcoRV. This plasmid (pUHL(PH)) was digested
with BgIII and SmaI, following the end-filling of protruding
ends, and ligated with a XbaI linker. The linker-ligated XbaI
fragment (0.9 kb) was replaced with the stuffer fragment of
pEF-BOS (Mizushima and Nagata, 1990), which contains the
human elongation factor la promoter. pSTneoB was obtained
from Japanese Cancer Research Resources Bank-Gene (Tokyo).
Transfection
A375 cells were transfected by the modified calcium phosphate method as reported by Chen and Okayama (1987), with
the pRSV-neo plasmid containing human mature IL-la cDNA
in both transcriptional orientations (MS and MAS). As a
control, transfection was performed with the pRSV-neo without an insert (neo). Transfectants were selected, 48 hr after
transfection, for acquisition of neomycin resistance by culturing in the presence of G418 (600 pg/ml). A375-R8 or -R19
cells were co-transfected with pEF-PH10 and pSTneoB by
lipofection using LipofectAMINE (GIBCO, Gaithersburg,
MD). Transfectants were selected as described above 48 hr
after transfection.
RNA extraction and Northern hybridization
Total RNA was extracted from A375 cells according to the
method of Chomczynski and Sacchi (1987). After being sizefractionated on agarose-formaldehyde gel and transferred to
nitrocellulose filter, the specific mRNA on the filter was
detected by hybridization with a 32P-labeled cDNA probe at
42°C for 18 hr in hybridization buffer containing 50% formamide, 5 x SSPE (1 x SSPE: 0.15 M NaCI, 10 mM NaH2P04,
10 mM EDTA, pH 7.4), 5 x Denhardt's solution, 1% SDS and
100 pg/ml denatured salmon sperm DNA. The following
probes were used: human IL-la (a 512-bp fragment, 372-875)
amplified by RT-PCR from THP-1 cell mRNA; human MnSOD (a 0.9-kbp fragment, full length); a Pst I-digested
1,300-bp fragment of human glyceroaldehyde phosphate dehydrogenase (GAPDH). These probes were labeled by random
priming (Multi Prime DNA labeling kit, Amersham, Aylesbury, UK). After hybridization, filters were washed at room
temperature in 2x SSC ( l x SSC: 0.15 M NaCl, 0.015 M
sodium citrate, pH 7.0) a few times. Filters were autoradiographed using a Bioimage analyzer (Fuji, BAS2000, Tokyo).
Assay for IL-1 activiq
IL-1 activity was determined by proliferation assay, with the
IL-1-dependent mouse T-cell line D10N4M provided by Dr.
S.J. Hopkins (Salford, U K Hopkins and Humphreys, 1989). In
brief, cells were cultured in RPMI 1640, 10 mM HEPES,
M 2-ME, 10% FBS, C o d (3 pg/ml), IL-2
antibiotics, 5 X
(40 U/ml) and standard IL-la or test samples. Cells were
cultured in wells of a flat-bottomed microtiter plate at 37°C in
A) RSV plasmids
EcoRl656.9.
e0R1605\
pRSV-hmlLlrx
6966 bp
I
pRSV-hmlLlaAS
""~.
6966b11
EcoRl 2676
RrnHl77R8
Xbal 2775
7
'Radii 3280
IEanHl3280
B) pEF plasmids
EF-la promolei
Xba I 3300
FIGURE
1 - Structures of human sense &la, anti-sense IL-la and neo expression vectors.
RESISTANCE TO IL-1 ANTI-PROLIFERATIVE EFFECT
807
5% C 0 2 in air. After 3-day culture, cell-proliferation activity
was assessed by the MTT method (Mosmann, 1983). After
solubilizing the formazan with 20% SDS, 50% dimethyl
formamide in water, absorbance at 595 nm was measured by an
ELISA autoreader (BioRad, Richmond, CA).
Assay for IL-6 activity
Biological activity of IL-6 was measured by its proliferative
action on the IL-6-dependent murine hybridoma clone
MH60 . BSF2, provided by Dr. T. Hirano (Osaka University,
Osaka, Japan; Kawano et al., 1988). Proliferation was measured by the M'IT method. IL-6 activity was expressed as the
equivalent amount of recombinant IL-6.
Assay for proliferation
Cells were detached from the culture dish with 0.02%
EDTA-PBS. Cells were washed with culture medium and 100
pl of cell suspension (2 x lo4 cells/ml) were added to each
flat-bottomed well of a 96-well microtiter plate (Falcon,
Lincoln, NJ). After 24-hr cultivation, 100 pI of medium
containing cytokines were added, and then cells were cultured
for another 72 hr. Proliferation of the cells was determined by
staining with crystal violet (Ruff and Gifford, 1980). After
solubilization of the dye-stained cells with 0.1% SDS, dye
uptake was caiculated by measuring the absorbance at 595 nm
by the ELISA autoreader.
RESULTS
Transfectwnof human IL-la expression plasmids
to A375-6 cells
IL-1-sensitive cells A375-6 were transfected with plasmids
containing human mature sense IL-la (pRSV-hmILla), antisense IL-la (pRSV-hmILlaAS) or control plasmid (pRSVneo) (Fig. 1) followed by selection with G418. To determine
IL-la mRNA level, total RNA was extracted from each
transfectant and various A375 clones and Northern hybridization performed (Fig. 2 ) . Endogenous IL-la mRNA was detected in IL-1-resistant clones, A375-R8 and -R19, but not in
IL-1-sensitive clone A375-6. IL-la transfectants in either
sense or anti-sense orientation expressed the smaller mRNA,
which can be distinguished from the endogenous IL-la mRNA.
Although the reason is not known, an additional mRNA signal
with smaller molecular weight was detected in the anti-sense
transfectant. We then determined IL-1 activity in culture
supernatants and cell lysates in each transfectant and various
A375 clones. As shown in Figure 3, sense IL-la transfectants
released IL-1 extracellularly and contained intracellular IL-1
at an equal level to one of the IL-1-resistant clones, R8 cells,
and higher than R19 cells.
IL-6production by A37.5-6 transfectant and other clones
It is known that IL-1 stimulation induces IL-6 production in
A375 cells and that IL-1-resistant clones were also resistant to
IL-6 and continuously produced IL-6 due to the autocrine
stimulation of IL-la (Araki et al., 1994). Therefore, we studied
whether there are any differences between the parent cells and
the transfectants in IL-6 production alone or in response to
IL-1. As shown in Figure 4a, similarly to IL-1 resistant cells,
mature IL-la sense transfectant constitutively released IL-6,
though the level is low compared with that of R8 cells but
almost equal to that of R19 cells. In the presence of IL-1Ra
(100 ng/ml), the IL-6 level produced by the transfectant
decreased by 22%. Therefore, IL-6 production in the transfectants appeared to be induced, at least partially, by endogenous
IL-1. In contrast, stimulation of the transfectant with IL-la
(100 U/ml) induced IL-6, but the level was lower than the 2
resistant cells and equal to those of parent and neo transfectant cells (Fig. 4b).
FIGURE2 - Expression of IL-la mRNA in A375 cell clones and
sense or anti-sense mature IL-la-transfected -75-6 cells. After
4-day culture in 90-mm dishes, total cellular RNA was isolated and
RNA samples were fractionated by agarose gel electrophoresis.
Northern blot analysis was conducted using 32P-labeledprobes for
human IL-la and GAPDH. Values were normalized based on the
density of GAPDH. Representative data of 3 experiments with
similar results are shown.
6) cell iyastas
A) culture sumatants
3"
501
40
T
FIGURE3 - IL-1 activities in culture supernatants and lysates of
A375 cell clones and neo sense or anti-sense mature IL-latransfected A375-6 cells. Cells (1.0 x 105/1 ml/well) were cultured in 24-well plates. After 24 hr, supernatants were obtained.
Cell lysates were prepared by sonication. IL-1 activities of the
culture supernatants (a) or cell lysates (b) were determined by
D10N4M in cell proliferation assay. Mean k SD based on
triplicate cultures is shown. Representative data of 3 experiments
with similar results are shown.
Mn-SOD mIWA expression in A3754 transfectants
and other clones
Expression of one of the anti-oxidative enzymes, Mn-SOD
mRNA, could be induced in A375 melanoma cells by IL-1
stimulation (Masuda et al., 1988). We then analyzed Mn-SOD
mRNA expression in the transfectant and other clones (Fig.
5). Both IL-1-resistant clones expressed Mn-SOD mRNA
ITOH ETAL.
808
A) untreated
400
6 )IL-la stimulation
r
4ooo
r
7
8
-C-
2
neo
MS
MAS A3156
RE
R19
L transfectants -Lanes 1
1000
neo
MS
MAS 1375-6
RB
R19
L transfectants i c l o n e s 1
FIGURE4 - IL-6 activities in culture supernatants of A375 cell
clones and neo sense or anti-sense mature IL-la-transfected
A375-6 cells treated with or without &la. Cells (1.0 X 105/1
mliwell) were cultured in 24-well plates without (a) or with (b)
IL-la (100 U/ml). After 24 hr, supernatants were obtained. IL-6
activity was determined by MH60. BSF2 cell proliferation assay.
Mean t SD based on triplicate cultures is shown.
o
B) 11-6
1 0 - 1 100 1 0 1
102
103
lo4
IL-la (Ulml)
MS
MAS
A375-6
R8
R19
100
-
g
75
8
'c
0
R19
i
lransfectants
clones
50
1
300 -
IL-6 (ng/ml)
C)TNF
CL
neo
MS
MAS
A37.5-6
100-
R8
transfectants
clones
R19
9
FIGURE5 - Expression of Mn-SOD mRNA in A375 cell clones
and sense or anti-sense mature IL-la-transfected A375-6 cells.
After culturing cells in 90-mm dishes for 4 days, culture media
were removed and fresh media added with or without IL-la (100
U/ml). After 2 hr, total cellular RNA was isolated and RNA
samples were fractionated b gel agarose electrophoresis. Northern blot analysis was con ucted using 32P-labeled probes for
human Mn-SOD and GAPDH. Values were normalized based on
the density of GAPDH. Representative data of 3 experiments with
similar results are shown.
B
without any stimulation. Mature IL-la sense transfectant
expressed about half of the level of IL-1-resistant clones but
twice as much as parental cells and anti-sense or neo transfectants. Upon stimulation with IL-1, Mn-SOD mRNA levels
increased in all cells except R8.
Cytokine sensitivity of A375-6 human I L - l a sense transfectants
and other clones
IL-la sense transfectants of A375-6 cells, parent cells and
IL-1-resistant cells were determined for their sensitivities to
IL-la, IL-6 and TNFa (Fig 6). As described previously (Araki
et al., 1994), parent cells A375-6 were sensitive to IL-la, IL-6
and TNFa. However, A375-R8 and -R19 cells were resistant to
IL-la and IL-6 but still sensitive to TNFa. IL-la sense and
FIGURE6 -Effects of IL-la, I L 6 and TNFa on the growth of
A375 cell clones and neo, sense or anti-sense mature IL-latransfected A375-6 cells. Cells (2.0 x 104/well)were cultured for 3
days with or without varying doses of IL-la (a), IL-6 (b) or TNFa
(c). After culture, cell proliferation was determined by staining
with crystal violet. Mean f SD based on triplicate cultures is
shown.
anti-sense transfectants of A375-6 cells exhibited the same
sensitivity as parent cells.
IL-1a anti-sense transfectants of Zl-1-resistant clones
We investigated whether the reduction of IL-1 production
may render the resistant cells sensitive to IL-1 by transfecting
the anti-sense IL-1 expression plasmid. As the anti-sense
expression plasmid under the control of the RSV-LTR promoter appeared to be insufficient to suppress IL-1 production,
we used the plasmid with more potent promoter EF-la,
pEF-PH10, which contains anti-sense of human precursor
IL-la. Transfectants of this anti-sense expression plasmid of
IL-1-resistant cells, R8PH10 and R19PH10, exhibited reductions of IL-1 production by 44% and 43% compared to neo
transfectants, respectively (Fig. 7). Reductions were more
remarkable (65% and 93%) in cell lysates. Continuous IL-6
production was also reduced in R8 but not in R19 (data not
shown). The sensitivities of these transfectants to IL-la, IL-6
and TNFa were not different from those of parent cells
(Fig. 8).
RESISTANCE TO IL-1 ANTI-PROLIFERATIVE EFFECT
A) supernatants
809
A) IL.1 a
-
R8-neo
R8-PHI0
R19-neo
---C
‘1 T
-0-C
-i
80
X
7
R19-PH10
IL-la entisense
transfectants
40
J
. ...
O10-l
-2
. ...- ......a
loo
lo1
.
lo2
8
,
IO~YY;.~~
IL-1a U/ml
RO-neO REPHIO
R19-neo R19-PH10
E) lLb
1201
80
B) lysates
30[
T
-
---cRE-neo
REPHlO
----C R19-neo
Rlg-PH,O
7
IL-la antisense
transfectants
X 40
T
C) TNF a
l2O1
---C
--.O-
REneo
REPH10
Re-neo
RB-PHlO
RlS-neO Rig-PH10
FIGURE 7 - IL-1 activity in culture supernatants and lysates of
anti-sense IL-la-transfected A375 cell clones and control transfectants. Cells (1.0 x 105/1ml/well) were cultured in 24-well plates.
After 24 hr, supernatants were obtained. Cell lysates were prepared by sonication. IL-1 activity was determined by DlON4M cell
proliferation assay, Mean f SD based on triplicate cultures is
shown. Differences in IL-1 activity in the supernatants and lysates
between neo transfectants and pEF-PH10 transfectants were
statisticallysignificant by unpaired t test: *p < 0.05, **p < 0.01.
DISCUSSION
FIGURE8 - Effects of IL-la, IL-6 and TNFa on the growth of
anti-sense IL-la-transfected A375 cell clones and control transfectants. Cells (2.0 x 104/well) were cultured for 3 days with or
without varying doses of IL-la (a), IL-6 (b) or TNFa (c). After
culture, cell proliferation was determined by staining with crystal
violet. Mean k SD based on triplicate cultures is shown.
These studies suggest that constitutive production of IL-la
in IL-1-resistant melanoma cells is not essential or enough to
their acquisition of resistance. Prior to this study, we compared
the characteristics of the IL-1-sensitive parent cells, A375-6,
with those of IL-1-resistant cells, R8 and R19. We noticed that
the major difference is the expression of IL-la at both protein
and mRNA levels (Araki et al., 1994). IL-la, but not IL-lp, is
constitutively produced by these resistant cells. Neither IL-la
nor -p is produced by the sensitive cells. Our first assumption,
therefore, is that the resistant cells are tolerant to IL-1 by
down-regulation of the IL-1 receptor (IL-1R) caused by
continuous exposure to endogenous IL-la. However, sensitive
and resistant cells expressed equal levels of IL-1R type I
mRNA, and they both produced IL-6 in response to exogenous
IL-1. Therefore, they appeared to express functional IL-lR, an
unlikely assumption.
We next hypothesized that the endogenous production of
IL-la may result in resistance. The close relation between
cytokine production by particular cell types and their sensitivity to cytokines is reported. Rat epithelial cells resistant to the
anti-proliferative effect of TGF-P showed increased TGF-P
mRNA expression compared to sensitive cells (Chapekar el al.,
1990). Long periods of treatment of TNF-sensitive tumor cells
with TNF induced resistance in these cells toward the cytokine, and the resistant cells exhibited the increased level of
TNFa mRNA (Spriggs et aZ., 1987). In addition, Himeno et al.
(1990) reported that TNF-sensitive murine fibrosarcoma L-M
cells acquired resistance to TNF by transfection of TNFa
expression plasmid and that TNF-resistant human cervical
cancer HeLa cells became sensitive to TNF by transfection of
anti-sense expression plasmid. There are a number of reports
showing that TNF shares biological functions with IL-1 (Bomford and Henderson, 1989), including anti-proliferative and
cytocidal effects on tumor cells. Therefore, we supposed that
spontaneous IL-la production in our IL-1-resistant cells is a
critical factor to acquire IL-1 resistance.
To investigate this hypothesis, we generated IL-la-producing cells from IL-1-sensitive A375-6 cells by transfection of
mature IL-la expression plasmid, pRSV-hmIL-la. IL-la production by the transfectant was confirmed at both mRNA and
IL-1 activity levels. Neither anti-sense nor neo transfectants
produced JL-1, eliminating the artifactual effect of transfec-
810
ITOH ETAL.
tion. However, the transfectant of mature sense IL-la to dependent kinase (Dinarello, 1994). At present, we do not
A375-6 did not mimic resistant cells with respect to the know which pathway is closely related to the IL-1 antisensitivities to IL-1 and IL-6, though their IL-1 production proliferative effect,As we found out that cells were arrested in
level is equal to that of one of the resistant cells. To know Go/GI phases (Morinaga et al., 1990), cell-cycle regulatory
whether there are any differences between transfectants and factors, including the cyclin-dependent kinase inhibitor, may
the resistant cells other than IL-1 production, these cells were be involved. Therefore, in resistant cells one of the downexamined for IL-6 production alone or in response to exog- stream pathways of post-receptor interaction with IL-1 will be
enous IL-1. The transfectant constitutively produced IL-6 at a deficient.
level equal to one of the resistant cells. As IL-6 production was
A second possibility is that resistant cells may express
inhibitable by IL-lRa, a competitive inhibitor of IL-1 binding protective molecules against the IL-1 effect. Over-expression
to their receptor, endogenous IL-1 appeared to induce IL-6 at of several protective molecules to stresses is reported to be
least partially in an autocrine manner. This characteristic is the induced by IL-1, including Mn-SOD, heat shock proteins and
same as resistant cells (Araki et al., 1994). However, the IL-6 autocrine production of some growth factors. One of the
level produced by the transfectant in response to exogenous factors, Mn-SOD, is reported to be responsible for the
IL-1 is usually low compared to those by resistant cells. In resistance to the IL-1 anti-proliferative effect in A375-6 cells
other words, the profile of IL-6 production in transfectant is (Hirose et al., 1993). In our experiment, although Mn-SOD
close to parent IL-1-sensitive cells rather than resistant cells. mRNA expression increased in the transfectant, the level was
When stimulated with LPS, the transfectant also produced an low compared to the resistant cells. Therefore, the low-level
increased level of IL-6, and the level was equal to that of expression of Mn-SOD may explain the inability to acquire
parent cells but less than that of resistant cells (data not resistance.
shown). We also examined these cells in Mn-SOD mRNA
A final explanation is that it is impossible to obtain a
expression alone or in response to IL-1. Mn-SOD is known to transfectant producing much more IL-1 because growth is
be induced by IL-1 or TNF (Masuda et a/., 1988) and is inhibited by IL-1. This is plausible because the level of
reported to be one of the mechanisms for their resistance to Mn-SOD in transfectants should be increased to the level of
either IL-1 or TNF (Hirose et al., 1993). Similar to resistant resistant cells if they produce much more IL-1. Actually, our
cells, Mn-SOD mRNA levels of transfectant increased without transfectant produced IL-1 at about 2 U / m l / l x lo5 cells for 3
stimulation, probably due to the autocrine effect of endog- days, which is not enough to inhibit the growth of A375 cells.
enous IL-1. However, the level is low compared to resistant Although the data are not shown, we also transfected A375-6
cells. In response to IL-1, the mRNA increased to a level cells with sense precursor IL-la expression plasmid with
similar to one of the resistant cells, R8. These results indicate promoter EF-la. In these experiments, the transfectants also
that the characteristics of the IL-la transfectant are similar, produced low levels of IL-1, and their sensitivities to cytokines
but not identical, to those of resistant cells and suggest that did not differ from parent cells, probably because either
expression of endogenous IL-la is unable to induce resistance processing of precursor IL-la is not sufficient in the cells or the
to IL-1.
transfectants producing more IL-1 were unable to proliferate.
As a next step to determine the role of endogenous IL-la in
Although the endogenous production of IL-la is not essenresistance, we inhibited production of IL-la in resistant cells tial for the acquired resistance to IL-1, the close association
by transfection of IL-la anti-sense expression plasmid, pE- between resistance and IL-1 production is intriguing considerF(PH)-10. In these resistant cells, constitutive production of ing the malignancy of tumor cells. It is reported that progresIL-1 decreased by anti-sense transfection by 4344% in super- sion of human melanoma cells often accompanies the producnatants and by 65-93% in cell lysates. However, the sensitivity tion of IL-1 or IL-6 and resistance to these cytokines (Lu and
of the transfectant to IL-1, IL-6 and TNF is equal to parent Kerbel, 1994; Kerbel, 1992). Indeed, our 2 resistant cells are
cells. These findings further strengthen the notion that sponta- resistant to both IL-1 and IL-6 and are less sensitive to
neous IL-la production is not essential or sufficient for oncostatin M than IL-1-sensitive cells (Onozaki et al., 1995).
acquisition of resistance to IL-1.
Such alterations will influence the relationship between tuThe mechanism of how A375-6 melanoma cells acquire mors and host, e.g., ectopic growth ability, regulation of matrix
resistance to IL-1 is not apparent. One of the resistant clones metalloproteinase, expression of adhesion molecules and host
(R19) lacks ILdR; thus, R19 does not transduce the IL-6 immunity. We believe that these cells are good models to
signal (Onozaki et al., 1995). However, another resistant clone, investigate the mechanism of progression of human melanoma
R8, expresses IL-6R, and IL-6-dependent tyrosine phosphory- cells.
lation was observed. Therefore, in R8 cells, the post-receptor
signaling pathway of IL-6 leading to growth inhibition may be
deficient. Therefore, it is possible that several pathways are
ACKNOWLEDGEMENTS
deficient in the resistant cells, though they both produce IL-la.
This work was supported in part by grants from the Ministry
There are many signaling pathways in IL-1, including activation of protein kinase A, protein kinase C, mitogen-activated of Education, Science and Culture of Japan, Kowa Life
protein (and its related) kinase, Tyr-kinase and ceramide- Science Foundation and Ajinomoto Co., Ltd.
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