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. 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