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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Interleukin-6 (IL-6) is involved in regulation of the immune response, acute phase reaction, and cell proliferation. The aim of this study was to investigate whether IL-6 is implicated in cell proliferation of human non-small-cell lung cancer (NSCLC) cell lines. We analyzed IL-6 messenger RNA (mRNA) and protein expression in eight NSCLC cell lines: A549, Calu3, Calu6, H23, H522, H810, H1155, and H1299. The A549, Calu3, Calu6, and H23 cell lines expressed IL-6 mRNA and protein. In these cell lines, fetal calf serum (FCS) significantly increased cell proliferation as assessed by thymidine incorporation. In the presence of IL-6 antisense oligonucleotides, both proliferation and IL-6 synthesis were downregulated. In contrast, IL-6 mRNA and protein could not be detected in the NSCLC cell lines H522, H810, H1155, and H1299. In these NSCLC cell lines, FCS only marginally increased cell proliferation and IL-6 antisense oligonucleotides did not affect cell proliferation. The addition of neither exogenous IL-6 nor neutralizing anti-IL-6 antibodies affected cell proliferation in any of the experiments. Our data thus provide evidence that intracellular IL-6 is required in the control of cell proliferation in a subset of human NSCLC cell lines. We suggest the existence of two subtypes of NSCLC, an IL-6-dependent and an IL-6-independent type.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Interleukin-6 (IL-6) is a glycoprotein produced by many
different cell types including monocytes, macrophages, fibroblasts, keratinocytes, and endothelial, mesangial, and
glial cells (1). IL-6 has a molecular weight of 20-30 kD, depending on posttranslational modifications such as N- or
O-glycosylation (2) or phosphorylation (3). Synthesis of
IL-6 is induced by a wide variety of mitogens such as lymphotoxin (4), IL-1, tumor necrosis factor (TNF) (5),
platelet-derived growth factor (PDGF) (1), platelet-activating factor (PAF) (10, 11), and granulocyte-inhibitory
protein (12). Expression of IL-6 is regulated by two different pathways: (1) an autocrine pathway; and (2) a paracrine, mitogen-mediated pathway via IL-1, TNF-
, or one
of the PDGF isoforms (13). In human lung fibroblasts,
PDGF induces transcription of IL-6 (17), whereas stimulation of IL-6 by IL-1 or TNF- is due to de novo synthesis
and stabilization of IL-6 messenger RNA (mRNA) (6).
Increasing evidence suggests that IL-6 is involved in the control of cell proliferation of nontransformed cells, such as pulmonary fibroblasts (17, 18), mesangial cells (17, 19), and vascular smooth muscle cells (VSMC), (17, 20). Recently, it has been reported that human fibroblasts, VSMC, and mesangial cells produce large amounts of IL-6 protein upon stimulation with PDGF isoforms, and that the mitogenic response to PDGF involves intracellular IL-6 (17).
Clinical investigations found increased serum levels of IL-6 in patients with lung cancer (39%), whereas IL-6 was not detected in the serum of patients with benign lung diseases (21). In addition, increased expression of IL-6 has been described in renal-cell carcinoma (22), ovarian carcinoma (23), pleural mesothelioma (24), parotid-gland adenoma (25), pheochromocytoma (26), glioblastoma (27), and prostate carcinoma (28). Antisense oligonucleotides to IL-6 mRNA were used in vitro to inhibit IL-6 production and to inhibit cell proliferation in several malignant cell lines, including lines of Kaposi's sarcoma (29), hairy-cell leukemia (13), malignant melanoma (30), and ovarian carcinoma (23) cells. Levy and colleagues reported that IL-6 antisense inhibition of cell proliferation could be reversed in myeloma cell lines by the addition of IL-6 protein (16).
In summary, IL-6 is involved in the regulation of cell proliferation in transformed and nontransformed cells. In this study, we investigated its regulatory potency on the proliferation of eight characterized human non-small-cell lung cancer (NSCLC) cell lines.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cell Culture
Eight NSCLC cell lines
A549, Calu3, Calu6, H23, H522,
H810, H1299, and H1155were obtained from the American Type Culture Collection (ATCC, Rockville, MD).
The six NSCLC cell lines A549, Calu3, Calu6, H23, H522,
and H1299 were cultivated in RPMI 1640 medium supplemented with 8 mM L-glutamine (GIBCO-BRL, Basel,
Switzerland) and 5% fetal calf serum (FCS) (Fakola,
Basel, Switzerland). The cell lines H810 and H1155 were
cultivated in RPMI 1640 medium supplemented with 2%
FCS and 0.001% serum extender MITO+TM (Becton Dickinson, Bedford, MA).
All experiments were performed with subconfluent cells (80%). Prior to stimulation, cells were growth-arrested by serum starvation in RPMI 1640 supplemented with 8 mM L-glutamine for 24 h. Starvation medium was replaced every 12 h to prevent autostimulation of the cells. Quiescent cells were stimulated with growth medium in the presence or absence of the indicated concentrations of phosphorothioated IL-6 sense or antisense oligonucleotides (17).
Phosphorothioate-Modified IL-6 Oligonucleotides
A 21-base IL-6 antisense phosphorothioated oligonucleotide (PTO) (5'-dGTG GGC TGC AGG GCA GGA TGA-3'), specific for a sequence in the first exon of the IL-6 gene, was used in all experiments on cell proliferation and IL-6 synthesis. The efficacy of this IL-6 antisense PTO (MWG-Biotechnology, Münchenstein, Switzerland) has previously been demonstrated (17). To exclude nonspecific effects of PTOs, IL-6 sense oligonucleotides, complementary to the IL-6 antisense PTO, were used at the same concentrations in all experiments. Three hours before cell stimulation, IL-6 PTOs (antisense or sense) were added at the indicated concentrations (1 µM, 2 µM, 5 µM, and 10 µM). After 12 h, half the initial concentration of IL-6 PTOs was added to the cultures.
Thymidine Incorporation
The mitogenic effect of FCS was determined by incorporation of [3H]thymidine (1 µCi/ml; Amersham, Little Chalfont, UK) following a standard protocol as originally described by Chesterman and colleagues (31). De novo synthesis of DNA was assessed by counting the incorporation of [3H]thymidine in the presence of IL-6 antisense PTOs or IL-6 sense PTOs. [3H]thymidine incorporation was determined 24 h after stimulation. All experiments were performed in triplicate for each cell line in at least three independent sets of experiments.
Reverse Transcription-Polymerase Chain Reaction Analysis of IL-6 mRNA
Total RNA was extracted from the NSCLC cell lines with
TrizolTM reagent (GIBCO BRL) according to the instructions of the manufacturer. Purity of RNA was measured
spectrophotometrically at 260 nm. All RNA preparations
had an A260/A280 ratio of > 1.75. Complementary DNA
(cDNA) was generated by reverse transcription (RT) of
600-ng aliquots of total RNA in a total volume of 20 µl
containing 1× polymerase chain reaction (PCR) buffer
(20 mM Tris HCl, pH 8.3; 50 mM KCl; 5 mM MgCl2; 1 mM
of each deoxynucleotide triphosphate [dNTP], 1 U ribonuclease [RNase] inhibitor, and 2.5 U murine leukemia virus
[MuLV] reverse transcriptase) at 42°C for 15 min (PCR
reagents were purchased from Perkin Elmer, Applied Biosystems Division, Foster City, CA). Ten microliters of the
RT reaction were used for PCR amplification in a total
volume of 50 µl, using 0.5 U Taq-polymerase and primers
(15 pmol each) corresponding to nucleotides 229-251 and
445-467 of the IL-6 cDNA (32), and to nucleotides 437-459
and 749-772 of 
-actin cDNA, respectively.
Amplification was done with 20 cycles for -actin cDNA
and 35 cycles for IL-6 cDNA, with denaturation at 98°C
for 15 s, primer annealing at 62°C for 15 s, and extension at
72°C for 30 s, followed by a final extension at 72°C for 5 min. Expected fragments from this process should exhibit
239 bp for IL-6 and 336 bp for -actin. Aliquots of 12 µl of
each PCR reaction mixture were size-fractionated by electrophoresis on 3.0% agarose gels (NuSieve GTG; FMC
BioProducts, Rockland, ME) in TBE-buffer (89 mM Tris
base, 89 mM boric acid, 2 mM ethylenediamine tetraacetic
acid [EDTA]).
Expression of IL-6 Protein
Secreted amounts of IL-6 were assessed with commercially available enzyme immunoassays (EIA; R&D Systems, Oxon, UK). In brief, cells were seeded onto 48-well cell-culture plates (Falcon, Basel, Switzerland) and growth-arrested by serum starvation for 24 h prior to stimulation. Aliquots of 100 µl of cell-culture media were collected at 24, 36, or 48 h after addition of growth medium. Unstimulated cells were used as a control at each time point. EIA was performed according to the manufacturer's instructions.
Statistical Analysis
The Mann-Whitney U test was used to compare the effects of FCS-treated and unstimulated cells, and the effects of antisense or sense oligonucleotides on FCS-stimulated cell proliferation. The null hypothesis was: (1) there is no difference between FCS-stimulated and nonstimulated cells, and (2) there is no effect of antisense treatment on FCS-stimulated cell proliferation.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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IL-6 Expression in NSCLC Cell Lines
NSCLC cell lines A549, Calu3, Calu6, H522, H1155, and
H1299 were screened for IL-6 expression at the mRNA
level with RT-PCR, and at the protein level with EIA of
cell-culture media. As shown in Figure 1, RT-PCR revealed the presence of IL-6 mRNA in the three NSCLC
cell lines A549, Calu3, and Calu6. IL-6 mRNA was not detected in the NSCLC cell lines H522, H1155, and H1299
(Figure 1). The 239-bp band (Figure 1, arrow b) corresponded to the amplified fragment of IL-6 mRNA, the
336-bp fragment (Figure 1, arrow a) corresponded to the
constitutively expressed -actin mRNA.
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These results were confirmed at the protein level by the demonstration through EIA of the presence of IL-6 protein in culture media of the cell lines A549, Calu3, and Calu6 (Table 1). A549 cells spontaneously secreted 12 ± 4 (mean ± SEM) pg/10,000 cells/24 h of IL-6 protein. IL-6 secretion was increased to 52 ± 10 pg/10,000 cells/24 h in the presence of 5% FCS. Spontaneous secretion of IL-6 protein by Calu3 cells was 12 ± 4 pg/10,000 cells/24 h, and was augmented in the presence of 5% FCS to 140 ± 15 pg/ 10,000 cells/24 h. The highest amount of IL-6 protein was secreted by Calu6 cells, with a spontaneous secretion of 1,300 ± 265 pg/10,000 cells/24 h. Addition of 5% FCS stimulated IL-6 secretion to 2,350 ± 271 pg/10,000 cells/24 h in these cells (Table 1). The NSCLC cell line H23 did not spontaneously secrete IL-6 protein within 24 h, whereas a spontaneous IL-6 secretion of 5 ± 2 pg/10,000 cells was detected at 48 h. Stimulation with 5% FCS induced the secretion of 15 ± 5 pg/10,000 cells/24 h. The increases in IL-6 synthesis in response to FCS in the four NSCLC cell lines A549, Calu3, Calu6, and H23 were statistically significant, at P < 0.01 (Mann-Whitney U test, two tailed).
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In contrast, IL-6 protein secretion was not detected in culture media of the cell lines H522, H810, H1155, and H1299, either spontaneously or in the presence of 5% FCS (Table 1).
IL-6 Antisense PTOs Reduced the Expression of IL-6 Protein
Expression of IL-6 protein in culture media was measured in the presence of IL-6 antisense or sense PTOs for FCS-activated cells.
IL-6 antisense PTOs significantly reduced IL-6 protein secretion as determined in culture media of the three cell lines A549, Calu3, and Calu6 (Table 2). In cell line A549, stimulated with 5% FCS, IL-6 antisense PTOs (5 µM) decreased IL-6 protein secretion from 52 ± 10 pg/10,000 cells/24 h to 14 ± 4 pg/10,000 cells/24 h. In cell line Calu3, stimulated with 5% FCS, IL-6 secretion decreased from 140 ± 15 pg/10,000 cells/24 h to 72 ± 22 pg/10,000 cells/ 24 h. In cell line Calu6, IL-6 protein secretion was only marginally reduced in the presence of 5 µM IL-6 antisense PTOs. However, upon increasing the concentration of IL-6 antisense PTOs to 20 µM, an inhibitory effect on IL-6 secretion from 2,350 ± 271 pg/10,000 cells/24 h to 1,765 ± 225 pg/10,000 cells/24 h (Table 2) was observed.
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FCS Increased the Proliferation of NSCLC Cell Lines A549, Calu3, Calu6, and H23
Cell proliferation in the NSCLC cell lines was evaluated by [3H]thymidine incorporation, both in unstimulated and in FCS-stimulated cells (Table 3).
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The A549 cell line spontaneously incorporated 843 ± 47 cpm of [3H]thymidine. In the presence of 5% FCS, the incorporation was significantly increased to 5,052 ± 268 cpm (P < 0.01). Unstimulated Calu3 cells incorporated 1,318 ± 91 cpm, and in the presence of FCS (5%) the [3H]thymidine incorporation was increased to 9,983 ± 644 cpm (P < 0.01). Calu6 exerted a spontaneous incorporation of 1,618 ± 396 cpm, and in the presence of FCS the incorporation increased to 5,519 ± 381 cpm (P < 0.01). Unstimulated H23 cells incorporated 2,723 ± 415 cpm, and in the presence of FCS the incorporation rose to 7,615 ± 725 cpm. The growth-stimulatory effect of FCS was dose-dependent in these four cell lines (data not shown). In contrast to the A549, Calu3, Calu6, and H23 cell lines, the four NSCLC cell lines H522, H810, H1155, and H1299 showed no significant increase in thymidine incorporation in response to FCS. However, they exhibited a high spontaneous incorporation of [3H]thymidine. The H522 cells spontaneously incorporated 4,190 ± 450 cpm, and in the presence of FCS (5%) incorporated 3,660 ± 662 cpm. The effect of FCS on H522 cells was not statistically significant (P > 0.05). H810 cells exhibited a spontaneous [3H]thymidine incorporation of 5,435 ± 762 cpm and an FCS-induced incorporation of 6,345 ± 846 cpm (statistically not different). H1155 cells spontaneously incorporated 10,516 ± 763 cpm, whereas in the presence of FCS (5%) the incorporation was increased to 13,349 ± 1,653 cpm, but the difference was not statistically significant (P > 0.05). H1299 cells exhibited a spontaneous incorporation of [3H]thymidine of 4,850 ± 492 cpm, and in the presence of FCS (5%) this increased to 4,925 ± 598 cpm (statistically not significant) (P > 0.05).
IL-6 Antisense PTOs Reduced the Cellular Proliferation of Four NSCLC Cell Lines
The effect of IL-6 antisense PTOs on cell proliferation was measured in both unstimulated and FCS-activated cells (Table 4).
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The spontaneous cell proliferation of NSCLC cell lines A549, Calu3, Calu6, and H23 was significantly inhibited in the presence of IL-6 antisense PTOs. In cell line A549, IL-6 antisense PTOs decreased spontaneous cell proliferation from 843 ± 47 to 459 ± 20 cells/24 h (P < 0.01). In cell line Calu3, IL-6 antisense PTOs decreased spontaneous cell proliferation from 1,318 ± 91 to 710 ± 57 cells/24 h (P < 0.01). In cell line Calu6, IL-6 antisense PTOs decreased spontaneous cell proliferation from 1,618 ± 396 cells/24 h to 825 ± 95 cells/24 h (P < 0.01). In cell line H23, IL-6 antisense PTOs decreased spontaneous cell proliferation from 2,723 ± 415 cells/ 24 h to 1,273 ± 357 cells/24 h (P < 0.01). In contrast, the NSCLC cell lines H522, H1155, and H1299 showed a high spontaneous cell proliferation that was not significantly affected by IL-6 antisense PTOs (P > 0.05, Table 4). IL-6 sense PTOs did not significantly affect the spontaneous proliferation in any of the eight NSCLC cell lines investigated (Table 4).
FCS-induced cell proliferation of the NSCLC cell lines A549, Calu3, Calu6, and H23 was inhibited in the presence of IL-6 antisense PTOs. In cell line A549, IL-6 antisense PTOs decreased FCS-induced cell proliferation from 5,052 ± 268 cells/24 h to 2,781 ± 178 cells/24 h (P < 0.01). In cell line Calu3, IL-6 antisense PTOs decreased FCS- induced cell proliferation from 9,983 ± 644 cells/24 h to 6,007 ± 290 cells/24 h (P < 0.01). In cell line Calu6, IL-6 antisense PTOs decreased FCS-induced cell proliferation from 5,519 ± 381 cells/24 h to 3,407 ± 312 cells/24 h (P < 0.01). In cell line H23, IL-6 antisense PTOs decreased FCS-induced cell proliferation from 7,615 ± 725 cells/24 h to 5,760 ± 79 cells/24 h (P < 0.05). FCS-induced [3H]thymidine incorporation in the three NSCLC cell lines H1299, H1155, and H522 was not significantly affected by addition of IL-6 antisense PTOs (all P > 0.05, Table 4). IL-6 sense PTOs did not significantly modulate [3H]thymidine incorporation in any of the eight NSCLC cell lines; all P values were > 0.12.
Figures 2a and 2b represent characteristic examples of the effect of IL-6 antisense PTOs on cell proliferation of unstimulated and stimulated cells in: (1) An NSCLC cell line expressing IL-6 (Calu3) (Figure 2a). FCS increased cell proliferation, and the IL-6 antisense PTOs inhibited proliferation of stimulated and unstimulated cells. (2) An NSCLC cell line not expressing IL-6 (H522) (Figure 2b). No significant effect on cell proliferation was observed in the presence of FCS or the IL-6 antisense PTOs.
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Effect of Exogenous IL-6 and IL-6 Antibodies
Addition of exogenous IL-6 at various concentrations (0.1-20 ng/ml) did not affect [3H]thymidine incorporation in any of the six cell lines in which this was investigated (data not shown). Neutralizing anti-IL-6 antibodies at various concentrations (1, 3, and 10 µg/ml) did not affect either spontaneous or FCS-induced cell proliferation in any of the six cell lines (data not shown).
All experiments described were performed on cells seeded at the same density.
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Discussion |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In the present study, we showed that four of eight NSCLC
cell linesA549, Calu3, Calu6, and H23endogenously
express IL-6 mRNA and protein. In reponse to 5% FCS,
an increase in IL-6 expression and cell proliferation was
observed in these NSCLC cell lines. The four other cell
linesH522, H810, H1155, and H1299lacking IL-6 expression, did not show an increase in cell proliferation in
response to 5% FCS. IL-6 antisense PTOs reduced IL-6
protein expression in IL-6-expressing NSCLC cell lines.
IL-6 antisense PTOs reduced both FCS-induced and spontaneous cell proliferation of A549, Calu3, Calu6, and H23
cells. No effect of IL-6 antisense PTOs on cell proliferation
was observed in the NSCLC cell lines H522, H810, H1155,
and H1299. These findings illustrate a central role of IL-6 in
the regulation of cellular proliferation of the NSCLC cell
lines A549, Calu3, Calu6, and H23. Results for the four
other NSCLC cell lines indicated that an IL-6-independent proliferation mechanism exists in some NSCLC cell lines.
The involvement of IL-6 in tumor progression has been suggested in various human tumors. Enhanced synthesis of IL-6 has been reported in cells of renal-cell carcinoma (22), ovarian carcinoma (23), pleural mesothelioma (24), parotid-gland adenoma (25), pheochromocytoma (26), and glioblastoma (27). IL-6 has been shown to regulate growth of Kaposi's sarcoma in patients with acquired immune deficiency syndrome (AIDS) (29), and AIDS-Kaposi's sarcoma-derived cell lines produced significant levels of IL-6. However, a direct correlation of enhanced IL-6 production and tumor proliferation has not been reported.
In this study we showed that IL-6 is secreted in four of eight NSCLC cell lines: A549, Calu3, Calu6, and H23. FCS significantly increased cell proliferation and the IL-6 protein level in these four NSCLC cell lines. The increase in cell proliferation and IL-6 secretion suggest the involvement of IL-6 in the regulation of cell proliferation in the IL-6-protein-secreting NSCLC cell lines.
The specificity of the antimitogenic action of IL-6 antisense PTOs in IL-8-expressing cells was further confirmed with a 24-bp IL-8 antisense PTO and the respective sense control oligonucleotide. In the presence of various concentrations (1-20 µM) of IL-6 antisense or sense PTOs, no antimitogenic effect or reduction of IL-6 synthesis was observed in FCS-stimulated human lung cell cultures (M. Roth, unpublished data).
We have previously shown that inhibition of IL-6 synthesis by specific IL-6 antisense PTOs results in downregulation of cell proliferation in untransformed human primary fibroblasts, VSMC, and mesangial cells (17). The antimitogenic effect of IL-6 antisense PTOs was paralleled by a decreased expression of two cell-cycle-related proteins: cdc2 and proliferating-cell nuclear antigen (PCNA). The antiproliferative effect of IL-6 antisense PTOs has been reported in several malignant cell lines, including those of Kaposi's sarcoma (29), hairy-cell leukemia (13), malignant melanoma (30), ovarian carcinoma (23) and prostate carcinoma (28). We demonstrate here that IL-6 antisense PTOs targeting IL-6 mRNA significantly inhibit IL-6 protein secretion in IL-6-secreting NSCLC cell lines. We also show an antiproliferative effect of IL-6 antisense oligonucleotides on both spontaneous and FCS-induced cell proliferation in the NSCLC cell lines A549, Calu3, Calu6, and H23. This inhibitory effect of IL-6 antisense oligonucleotides on cell proliferation or on IL-6 protein secretion was dose dependent. We conclude that IL-6 plays a key role in proliferative regulation of the NSCLC cell lines A549, Calu3, Calu6, and H23.
The observations that neither the addition of monoclonal neutralizing anti-IL-6 antibodies to cell-culture media nor the addition of exogenous IL-6 affected cell proliferation or IL-6 synthesis further characterize the IL-6-dependent proliferative pathway as intracellular. These findings agree with those of Lu and colleagues, who reported that melanoma cell lines constitutively producing IL-6 were significantly growth inhibited by IL-6 antisense PTOs but not by anti-IL-6 antibodies (30).
However, we also found that the regulation of proliferation in some NSCLC cell lines is IL-6 independent. The NSCLC cell lines H522, H810, H1155, and H1299 showed only a marginal increase in proliferation due to activation by FCS, and IL-6 antisense oligonucleotides did not affect the cell proliferation of these NSCLC cell lines. Porgador and coworkers reported that metastatic tumor cells did not produce IL-6 protein, and that exogenous IL-6 protein did not alter these cells' proliferation (33). Our results and those of Porgador and coworkers illustrate the existence of IL-6-independent cell lines.
Wu and associates reported that serum IL-6 levels reflect disease status of gastric cancer (34). It is not yet known whether the level of IL-6 protein secretion in certain lung-tumor cell lines can be linked with tumor progression or could be correlated with the tumor stage. However it has to be noted that each of the NSCLC cell lines investigated in the present study exerted a distinct response to FCS and IL-6 antisense PTOs. It remains unclear whether these distinct responses can be linked to the passage number of the NSCLC cell lines, or whether they reflect specific biologic properties of the original carcinoma. Investigations are needed to characterize the role of IL-6 in the proliferation control mechanism in primary tumor-cell culture.
In conclusion, we investigated the role of IL-6 in cell proliferation in eight NSCLC cell lines. We demonstrated that in the four IL-6 protein-expressing NSCLC cell lines, IL-6 protein is involved in cell proliferation. This approach allowed us to consider two types of NSCLC cell lines on the basis of their ability to secrete IL-6 protein. Circumvention of the IL-6 pathway in non-IL-6-secreting NSCLC cell lines probably constitutes an essential step in the progression of tumor development, correlated with a high spontaneous proliferative capacity.
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Footnotes |
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Address correspondence to: Michael Roth, Ph.D., Division of Pneumology, Department of Internal Medicine and Research, University Hospital Basel, Hebelstrasse 20, CH-4031 Basel, Switzerland. E-mail: rothm{at}ubaclu.unibas.ch
(Received in original form November 21, 1997 and in revised form February 2, 1998).
Acknowledgments: This research was supported by the Krebsliga beider Basel through grant F1/95. The authors particularly thank Victoria Bruce and Dr. Oliver Eickelberg for helpful advice and stimulating discussion. This work was presented in part at Minisymposium C19, "Cellular and Molecular Aspect of Lung Cancer," of the annual meeting of the American Thoracic Society, May 17-22, 1997, in San Francisco.
Abbreviations dNTP, deoxynucleotide triphosphate; FCS, fetal calf serum; IL-6, interleukin-6; NSCLC, non-small-cell lung cancer; PDGF, platelet- derived growth factor; PTO, phosphorothioated oligonucleotide; RT-PCR, reverse transcription-polymerase chain reaction; VSMC, vascular smooth-muscle cells.
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References |
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Materials and Methods
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Discussion
References
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