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Interleukin-6 sensitizes multiple myeloma cell lines for apoptosis induced by interferon-α
Experimental Hematology 28 (2000) 244–255
Interleukin-6 sensitizes multiple myeloma cell lines for apoptosis induced by interferon-␣ Rumi Minamia, Koichiro Mutaa, Choi Ilseunga, Yasunobu Abea, Junji Nishimurab, and Hajime Nawataa a Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan; Department of Clinical Immunology, Medical Institute of Bioregulation, Kyushu University, Beppu, Japan
b
(Received 24 May 1999; revised 3 October 1999; accepted 2 November 1999)
Objective. Interleukin-6 (IL-6) is a multifunctional cytokine affecting growth and survival of normal B cell lineage and multiple myeloma cells. To test the hypothesis that IL-6, as well as other hematopoietic growth factors, may enhance apoptosis of target cells, we investigated the effect of IL-6 on myeloma cells in the presence of IFN-␣, which is prescribed for patients with multiple myeloma. Materials and Methods. Four myeloma cell lines, PCM6, NOP-2, U266, RPMI8226 were tested. We determined the induction of apoptosis by flow cytometry, using an FITC-Annexin V. Results. IFN-␣ induced apoptosis on myeloma cell lines, and this apoptosis was further enhanced in the presence of IL-6, via activation of caspase 3. During induction of this apoptosis, the expression of c-Myc and Fas increased. The addition of IL-6 further increased the expression of Fas, but not that of c-Myc. Bcl-2, Bcl-x, and p53 were not affected by the addition of IL-6 and/or IFN-␣. Addition of a PI-3-K inhibitor interfered with the enhancing effect of IL-6 on the apoptosis induced by IFN-␣. Conclusion. We propose that IL-6 has the death signal, as well as growth promoting effects, and that PI-3-K may play a key role in the induction of apoptosis by IL-6. © 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc. Keywords: Interleukin-6—Interferon-␣—Myeloma cell—Apoptosis— Phosphatidylinositol-3-kinase
Introduction IFN-␣, which inhibits cell growth and induces cell death, has been prescribed for treatment of multiple myeloma. It was reported that IFN-␣ induces G1 phase arrest of the B cell lineage such as Burkitt lymphoma cells and multiple myeloma cells by suppressing G1 cyclins [1] and/or by upregulating expression of CDK inhibitors [2]. In some myeloma cell lines, functional interleukin-6 (IL-6) receptors are downregulated by IFN-␣ [3,4]. IFN-␣ has also been reported to upregulate FAS on multiple myeloma cell lines such as U266 [5]. Fas is expressed on activated lymphoid cells, on normal CD34⫹ cells, and in several hematologic malignancies [6–8]. Fas triggering leads to activation of caspase 8 [9] and other caspases such as caspase 3 [10], then subsequently apoptosis. In some cell types, Bcl-2 and Bcl-x inhibit this apoptotic pathway [11,12]. Offprint requests to: Rumi Minami, M.D., Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan; E-mail: [email protected]
IL-6 is a multifunctional cytokine that acts on various kinds of target cells, and modulates immune responses and hematopoiesis. It has been reported that IL-6 enhances proliferation [13] and survival [14] of myeloma cells in an autocrine [15] and paracrine fashion [16]. It is considered that the effects of IL-6 on proliferation and survival are dissociable and are likely to be mediated by distinct mechanisms [17]. After specifically binding to a cell surface receptor, which contains at least one subunit of the signal transducting protein, gp130 [18], IL-6 activates the Ras-MAPK (for mitogen-activated protein kinase) pathway [19,20]and JAK (for Janus kinase) -STAT ( for signal transducers and activators of transcription) pathway [21]. The proliferation signal is probably transmitted though the Ras pathway [22]. The mechanism by which IL-6 influences survival of myeloma cells has not been well characterized. It was reported that hematopoietic growth factor induces cell death of acute myeloblastic leukemia cells [23,24]. Stahnke et al. [25] reported the upregulation of Fas and increased Fas-induced apoptosis in bone marrow precursor cells exposed to IL-3, GM-CSF, or G-CSF. All this evidence suggests that cell
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death is linked to cell proliferation and that hematopoietic growth factors possibly enhance apoptosis of the cells under certain condition. To test the hypothesis that IL-6, as well as other hematopoietic growth factors, may enhance apoptosis of target cells, we investigated the effect of IL-6 on myeloma cells in the presence of IFN-␣. We report here that IL-6 has an sensitizing effect on apoptosis of myeloma cells. When examining four IFN-␣ responsive myeloma cell lines, we also found that the magnitude of apoptosis-sensitizing effect correlated with that of growth-promoting effects of IL-6, and that phosphatidylinositol 3-kinase (PI-3-K) plays a pivotal role in determining the function of IL-6.
Materials and methods Reagents Recombinant IL-6 (rIL-6, specific activity, ⬎107 U/mg) was kindly provided by Kirin-Brewery Co Ltd. (Tokyo, Japan) and natural human IFN-␣ (specific activity, 102 U/ng) was kindly provided by Sumitomo Pharmaceuticals (Tokyo, Japan). Anti-humanFas monoclonal antibody, which stimulates Fas-mediated apoptotic pathway (anti-Fas) CH-11 (Beckman Coulter Co., Miami, FL), mouse IgM GC323 (Immunotech, Marseille, France), a peptide caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD-fmk; Medical & Biological Laboratories Co., Nagoya, Japan), fetal calf serum (FCS; Commonwealth Serum Laboratories, Melbourne, Australia), detoxified bovine serum albumin (BSA; Stem Cell Technologies Inc, Vancouver, BC), ironsaturated transferrin (Sigma Chemical Co., St. Louis, MO) were added to cultures, as indicated. LY294002 [26], a PI-3-K inhibitor, was purchased from Calbiochem (Nottingham, UK) and stock solutions (100 mM) were prepared in DMSO, and kept at ⫺20⬚C. Final dilutions were prepared immediately prior to use. Myeloma cell lines PCM6 is a human myeloma cell line cultivated in our laboratory, and which derived from peripheral blood of a patient with advanced IgG myeloma [27]. The status of mycoplasma infection is negative. The viability and growth of this cell line are suppressed by removing rIL-6 from the medium when cells are growing exponentially in serum-containing media, therefore we consider this cell line to be IL-6 dependent. Myeloma cell lines, NOP-2 [28], U266, and RPMI 8226 were also used. NOP-2 is a human myeloma cell line, established from a Bence-Jones type myeloma patient in Nagoya University School of Medicine (Japan). U266 was obtained from American Type Culture Collection (ATCC) and RPMI 8226 was obtained from the Japanese Cancer Research Resources Bank. Cell culture PCM6 cells were grown in McCoy’s 5A modified medium (Life Technologies, Inc., Grand Island, NY) containing 20% FCS and 3 ng/mL rIL-6. U266, RPMI 8226, and NOP-2 were grown in RPMI 1640 medium (GIBCO BRL, Tokyo, Japan) containing 10% FCS. The cell cultures were performed at 37⬚C, 100% humidity, and 5% CO2 in air. Prior to experiments, to investigate the effects of rIL-6
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and/or IFN-␣ on apoptosis, the cells were washed in Daigo’s T medium (Nihon Pharmaceutical Co., Osaka, Japan) and pre-incubated for 60 hours in serum-free media consisting of Daigo’s T medium supplemented with 0.1% BSA and 300 g/mL transferrin in order to eliminate the effects of unknown growth factors in serum, and to synchronize cells in the G0/G1 phase. Viability of the cells after 60-hour incubation exceeded 90% in each experiment. The cells were then washed twice in McCoy’s 5A modified medium and resuspended in culture media containing 20% FCS, with or without rIL-6 (3 ng/mL), and/or IFN-␣ as indicated. The effects of Z-VAD-fmk, anti-Fas antibodies; and LY294002 were also tested, under these experimental conditions. To investigate the effects of IL-6 and the PI-3-K inhibitor on apoptosis in serum starved conditions, the cells were grown in culture media containing 20% FCS without rIL-6 for 60 hours, and then washed twice in Daigo’s T medium and transferred to serum-free media, prepared as described above, with or without rIL-6 and/or LY294002 as indicated. [3H] Thymidine incorporation Effects of rIL-6 or IFN-␣ on proliferation of PCM6 were determined by measuring DNA synthesis, using [3H] thymidine, as described [29]. Briefly, 0.5 mL of serum-free cultures of 105 PCM6 cells were cultured with or without cytokine (rIL-6 or IFN-␣), then incubated with 1.25 Ci of [3H] thymidine (6.7 Ci/mmol; New England Nuclear Corp.) for 1 hour. The cells were then collected by centrifugation, washed with MacCoy 5A, and placed in 10% icecold trichloroacetic acid (TCA). The cell precipitates were collected on glass filter discs (type GF/A;Whatman, Maidstone, England), washed with 5% ice-cold TCA, followed by 95% ethanol, air dried, placed in scintillant and counted in a scintillation counter. [3H] thymidine incorporation was expressed as a percentage of cellular radioactivity in cultures in the absence of cytokines. Assays for detection of apoptosis Viability of the cells was determined by trypan blue exclusion. To determine the effect of IFN-␣ on DNA cleavage during apoptosis of PCM6, the amount of fragmented DNA was measured, as described [30]. [3H] thymidine (0.5 Ci/mL) was added to 2.5⫻106 cells and incubation was carried out for a further 30 minutes. Replicate [3H]thymidine-labeled cells (5⫻106) were then incubated in 1 mL of culture medium containing 2 mol/L thymidine and 2 mol/L deoxycytidine (Sigma Chemical Co.), with or without IFN-␣. After a 16-hour culture, cell replicates were collected and DNA was extracted with phenol:chloroform (1:1 v/v) and analyzed by electrophoresis on an alkaline pH, 0.6% agarose gel, as described [30]. Each lane was cut into eight 10-mm fractions and the radioactivity of each fraction was expressed as a percentage of the total radioactivity. Apoptotic cells were identified, using FITC-conjugated annexin V (FITC-annexin V; Immunotech), which has a high affinity for phosphatidylserine present in the outer membrane of apoptotic cells [31]. The assay was done according to recommendations of the manufacturer. Briefly, cells were washed twice in PBS and resuspended in 500 L of binding buffer containing 5 L of annexin V and 5 L of propidium iodide (PI) (250 g/mL) for 10 minutes on ice and in the dark. The fluorescence of FITC (FLT 1 LOG) and PI (FLT 4 LOG) was analyzed using flow cytometry (Epics ELITE; Beckman Coulter Co.).
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Cell cycle analysis After 60 hours of serum deprivation, we added FCS, rIL-6 and/or IFN-␣, and the proportion of the cells in each phase of cell cycle was determined, as described [32]. Briefly, the cells were washed with PBS, fixed with 50% methanol, treated with RNase A (1 mg/ mL) (USB Co., Cleveland, OH), and stained with PI (0.1% sodium citrate, propidium iodide 50 g/mL) (Sigma Chemical Co.). Cellular DNA content was then analyzed using a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). The percentages of cells in different phases of the cell cycle were determined after PI staining, using ModFit LT software. Western blot analysis Cells were lysed in buffer containing 62.5mM Tris-HCl (pH 6.8), 100mM dithothreitol, 2% (W/V) SDS, and 10% glycerol. Samples containing 100 g of protein were fractionated on sodium dodecyl sulfate (SDS) polyacrylamide gels and electroblotted onto a polyvinylene difluoride (PVDF) (ImmobilonTM; Millipore, Bedford, MA) filter in blotting buffer (48 mM Tris, 39 mM glycine, 20% [v/ v] methanol, 1.3 mM SDS); 7.5% acrylamide gel was used for the detection of Rb and c-Myc, 10% gel for p53, and 12.5% gel for Bcl-x, Bcl-2, and caspase3. The filters were blocked for 2 hours in blocking solution (5% low fat milk in PBS containing 0.5% Tween 20) and then were incubated for an additional 1 hour with specific antibodies. The antibodies used in this article were as follows: anti-human Rb antibody (G3-349) purchased from Pharmingen (San Diego, CA); anti-human c-Myc antibody (A-14) and antihuman Bcl-2 (N-19) purchased from Santa Cruz Biotechnology (Santa Cruz, CA); anti-human p53 antibody (AB-2) purchased from Calbiochem (Cambridge, MA); anti-human Bcl-x antibody and anti-human CPP/Caspase-3 antibody (19) purchased from Transduction Laboratories (Lexington, KY); and anti-human -actin antibody (N3509) purchased from Amersham Life Science (Buckinghamshire, England). Following extensive washing with PBS containing 0.5% Tween 20 (PBS-T), the filter was incubated for 30 minutes with horseradish peroxidase-conjugated anti-rabbit antibodies (whole Ig, NA934; Amersham Life Science) for c-Myc and Bcl-2, or anti-mouse antibodies (whole Ig, NA931; Amersham Life Science) for Rb, Bcl-x, p53, caspase3, and -actin. The filter was extensively washed in PBS-T, and bound antibodies were visualized using the enhanced chemiluminescene Western blotting system (ECL; Amersham Life Science). Blots were then stripped and reblotted with anti--actin Ab to ensure that equivalent levels of proteins were present in each lane. Immunofluorescence analysis of Fas/Apo-1 expression The cells were harvested at the indicated times and resuspended in 50 L of PBS containing 5% FCS, then incubated with 5 L of FITC-conjugated mouse anti-Fas antibody (clone UB2; Immunotech) on ice for 30 minutes and in the dark. Isotype-matched FITC conjugated mouse IgG (Immunotech) was used as a negative control. The cells were washed with PBS and analyzed using a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems). Caspase 3 activity assay The cell-permeable fluorigenic substrate (PhiPhilux-G1D2 caspase 3 activity detection kit; Onco Immunin, Inc., Paint Branch Drive Park, MD) was used to assay caspase 3 activity. This substrate is cleaved by activated caspase3 and produces fluorescence
at 505 nm, and the fluorescence peak is 530 nm. The cells were harvested and washed twice with PBS. The cell replicate (106 cells) was resuspended in 50 L of substrate solution containing 10% FCS, then incubation was carried out for 1 hour at 37⬚C in the dark. After incubation, the cells were washed once in flowcytometry dilution buffer and suspended in 500 L of flow cytometry dilution buffer. The fluorescence emission was determined using the FL-2 channel of a FACScan flow cytometer. Statistical analysis Each experiment was repeated three times to compare data on different groups, and paired t-test was used to determine the statistical significance.
Results IFN-␣ induces apoptosis on PCM6 cells To investigate the effect of IFN-␣ on apoptosis of PCM6, we examined the viability of cells cultured with IFN-␣. When we added IFN-␣ on exponential growing PCM6, the cells gradually died and the viability was 70.8% after culture for 72 hours. When we added IFN-␣ to synchronized cells conditioned by serum-free culture for 60 hours as described in the materials and methods section, the cells died more rapidly and the viability was 58.6% after culture for 48 hours. Thus, we did the following examinations under the latter condition. To investigate the effect of IFN-␣ on apoptosis of PCM6, binding of annexin V was determined, as described above (Fig. 1A). Early apoptotic cells were stained with FITC-annexin V alone (fraction 4), whereas late apoptotic or secondary necrotic cells were stained by both FITC-annexin V and PI (fraction 3). We defined FITCannexin V positive cells as apoptotic cells. When the cells were cultured with IFN-␣ (1000 U/mL), the percentage of the apoptotic cells (Fig. 1A[b]; 56.2%) exceeded than that seen in the culture without IFN-␣ (Fig.1A[a]; 12.6%). To confirm that IFN-␣ induced apoptosis, the pattern of DNA breakdown was also determined (Fig. 1B). DNA isolated from cells cultured with IFN-␣ showed a marked increase in DNA fragmentation, compared with the case without IFN-␣. The effect of IFN-␣ on proliferation, viability, and apoptosis varied with its concentration. As shown in Table 1, stimulation of cell proliferation was observed only with low concentrations of IFN-␣ (0.1 U/mL, 1 U/mL), while apoptosis, determined using annexin V, was dose-dependent (Table 1, Fig. 1C). IL-6 enhanced apoptosis induced by IFN-␣ To determine the effect of IL-6 on the apoptosis induced by IFN-␣, we examined the viability of PCM6 by trypan blue exclusion (Fig. 2A). While 58.6% ⫾ 4.2% of the cells cultured with IFN-␣ were viable after 48 hours, the percentage of viable cells decreased to 41.9% ⫾ 3.8% in the presence of IFN-␣ ⫹ rIL-6 (p ⬍ 0.01). When the percentage of apop-
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Figure 1. Apoptosis of PCM6 induced by IFN-␣. The cells were cultured in serum-free media for 60 hours, then incubated in culture media containing 20% FCS either with ([b] and 䊉) or without IFN-␣ ([a] and 䊊) [1000 U/mL, (A) and (B); various concentrations, (C)]. (A) Apoptosis was determined after 24 hours by labeling with FITC-annexin V and PI. Early apoptotic cells were stained with FITC-annexin V alone (Fraction 4), whereas late apoptotic cells were stained with both FITC-annexin V and PI (Fraction 2). (B) Effect of IFN-␣ on DNA breakdown of PCM6. DNA was extracted from cells that had been labeled with [3H] thymidine and incubated for 24 hours at 37⬚C in culture medium with (䊉) or without IFN-␣ (䊊), and analyzed on alkaline pH, 0.6% agarose gels. The radioactivity of each fraction is expressed as a percent of the total radioactivity in each lane. Numbered arrows indicate the migration of DNA marker fragments with the number of kilobases shown. (C) Flow cytometric analysis using FITC-annexin V was also done after culture for 24 hours under various concentrations of IFN-␣.
totic cells was measured, based on annexin V staining, the addition of rIL-6 significantly increased the number of apoptotic cells induced by IFN-␣ (Fig. 2B, p ⬍ 0.01). The effect of rIL-6 and IFN-␣ on apoptosis was synergistic. The number of apoptotic cells slightly increased, but not significantly, when cultured with rIL-6 alone, compared with the case without exogenous cytokines (Fig. 2B). The effect of IL-6 on IFN-␣ induced apoptosis was to a lesser degree and
appeared more slowly when IFN-␣, with or without rIL-6 was added to exponential growing cells (data not shown). When we examined the effect of IL-6 on the apoptosis induced by various concentrations of IFN-␣ (Table 1), the apoptosis sensitizing effect of IL-6 was obtained with all concentrations used, but as the effect was maximum with 1000 U/mL of IFN-␣, further examinations were made using the concentration of 1000 U/mL.
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Table 1. Effect of low concentration of IFN-␣ on proliferation, viability, and apoptosis Proliferation IFN-␣ (U/mL) 0 0.1 1 10 100 1000
Viability (%)
Apoptosis (%)
IL-6 (⫺)
IL-6 (⫹)
IL-6 (⫺)
IL-6 (⫹)
IL-6 (⫺)
IL-6 (⫹)
1.13 ⫾ 0.03 1.43 ⫾ 0.05 1.37 ⫾ 0.10 1.08 ⫾ 0.03 0.98 ⫾ 0.06 0.85 ⫾ 0.04
2.01 ⫾ 0.04 1.70 ⫾ 0.10 1.10 ⫾ 0.05 1.01 ⫾ 0.06 0.90 ⫾ 0.02 0.80 ⫾ 0.04
89.5 ⫾ 0.8 89.2 ⫾ 1.6 90.3 ⫾ 0.7 87.9 ⫾ 1.1 73.3 ⫾ 3.3 69.5 ⫾ 0.9
87.6 ⫾ 1.3 89.4 ⫾ 1.3 77.2 ⫾ 1.9 75.4 ⫾ 1.8 65.1 ⫾ 2.5 56.5 ⫾ 3.5
16.2 16.9 22.6 31.6 34.1 39.1
33.6 28.6 36.1 55.4 59.4 68.3
After culture in serum free media for 60 hours, the cells were suspended in culture media with rIL-6 and various concentrations of IFN-␣ (0, 0.1, 1.0, 10, 100, and 1000 U/mL). Cell proliferation was estimated as follows: the number of cells after 48-hour culture/the number of cells at start of the culture. After 24 hours, cells were harvested and viability and apoptosis were estimated. Viability was determined by trypan blue exclusion and apoptotic cells were detected by analysis of FITC-annexin V-stained cells on FACScan analysis. Results are the mean ⫾ SEM of three separate experiments.
Because the pattern of response to IFN-␣ differs with the cell line, we studied apoptosis of three other cell lines, U266, NOP-2, and RPMI 8226 by annexin V staining (Table 2). We concomitantly examined the effect of IL-6 on cellular proliferation by determining SI as indicated in the Table 2. Proliferation of these cell lines responded variably to rIL-6. PCM6 was most responsive and cells cultured with rIL-6 had approximately a twofold number over that without rIL-6. NOP-2 was most sensitive to IFN-␣ and apoptosis was induced within 24 hours. U266 and RPMI 8226 were less sensitive to IFN-␣ and apoptosis was induced more slowly (data not shown). Similar to findings with PCM6, apoptosis was enhanced in the presence of rIL-6 in U266. Also in NOP-2 and RPMI 8226, apoptosis was slightly increased in the presence of rIL-6, albeit not significantly. Therefore, the level of IL-6 mediated enhancement of IFN-␣-induced apoptosis by IL-6 paralleled SI, indicating that the degree of sensitization of apoptosis was closely related to the extent of proliferation response by rIL-6.
Figure 2. Effect of rIL-6 on apoptosis induced by IFN-␣. After culture in serum-free media for 60 hours, the cells were suspended in media containing 20% FCS either without exogenous cytokine (䊊) or with rIL-6 (䊉), IFN-␣ (1000 U/mL) (䊐), rIL-6 and IFN-␣ (䊏). (A) Viable cells were determined by trypan blue dye exclusion. (B) Apoptotic cells were detected by analysis of FITC-annexin V-stained cells on FACScan analysis. Results are the mean ⫾ SEM of three separate experiments. *Significantly different (p ⬍ 0.01) from corresponding values in cultures with IFN-␣ alone; † significantly different (p ⬍ 0.05) from corresponding values in cultures with IFN-␣ alone.
Effect of IL-6 and IFN-␣ on cell cycle To investigate the mechanism by which IL-6 enhances apoptosis of PCM6, we examined the effect of IL-6 and IFN-␣ on cell cycle progression (Fig. 3A) and DNA synthesis (Fig. 3B), using PCM6. At start of the culture, 64.9% ⫾ 2.7% of the cells were in G0/G1 phase (Fig. 3A[a]). In the absence of IFN-␣, the number of cells in S phase increased to 44.4% ⫾ 1.4% after a 24-hour culture. Addition of rIL-6 significantly increased the percentage of the cells in S-phase (54.0% ⫾ 1.9%, p ⬍ 0.05) (Fig. 3A[b]), but decreased the percentage in G0/G1 phase (Fig. 3A[a]). Stimulation of cell cycle progression by rIL-6 was also evident when we measured the extent of DNA synthesis, using [3H]thymidine (Fig. 3B). Addition of IFN-␣ resulted in G0/G1 arrest (Fig. 3A) with a remarkable decrease in DNA synthesis (Fig. 3B). Addition of rIL-6 to the culture together with IFN-␣ did not alter the pattern of cell cycle distributions based on findings seen with IFN-␣ alone (Fig. 3A). We also investigated some of the cell cycle regulating proteins, Rb, cyclinD1, and cyclinE (data not shown) by
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Table 2. Effect of rIL-6 on proliferation and apoptosis Apoptotic cells (%) (addition to culture)
PCM6 U266-B NOP-2 PRMI 8226
Cell proliferation
None
rIL-6
IFN-␣
IFN-␣ ⫹ rIL-6
1.90 ⫾ 0.01 1.37 ⫾ 0.03 1.17 ⫾ 0.02 1.03 ⫾ 0.07
20.7 ⫾ 4.7 14.4 ⫾ 4.5 17.4 ⫾ 2.1 24.0 ⫾ 2.2
27.0 ⫾ 5.4 15.0 ⫾ 5.1 22.4 ⫾ 1.3 28.1 ⫾ 0.3
45.2 ⫾ 9.2 42.0 ⫾ 5.7 47.7 ⫾ 1.4 40.4 ⫾ 1.7
62.4 ⫾ 8.4* 53.9 ⫾ 1.5* 51.5 ⫾ 1.1 42.0 ⫾ 0.8
After culture in serum free media for 60 hours, the cells were suspended in culture media either without exogenous cytokine or with rIL-6, IFN-␣ (1000 U/ mL), rIL-6 and IFN-␣. Cell proliferation was estimated as follows: the number of cells cultured in media with rIL-6 for 48 hours/the number of cells cultured in media alone for 48 hours. After 24-hour (PCM6), 48-hour (NOP-2), or 72-hour (U266, RPMI 8226) culture, cells were harvested and apoptotic cells were detected by analysis of FITC-annexin V-stained cells on FACScan analysis. Results are the mean ⫾ SEM of three separate experiments. Percent apoptosis in each group was compared using a paired t-test. * Significantly different (p ⬍ 0.01) from corresponding values in cultures with IFN-␣ alone.
western blot. Cells showed a transition from the hypophosphorylated to the hyperphosphorylated form of Rb when cells were treated without IFN-␣, and rIL-6 slightly upregulated the protein level of hyperphosphorylated Rb (Fig. 4). There was no remarkable difference in the protein level of cyclinD1 and cyclinE between cells treated without any cytokines or with IFN-␣, rIL-6, or IFN-␣⫹rIL-6 (data not shown). c-Myc was increased in the presence of IFN-␣ To investigate the molecular mechanism by which rIL-6 and IFN-␣ influence the apoptosis of PCM6, we examined expression of apoptosis-related proteins by western blotting (Fig. 4). Under serum-free conditions, c-Myc was not detected by western blotting (data not shown) but was induced within 12 hours after exposure to FCS, with or without exogenous cytokines. Addition of IFN-␣ remarkably increased the amount of c-Myc, which was most evident after a 24-hour culture. Addition of rIL-6 did not influence c-Myc expression. Neither IFN-␣ nor rIL-6 influenced the expression of p53, Bcl-2, and Bcl-x protein. Fas-related apoptosis pathway was enhanced by IFN-␣ and IL-6 PCM6 expressed Fas under regular culture conditions with FCS ⫹ rIL-6 (data not shown). When IFN-␣ was added to the culture, expression of Fas was significantly increased, compared to findings in cultures without IFN-␣ (Fig. 5A[a] and [c], and 5B). Addition of rIL-6 to the culture with IFN-␣ significantly increased the expression of Fas, compared with observations of cultures with IFN-␣ alone (Fig. 5A[c] and [d], and 5B). In the absence of IFN-␣, expression of Fas was not significantly increased by rIL-6 (Fig. 5A[a] and [b], and 5B). Next, we examined the activity of caspase3, which is activated by the stimulation of Fas and plays critical roles as a downstream mediator in Fas mediated apoptosis [10,12]. We preformed western blot analysis for caspase3 (Fig. 5D), and for the p17 fragment of caspase3, that is the activated form of caspase3, obtained from cells treated with
IFN-␣, and those treated with IFN-␣ plus IL-6. We wanted to determine if caspase3 would be activated in intact cells treated with exogenous cytokines by a cell-permeable fluorigenic caspase substrate, PhiPhilux G1D2. Addition of IFN-␣ significantly increased the activity of caspase3, as compared with findings without IFN-␣ (Fig. 5C[a] and [c], and 5D). When rIL-6 was added together with IFN-␣, activity of caspase3 was further increased over that seen with IFN-␣ alone (p ⬍ 0.05, Fig. 5C[c] and [d], and 5D). To confirm that caspases are involved in the apoptosis induced by IFN-␣, with or without IL-6, we examined the effect of the caspase inhibitor, Z-VAD-fmk, on apoptosis of PCM6 (Table 3). In cells treated with Z-VAD-fmk, apoptosis induced by IFN-␣ was inhibited and the apoptosis sensitizing effect of IL-6 was not observed. These results indicate that IFN-␣ induces a caspase dependent death and the apoptosis sensitizing effect of IL-6 also depends on caspases. To determining IL-6 influences the Fas-mediated signal to enhance apoptosis, we examined the effect of rIL-6 on apoptosis and caspase3 activity in the presence of anti-Fas (Fig. 6A and 6B) instead of IFN-␣. Addition of anti-Fas Ab increased the percentage of apoptotic cells, compared with findings without Ab, and the addition of rIL-6 plus anti-Fas Ab further increased the percentage of apoptotic cells, compared to findings with Ab alone (Fig. 6A). When the activity of caspase3 was measured, addition of rIL-6 plus anti-Fas Ab resulted in greater activity than that seen with anti-Fas Ab alone (Fig. 6B). These results suggest that IFN-␣ may induce apoptosis of PCM6 partially via a Fas-related pathway, and that IL-6 interacts and modulates the members of Fas signaling pathway such as Fas and caspase3. Apoptosis induced by serum deprivation was inhibited by IL-6 While IL-6 enhanced the apoptosis induced by IFN-␣, IL-6 is also a factor in survival of multiple myeloma cells. To investigate whether IL-6 has an anti-apoptotic function on PCM6 cells, we studied the effect of IL-6 on apoptosis in-
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Figure 4. Western blot analysis of Rb, c-Myc, Bcl-2, Bcl-x, p53 of PCM6. After culture in serum-free media for 60 hours, the cells were incubated in media containing 20% FCS either without exogenous cytokine (lane 1) or with rIL-6 (lane 2), IFN-␣ (1000 U/mL) (lane 3), rIL-6 and IFN-␣ (lane 4) for 12 hours (Rb), or 24 hours (c-Myc, Bcl-2, Bcl-x, p53). Lysates of cells of each group were subjected to SDS-PAGE electrophoresis and immunoblotted with Rb, c-Myc, Bcl-2, Bcl-x, p53 Abs. Blots were then stripped and reprobed with anti-actin Ab to ensure that equivalent levels of proteins were present in each lane.
Figure 3. Effect of rIL-6 and IFN-␣ on cell cycle status and DNA synthesis. After culture in serum-free media for 60 hours, PCM6 were suspended in media containing 20% FCS either without exogenous cytokine (䊊) or with rIL-6 (䊉), IFN-␣ (1000 U/mL) (䊐), rIL-6 and IFN-␣ (䊏). The percentages of cells in different phases of the cell cycle were determined after PI staining using ModFit LT software. (B) PCM6 (105 cells) were incubated in serumfree medium with or without a cytokine (rIL-6 or IFN-␣). Cells were pulsed with [3H] thymidine (1.25 Ci) for 1 hour, and the incorporated radioactivity was measured, as described in the materials and methods section. Results are the mean ⫾ SEM of three separate experiments. The p values were determined by using paired t-test. Percent apoptosis in each group was compared using paired t-test. *Significantly different (p ⬍ 0.01) from corresponding values in cultures without exogenous cytokines; †significantly different (p ⬍ 0.05) from corresponding values in cultures without exogenous cytokine.
duced by serum deprivation (Fig. 7A). When PCM6 cells cultured in media with FCS were transferred to serum-free media, apoptotic cells gradually increased and the percentage of apoptotic cells was smaller in the culture with than
without rIL-6 after culture for 72 hours (Fig. 7A; 21.9% ⫾ 0.3% with rIL-6 vs 42.3% ⫾ 2.4% without rIL-6). Similar results were obtained when the viability of the cells was determined by trypan blue exclusion (data not shown). Therefore, rIL-6 apparently protects PCM6 cells from the apoptosis induced by serum deprivation.
Effects of IL-6 on apoptosis as influenced by a PI-3-K inhibitor (LY294002) To examine the role of PI-3-K in the regulation of apoptosis by rIL-6 and IFN-␣, the effect of the PI-3-K inhibitor, LY294002, on apoptosis of PCM6 was determined. When LY294002 was added to the culture with IFN-␣ and/or rIL-6, PCM6 were protected from the apoptosis induced by IFN-␣, and the apoptosis-promoting effect of rIL-6 was abolished (Fig. 7B). In addition, LY294002 blocked cell cycle progression after exposure to FCS ⫹ rIL-6 at G0/G1 phase (data not shown). When the effect of LY294002 on apopto-
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Figure 5. Effect of IFN-␣ and rIL-6 on Fas expression and activity of caspase 3. After culture in serum-free media for 60 hours, the PCM6 cells were suspended in media containing 20% FCS either without exogenous cytokine [(a) and 䊊] or with rIL-6 [(b) and 䊉]; IFN-␣ (1000 U/mL) [(c) and 䊐]; rIL-6 and IFN-␣ [(d) and 䊏]. (A) Cells were stained with FITC-conjugated anti-Fas mAb after 0-hour (—) and 48-hour (—) culture. The population of Fas-positive cells was gated using mouse anti-IgG1 antibody (· · ·). The cells were analyzed by FACScan. The fluorescence intensity is given in logarithmic scale. Data are representative of one of three separate experiments. (B) Time course of Fas expression. Results are the mean ⫾ SEM of three separate experiments; p values were determined by using paired t-test. *Significantly different (p ⬍ 0.01) from corresponding values in cultures with IFN-␣ alone; †significantly different (p ⬍ 0.05) from corresponding values in cultures with IFN-␣ alone. (C) After 48 hours, cells were incubated with a fluorigenic caspase3 substrate PhiPhilux-G1D2 for 1 hour and then analyzed by FACScan (—). The fluorescence intensity is given in logarithmic scale. The percentages of cells with caspase3 activation are indicated. Nonapoptotic cells prestimulated by any cytokine we used served as negative controls, the absence of caspase3 activation (—). (D) Time course of caspase3 activation. Results are the mean ⫾ SEM of three separate experiments; p values were determined by using paired t-test. *Significantly different (p ⬍ 0.01) from corresponding values in cultures with IFN-␣ alone; †significantly different (p ⬍ 0.05) from corresponding values in cultures with IFN-␣ alone. The inset shows western blot analysis of caspase3. Cell lysates were obtained after 48-hour culture without exogenous cytokine (lane 1) or with rIL-6 (lane 2), IFN-␣ (1000 U/mL) (lane 3), and rIL-6 and IFN-␣ (lane 4).
Table 3. Effect of Z-VAD-fmk on apoptosis by IFN-␣ Apoptotic cell (%)
None IFN-␣ IFN-␣ ⫹ rIL-6
Z-VAD-fmk (⫺)
Z-VAD-fmk (⫹)
5.51 28.2 45.8
5.06 5.25 5.67
After culture is serum free media for 60 hours, PCM6 were incubated for 24 hours in media containing 20% FCS in the absence or presence of Z-VAD-fmk (20 M), without any cytokines, or with IFN-␣ (1000 U/mL), rIL-6 (3 ng/mL) plus IFN-␣. Apoptosis was detected by analysis of FITCannexin V-stained cells on FACScan.
sis induced by serum starvation was examined, addition of LY294002 led to an increase in the percentage of apoptotic cells, with or without rIL-6 (Fig. 7A). Because the absence of rIL-6 augmented the apoptosis, the anti-apoptotic effect of rIL-6 was not affected by LY294002. These data suggest that the function of IL-6, as a growth and apoptosis-promoting factor, is mediated by the activity of PI-3-K, however, the function as a survival factor is not related to PI-3-K.
Discussion We obtained evidence that IL-6 sensitized IL-6-responsive myeloma cells for apoptosis and stimulated cellular proliferation. We used four IFN-␣ sensitive myeloma cell lines,
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Figure 6. Effect of rIL-6 on Fas-induced apoptosis. After culture in serum-free media for 60 hours, PCM6 were suspended in media containing 20% FCS with control mouse IgM [(a)]; rIL-6 and control mouse IgM, anti-Fas (50 ng/mL) [(b)]; and rIL-6 and anti-Fas (50 ng/mL) [(c)]. (A) After 24 and 48 hours, the cells were harvested. Apoptotic cells were detected by analysis of FITC-annexin V-stained cells on FACScan analysis. Results are the mean ⫾ SEM of three separate experiments. Percent apoptosis in each group was compared using paired t-test. *Significantly different (p ⬍ 0.01) from corresponding values in cultures with IFN-␣ alone. (B) After a 24-hour culture, PCM6 were incubated with a fluorigenic caspase3 substrate PhiPhilux-G1D2 for 1 hour and then analyzed using FACScan. The fluorescence intensity is given in logarithmic scale. The percentages of cells with caspase3 activation are indicated. Nonapoptotic cells prestimulated by any cytokine we used served as negative controls, the absence of caspase3 activation (—). The percentages of cells with caspase3 activation are indicated. Nonapoptotic cells used served as negative controls, the absence of caspase3 activation. Data are representative of three separate experiments.
all of which respond to IL-6 to a different degree. Apoptosis was more strongly enhanced by rIL-6 when the cell line used was the more sensitive to rIL-6 in proliferative response. This suggests that signaling pathways to initiate apoptosis and cellular proliferation are simultaneously activated through the IL-6 receptor. It was recently reported that hematopoietic growth factors such as IL-3, G-CSF, or GM-CSF increased Fas-induced apoptosis of bone marrow precursor cells [25]. Together with our data, this suggests that cytokines that promote cellular proliferation also possess the “death signal,” related to the Fas/FasL system, as a intracellular negative-feed back mechanism. The role of the Fas system has been demonstrated primarily in cases of activation-induced cell death (AICD), which closely relates to the cellular proliferation of T lymphocytes. Expression of Fas and c-myc is required for T cells to
undergo AICD [33]. In the myeloma cell line PCM6, the addition of IFN-␣ increased expression of Fas and c-Myc prior to the initiation of apoptosis, as well as AICD. The activity of caspase3, one of the downstream components in Fas/Fas ligand (FasL) signaling pathway, also increased in the presence of IFN-␣. Addition of rIL-6 influenced this cascade by further enhancing Fas expression and caspase3 activity. Our data obtained using anti-Fas confirmed that IL-6 influences the apoptotic signal related to the Fas/FasL system, therefore, this system seems to play a key role in the signaling pathway of apoptosis induced by “growth factors.” Our finding that IL-6 enhanced Fas related apoptosis differs from the data of Chauhan et al. [34], which IL-6 inhibits Fas-induced apoptosis in the myeloma cell line, RPMI 8226. This discrepancy might relate to differences in experimental systems. The most obvious difference is that
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Figure 7. Effect of LY294002 on apoptosis induced by serum starvation and IFN-␣. (A) After culture in media containing 20% FCS without exogenous cytokine for 60 hours, PCM6 were incubated in serum-free media in the absence or presence of LY294002 (50 ng/mL) with or without rIL-6 (3 ng/mL). At the indicated times cells were harvested and apoptotic cells were detected by analysis of FITC-annexin V-stained cells on FACScan. (B) After culture in serumfree media for 60 hours, PCM6 were incubated in media containing 20% FCS in the absence or presence of LY294002 (50 ng/mL) without any cytokine we used or with rIL-6 (3 ng/mL), IFN-␣ (1000 U/mL), rIL-6 (3 ng/mL), and IFN-␣. Results are the mean ⫾ SEM of three separate experiments. Percent apoptosis in each group was compared using paired t-test. *Significantly different (p ⬍ 0.05) from corresponding values in cultures without exogenous cytokines; † significantly different (p ⬍ 0.05) from corresponding values in cultures with LY294002 alone; †significantly different (p ⬍ 0.05) from corresponding values in cultures with IFN-␣ alone.
an IL-6 independent myeloma cell line was used in the previous study [34]. Furthermore, unlike our culture condition, the cells were not synchronized in G0/G1 phase and were preincubated with rIL-6 prior to stimulation with anti-Fas. In IL-6 responsive PCM6 cells, IL-6 has a multifunction, such as stimulating proliferation, reducing apoptosis, and, as described here, enhancing apoptosis. It has been reported that different signals induced through gp130 may have distinct biologic functions; growth stimulation via the MAPK cascade and anti-apoptosis via other cascades including the STAT 1/3 pathway [35]. IL-6 may trigger proliferation, survival, and apoptosis-enhancing effects through different sig-
naling pathways. We examined the association of the effects of IL-6 with PI-3-K, which plays a critical role in signaling from gp130 subunit of IL-6 receptor [36]. PI-3-K is a downstream target of Ras [37] and seems to be important for differentiation or proliferation of various cells. On the other hand, it has been reported that Fas induces apoptosis by Ras-regulated PI-3-K activation in Jurkat cells [38], and Uddin et al. [39] reported the activation of PI-3-K serine kinase by IFN-␣ in U266 and Daudi cells. Our experiments clearly indicated that PI-3-K mediated the apoptosisenhancing effects of IL-6. All these observations taken together suggest that PI-3-K might play an essential role in
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the signaling pathway for the proliferation and apoptosis induced by cytokine stimuli, or activation of Fas. In conclusion, IL-6 is multifunctional and has distinct effects on both apoptosis and proliferation of myeloma cells. PI-3-K is likely to be involved in the function of IL-6. Further studies on precise mechanisms determining the direction of IL-6 action are warranted and may yield pertinent findings, which may aid in developing protocols for treating subjects with multiple myeloma.
Acknowledgments We are grateful to Michio Kawano, M.D., for critical comments on the manuscript. We also thank Seiichi Motomura, M.D., for helpful advice; Ms. Shizuko Aoki and Ms. Shiho Isewaki for technical support; Sumitomo Pharmaceuticals for human IFN-␣; and Ms. M. Ohara provided language assistance.
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Interleukin-6 sensitizes multiple myeloma cell lines for apoptosis induced by interferon-␣ Rumi Minamia, Koichiro Mutaa, Choi Ilseunga, Yasunobu Abea, Junji Nishimurab, and Hajime Nawataa a Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan; Department of Clinical Immunology, Medical Institute of Bioregulation, Kyushu University, Beppu, Japan
b
(Received 24 May 1999; revised 3 October 1999; accepted 2 November 1999)
Objective. Interleukin-6 (IL-6) is a multifunctional cytokine affecting growth and survival of normal B cell lineage and multiple myeloma cells. To test the hypothesis that IL-6, as well as other hematopoietic growth factors, may enhance apoptosis of target cells, we investigated the effect of IL-6 on myeloma cells in the presence of IFN-␣, which is prescribed for patients with multiple myeloma. Materials and Methods. Four myeloma cell lines, PCM6, NOP-2, U266, RPMI8226 were tested. We determined the induction of apoptosis by flow cytometry, using an FITC-Annexin V. Results. IFN-␣ induced apoptosis on myeloma cell lines, and this apoptosis was further enhanced in the presence of IL-6, via activation of caspase 3. During induction of this apoptosis, the expression of c-Myc and Fas increased. The addition of IL-6 further increased the expression of Fas, but not that of c-Myc. Bcl-2, Bcl-x, and p53 were not affected by the addition of IL-6 and/or IFN-␣. Addition of a PI-3-K inhibitor interfered with the enhancing effect of IL-6 on the apoptosis induced by IFN-␣. Conclusion. We propose that IL-6 has the death signal, as well as growth promoting effects, and that PI-3-K may play a key role in the induction of apoptosis by IL-6. © 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc. Keywords: Interleukin-6—Interferon-␣—Myeloma cell—Apoptosis— Phosphatidylinositol-3-kinase
Introduction IFN-␣, which inhibits cell growth and induces cell death, has been prescribed for treatment of multiple myeloma. It was reported that IFN-␣ induces G1 phase arrest of the B cell lineage such as Burkitt lymphoma cells and multiple myeloma cells by suppressing G1 cyclins [1] and/or by upregulating expression of CDK inhibitors [2]. In some myeloma cell lines, functional interleukin-6 (IL-6) receptors are downregulated by IFN-␣ [3,4]. IFN-␣ has also been reported to upregulate FAS on multiple myeloma cell lines such as U266 [5]. Fas is expressed on activated lymphoid cells, on normal CD34⫹ cells, and in several hematologic malignancies [6–8]. Fas triggering leads to activation of caspase 8 [9] and other caspases such as caspase 3 [10], then subsequently apoptosis. In some cell types, Bcl-2 and Bcl-x inhibit this apoptotic pathway [11,12]. Offprint requests to: Rumi Minami, M.D., Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan; E-mail: [email protected]
IL-6 is a multifunctional cytokine that acts on various kinds of target cells, and modulates immune responses and hematopoiesis. It has been reported that IL-6 enhances proliferation [13] and survival [14] of myeloma cells in an autocrine [15] and paracrine fashion [16]. It is considered that the effects of IL-6 on proliferation and survival are dissociable and are likely to be mediated by distinct mechanisms [17]. After specifically binding to a cell surface receptor, which contains at least one subunit of the signal transducting protein, gp130 [18], IL-6 activates the Ras-MAPK (for mitogen-activated protein kinase) pathway [19,20]and JAK (for Janus kinase) -STAT ( for signal transducers and activators of transcription) pathway [21]. The proliferation signal is probably transmitted though the Ras pathway [22]. The mechanism by which IL-6 influences survival of myeloma cells has not been well characterized. It was reported that hematopoietic growth factor induces cell death of acute myeloblastic leukemia cells [23,24]. Stahnke et al. [25] reported the upregulation of Fas and increased Fas-induced apoptosis in bone marrow precursor cells exposed to IL-3, GM-CSF, or G-CSF. All this evidence suggests that cell
0301-472X/00 $–see front matter. Copyright © 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc. PII S0301-472X(99)0 0 1 5 6 - 3
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death is linked to cell proliferation and that hematopoietic growth factors possibly enhance apoptosis of the cells under certain condition. To test the hypothesis that IL-6, as well as other hematopoietic growth factors, may enhance apoptosis of target cells, we investigated the effect of IL-6 on myeloma cells in the presence of IFN-␣. We report here that IL-6 has an sensitizing effect on apoptosis of myeloma cells. When examining four IFN-␣ responsive myeloma cell lines, we also found that the magnitude of apoptosis-sensitizing effect correlated with that of growth-promoting effects of IL-6, and that phosphatidylinositol 3-kinase (PI-3-K) plays a pivotal role in determining the function of IL-6.
Materials and methods Reagents Recombinant IL-6 (rIL-6, specific activity, ⬎107 U/mg) was kindly provided by Kirin-Brewery Co Ltd. (Tokyo, Japan) and natural human IFN-␣ (specific activity, 102 U/ng) was kindly provided by Sumitomo Pharmaceuticals (Tokyo, Japan). Anti-humanFas monoclonal antibody, which stimulates Fas-mediated apoptotic pathway (anti-Fas) CH-11 (Beckman Coulter Co., Miami, FL), mouse IgM GC323 (Immunotech, Marseille, France), a peptide caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD-fmk; Medical & Biological Laboratories Co., Nagoya, Japan), fetal calf serum (FCS; Commonwealth Serum Laboratories, Melbourne, Australia), detoxified bovine serum albumin (BSA; Stem Cell Technologies Inc, Vancouver, BC), ironsaturated transferrin (Sigma Chemical Co., St. Louis, MO) were added to cultures, as indicated. LY294002 [26], a PI-3-K inhibitor, was purchased from Calbiochem (Nottingham, UK) and stock solutions (100 mM) were prepared in DMSO, and kept at ⫺20⬚C. Final dilutions were prepared immediately prior to use. Myeloma cell lines PCM6 is a human myeloma cell line cultivated in our laboratory, and which derived from peripheral blood of a patient with advanced IgG myeloma [27]. The status of mycoplasma infection is negative. The viability and growth of this cell line are suppressed by removing rIL-6 from the medium when cells are growing exponentially in serum-containing media, therefore we consider this cell line to be IL-6 dependent. Myeloma cell lines, NOP-2 [28], U266, and RPMI 8226 were also used. NOP-2 is a human myeloma cell line, established from a Bence-Jones type myeloma patient in Nagoya University School of Medicine (Japan). U266 was obtained from American Type Culture Collection (ATCC) and RPMI 8226 was obtained from the Japanese Cancer Research Resources Bank. Cell culture PCM6 cells were grown in McCoy’s 5A modified medium (Life Technologies, Inc., Grand Island, NY) containing 20% FCS and 3 ng/mL rIL-6. U266, RPMI 8226, and NOP-2 were grown in RPMI 1640 medium (GIBCO BRL, Tokyo, Japan) containing 10% FCS. The cell cultures were performed at 37⬚C, 100% humidity, and 5% CO2 in air. Prior to experiments, to investigate the effects of rIL-6
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and/or IFN-␣ on apoptosis, the cells were washed in Daigo’s T medium (Nihon Pharmaceutical Co., Osaka, Japan) and pre-incubated for 60 hours in serum-free media consisting of Daigo’s T medium supplemented with 0.1% BSA and 300 g/mL transferrin in order to eliminate the effects of unknown growth factors in serum, and to synchronize cells in the G0/G1 phase. Viability of the cells after 60-hour incubation exceeded 90% in each experiment. The cells were then washed twice in McCoy’s 5A modified medium and resuspended in culture media containing 20% FCS, with or without rIL-6 (3 ng/mL), and/or IFN-␣ as indicated. The effects of Z-VAD-fmk, anti-Fas antibodies; and LY294002 were also tested, under these experimental conditions. To investigate the effects of IL-6 and the PI-3-K inhibitor on apoptosis in serum starved conditions, the cells were grown in culture media containing 20% FCS without rIL-6 for 60 hours, and then washed twice in Daigo’s T medium and transferred to serum-free media, prepared as described above, with or without rIL-6 and/or LY294002 as indicated. [3H] Thymidine incorporation Effects of rIL-6 or IFN-␣ on proliferation of PCM6 were determined by measuring DNA synthesis, using [3H] thymidine, as described [29]. Briefly, 0.5 mL of serum-free cultures of 105 PCM6 cells were cultured with or without cytokine (rIL-6 or IFN-␣), then incubated with 1.25 Ci of [3H] thymidine (6.7 Ci/mmol; New England Nuclear Corp.) for 1 hour. The cells were then collected by centrifugation, washed with MacCoy 5A, and placed in 10% icecold trichloroacetic acid (TCA). The cell precipitates were collected on glass filter discs (type GF/A;Whatman, Maidstone, England), washed with 5% ice-cold TCA, followed by 95% ethanol, air dried, placed in scintillant and counted in a scintillation counter. [3H] thymidine incorporation was expressed as a percentage of cellular radioactivity in cultures in the absence of cytokines. Assays for detection of apoptosis Viability of the cells was determined by trypan blue exclusion. To determine the effect of IFN-␣ on DNA cleavage during apoptosis of PCM6, the amount of fragmented DNA was measured, as described [30]. [3H] thymidine (0.5 Ci/mL) was added to 2.5⫻106 cells and incubation was carried out for a further 30 minutes. Replicate [3H]thymidine-labeled cells (5⫻106) were then incubated in 1 mL of culture medium containing 2 mol/L thymidine and 2 mol/L deoxycytidine (Sigma Chemical Co.), with or without IFN-␣. After a 16-hour culture, cell replicates were collected and DNA was extracted with phenol:chloroform (1:1 v/v) and analyzed by electrophoresis on an alkaline pH, 0.6% agarose gel, as described [30]. Each lane was cut into eight 10-mm fractions and the radioactivity of each fraction was expressed as a percentage of the total radioactivity. Apoptotic cells were identified, using FITC-conjugated annexin V (FITC-annexin V; Immunotech), which has a high affinity for phosphatidylserine present in the outer membrane of apoptotic cells [31]. The assay was done according to recommendations of the manufacturer. Briefly, cells were washed twice in PBS and resuspended in 500 L of binding buffer containing 5 L of annexin V and 5 L of propidium iodide (PI) (250 g/mL) for 10 minutes on ice and in the dark. The fluorescence of FITC (FLT 1 LOG) and PI (FLT 4 LOG) was analyzed using flow cytometry (Epics ELITE; Beckman Coulter Co.).
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Cell cycle analysis After 60 hours of serum deprivation, we added FCS, rIL-6 and/or IFN-␣, and the proportion of the cells in each phase of cell cycle was determined, as described [32]. Briefly, the cells were washed with PBS, fixed with 50% methanol, treated with RNase A (1 mg/ mL) (USB Co., Cleveland, OH), and stained with PI (0.1% sodium citrate, propidium iodide 50 g/mL) (Sigma Chemical Co.). Cellular DNA content was then analyzed using a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). The percentages of cells in different phases of the cell cycle were determined after PI staining, using ModFit LT software. Western blot analysis Cells were lysed in buffer containing 62.5mM Tris-HCl (pH 6.8), 100mM dithothreitol, 2% (W/V) SDS, and 10% glycerol. Samples containing 100 g of protein were fractionated on sodium dodecyl sulfate (SDS) polyacrylamide gels and electroblotted onto a polyvinylene difluoride (PVDF) (ImmobilonTM; Millipore, Bedford, MA) filter in blotting buffer (48 mM Tris, 39 mM glycine, 20% [v/ v] methanol, 1.3 mM SDS); 7.5% acrylamide gel was used for the detection of Rb and c-Myc, 10% gel for p53, and 12.5% gel for Bcl-x, Bcl-2, and caspase3. The filters were blocked for 2 hours in blocking solution (5% low fat milk in PBS containing 0.5% Tween 20) and then were incubated for an additional 1 hour with specific antibodies. The antibodies used in this article were as follows: anti-human Rb antibody (G3-349) purchased from Pharmingen (San Diego, CA); anti-human c-Myc antibody (A-14) and antihuman Bcl-2 (N-19) purchased from Santa Cruz Biotechnology (Santa Cruz, CA); anti-human p53 antibody (AB-2) purchased from Calbiochem (Cambridge, MA); anti-human Bcl-x antibody and anti-human CPP/Caspase-3 antibody (19) purchased from Transduction Laboratories (Lexington, KY); and anti-human -actin antibody (N3509) purchased from Amersham Life Science (Buckinghamshire, England). Following extensive washing with PBS containing 0.5% Tween 20 (PBS-T), the filter was incubated for 30 minutes with horseradish peroxidase-conjugated anti-rabbit antibodies (whole Ig, NA934; Amersham Life Science) for c-Myc and Bcl-2, or anti-mouse antibodies (whole Ig, NA931; Amersham Life Science) for Rb, Bcl-x, p53, caspase3, and -actin. The filter was extensively washed in PBS-T, and bound antibodies were visualized using the enhanced chemiluminescene Western blotting system (ECL; Amersham Life Science). Blots were then stripped and reblotted with anti--actin Ab to ensure that equivalent levels of proteins were present in each lane. Immunofluorescence analysis of Fas/Apo-1 expression The cells were harvested at the indicated times and resuspended in 50 L of PBS containing 5% FCS, then incubated with 5 L of FITC-conjugated mouse anti-Fas antibody (clone UB2; Immunotech) on ice for 30 minutes and in the dark. Isotype-matched FITC conjugated mouse IgG (Immunotech) was used as a negative control. The cells were washed with PBS and analyzed using a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems). Caspase 3 activity assay The cell-permeable fluorigenic substrate (PhiPhilux-G1D2 caspase 3 activity detection kit; Onco Immunin, Inc., Paint Branch Drive Park, MD) was used to assay caspase 3 activity. This substrate is cleaved by activated caspase3 and produces fluorescence
at 505 nm, and the fluorescence peak is 530 nm. The cells were harvested and washed twice with PBS. The cell replicate (106 cells) was resuspended in 50 L of substrate solution containing 10% FCS, then incubation was carried out for 1 hour at 37⬚C in the dark. After incubation, the cells were washed once in flowcytometry dilution buffer and suspended in 500 L of flow cytometry dilution buffer. The fluorescence emission was determined using the FL-2 channel of a FACScan flow cytometer. Statistical analysis Each experiment was repeated three times to compare data on different groups, and paired t-test was used to determine the statistical significance.
Results IFN-␣ induces apoptosis on PCM6 cells To investigate the effect of IFN-␣ on apoptosis of PCM6, we examined the viability of cells cultured with IFN-␣. When we added IFN-␣ on exponential growing PCM6, the cells gradually died and the viability was 70.8% after culture for 72 hours. When we added IFN-␣ to synchronized cells conditioned by serum-free culture for 60 hours as described in the materials and methods section, the cells died more rapidly and the viability was 58.6% after culture for 48 hours. Thus, we did the following examinations under the latter condition. To investigate the effect of IFN-␣ on apoptosis of PCM6, binding of annexin V was determined, as described above (Fig. 1A). Early apoptotic cells were stained with FITC-annexin V alone (fraction 4), whereas late apoptotic or secondary necrotic cells were stained by both FITC-annexin V and PI (fraction 3). We defined FITCannexin V positive cells as apoptotic cells. When the cells were cultured with IFN-␣ (1000 U/mL), the percentage of the apoptotic cells (Fig. 1A[b]; 56.2%) exceeded than that seen in the culture without IFN-␣ (Fig.1A[a]; 12.6%). To confirm that IFN-␣ induced apoptosis, the pattern of DNA breakdown was also determined (Fig. 1B). DNA isolated from cells cultured with IFN-␣ showed a marked increase in DNA fragmentation, compared with the case without IFN-␣. The effect of IFN-␣ on proliferation, viability, and apoptosis varied with its concentration. As shown in Table 1, stimulation of cell proliferation was observed only with low concentrations of IFN-␣ (0.1 U/mL, 1 U/mL), while apoptosis, determined using annexin V, was dose-dependent (Table 1, Fig. 1C). IL-6 enhanced apoptosis induced by IFN-␣ To determine the effect of IL-6 on the apoptosis induced by IFN-␣, we examined the viability of PCM6 by trypan blue exclusion (Fig. 2A). While 58.6% ⫾ 4.2% of the cells cultured with IFN-␣ were viable after 48 hours, the percentage of viable cells decreased to 41.9% ⫾ 3.8% in the presence of IFN-␣ ⫹ rIL-6 (p ⬍ 0.01). When the percentage of apop-
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Figure 1. Apoptosis of PCM6 induced by IFN-␣. The cells were cultured in serum-free media for 60 hours, then incubated in culture media containing 20% FCS either with ([b] and 䊉) or without IFN-␣ ([a] and 䊊) [1000 U/mL, (A) and (B); various concentrations, (C)]. (A) Apoptosis was determined after 24 hours by labeling with FITC-annexin V and PI. Early apoptotic cells were stained with FITC-annexin V alone (Fraction 4), whereas late apoptotic cells were stained with both FITC-annexin V and PI (Fraction 2). (B) Effect of IFN-␣ on DNA breakdown of PCM6. DNA was extracted from cells that had been labeled with [3H] thymidine and incubated for 24 hours at 37⬚C in culture medium with (䊉) or without IFN-␣ (䊊), and analyzed on alkaline pH, 0.6% agarose gels. The radioactivity of each fraction is expressed as a percent of the total radioactivity in each lane. Numbered arrows indicate the migration of DNA marker fragments with the number of kilobases shown. (C) Flow cytometric analysis using FITC-annexin V was also done after culture for 24 hours under various concentrations of IFN-␣.
totic cells was measured, based on annexin V staining, the addition of rIL-6 significantly increased the number of apoptotic cells induced by IFN-␣ (Fig. 2B, p ⬍ 0.01). The effect of rIL-6 and IFN-␣ on apoptosis was synergistic. The number of apoptotic cells slightly increased, but not significantly, when cultured with rIL-6 alone, compared with the case without exogenous cytokines (Fig. 2B). The effect of IL-6 on IFN-␣ induced apoptosis was to a lesser degree and
appeared more slowly when IFN-␣, with or without rIL-6 was added to exponential growing cells (data not shown). When we examined the effect of IL-6 on the apoptosis induced by various concentrations of IFN-␣ (Table 1), the apoptosis sensitizing effect of IL-6 was obtained with all concentrations used, but as the effect was maximum with 1000 U/mL of IFN-␣, further examinations were made using the concentration of 1000 U/mL.
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Table 1. Effect of low concentration of IFN-␣ on proliferation, viability, and apoptosis Proliferation IFN-␣ (U/mL) 0 0.1 1 10 100 1000
Viability (%)
Apoptosis (%)
IL-6 (⫺)
IL-6 (⫹)
IL-6 (⫺)
IL-6 (⫹)
IL-6 (⫺)
IL-6 (⫹)
1.13 ⫾ 0.03 1.43 ⫾ 0.05 1.37 ⫾ 0.10 1.08 ⫾ 0.03 0.98 ⫾ 0.06 0.85 ⫾ 0.04
2.01 ⫾ 0.04 1.70 ⫾ 0.10 1.10 ⫾ 0.05 1.01 ⫾ 0.06 0.90 ⫾ 0.02 0.80 ⫾ 0.04
89.5 ⫾ 0.8 89.2 ⫾ 1.6 90.3 ⫾ 0.7 87.9 ⫾ 1.1 73.3 ⫾ 3.3 69.5 ⫾ 0.9
87.6 ⫾ 1.3 89.4 ⫾ 1.3 77.2 ⫾ 1.9 75.4 ⫾ 1.8 65.1 ⫾ 2.5 56.5 ⫾ 3.5
16.2 16.9 22.6 31.6 34.1 39.1
33.6 28.6 36.1 55.4 59.4 68.3
After culture in serum free media for 60 hours, the cells were suspended in culture media with rIL-6 and various concentrations of IFN-␣ (0, 0.1, 1.0, 10, 100, and 1000 U/mL). Cell proliferation was estimated as follows: the number of cells after 48-hour culture/the number of cells at start of the culture. After 24 hours, cells were harvested and viability and apoptosis were estimated. Viability was determined by trypan blue exclusion and apoptotic cells were detected by analysis of FITC-annexin V-stained cells on FACScan analysis. Results are the mean ⫾ SEM of three separate experiments.
Because the pattern of response to IFN-␣ differs with the cell line, we studied apoptosis of three other cell lines, U266, NOP-2, and RPMI 8226 by annexin V staining (Table 2). We concomitantly examined the effect of IL-6 on cellular proliferation by determining SI as indicated in the Table 2. Proliferation of these cell lines responded variably to rIL-6. PCM6 was most responsive and cells cultured with rIL-6 had approximately a twofold number over that without rIL-6. NOP-2 was most sensitive to IFN-␣ and apoptosis was induced within 24 hours. U266 and RPMI 8226 were less sensitive to IFN-␣ and apoptosis was induced more slowly (data not shown). Similar to findings with PCM6, apoptosis was enhanced in the presence of rIL-6 in U266. Also in NOP-2 and RPMI 8226, apoptosis was slightly increased in the presence of rIL-6, albeit not significantly. Therefore, the level of IL-6 mediated enhancement of IFN-␣-induced apoptosis by IL-6 paralleled SI, indicating that the degree of sensitization of apoptosis was closely related to the extent of proliferation response by rIL-6.
Figure 2. Effect of rIL-6 on apoptosis induced by IFN-␣. After culture in serum-free media for 60 hours, the cells were suspended in media containing 20% FCS either without exogenous cytokine (䊊) or with rIL-6 (䊉), IFN-␣ (1000 U/mL) (䊐), rIL-6 and IFN-␣ (䊏). (A) Viable cells were determined by trypan blue dye exclusion. (B) Apoptotic cells were detected by analysis of FITC-annexin V-stained cells on FACScan analysis. Results are the mean ⫾ SEM of three separate experiments. *Significantly different (p ⬍ 0.01) from corresponding values in cultures with IFN-␣ alone; † significantly different (p ⬍ 0.05) from corresponding values in cultures with IFN-␣ alone.
Effect of IL-6 and IFN-␣ on cell cycle To investigate the mechanism by which IL-6 enhances apoptosis of PCM6, we examined the effect of IL-6 and IFN-␣ on cell cycle progression (Fig. 3A) and DNA synthesis (Fig. 3B), using PCM6. At start of the culture, 64.9% ⫾ 2.7% of the cells were in G0/G1 phase (Fig. 3A[a]). In the absence of IFN-␣, the number of cells in S phase increased to 44.4% ⫾ 1.4% after a 24-hour culture. Addition of rIL-6 significantly increased the percentage of the cells in S-phase (54.0% ⫾ 1.9%, p ⬍ 0.05) (Fig. 3A[b]), but decreased the percentage in G0/G1 phase (Fig. 3A[a]). Stimulation of cell cycle progression by rIL-6 was also evident when we measured the extent of DNA synthesis, using [3H]thymidine (Fig. 3B). Addition of IFN-␣ resulted in G0/G1 arrest (Fig. 3A) with a remarkable decrease in DNA synthesis (Fig. 3B). Addition of rIL-6 to the culture together with IFN-␣ did not alter the pattern of cell cycle distributions based on findings seen with IFN-␣ alone (Fig. 3A). We also investigated some of the cell cycle regulating proteins, Rb, cyclinD1, and cyclinE (data not shown) by
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Table 2. Effect of rIL-6 on proliferation and apoptosis Apoptotic cells (%) (addition to culture)
PCM6 U266-B NOP-2 PRMI 8226
Cell proliferation
None
rIL-6
IFN-␣
IFN-␣ ⫹ rIL-6
1.90 ⫾ 0.01 1.37 ⫾ 0.03 1.17 ⫾ 0.02 1.03 ⫾ 0.07
20.7 ⫾ 4.7 14.4 ⫾ 4.5 17.4 ⫾ 2.1 24.0 ⫾ 2.2
27.0 ⫾ 5.4 15.0 ⫾ 5.1 22.4 ⫾ 1.3 28.1 ⫾ 0.3
45.2 ⫾ 9.2 42.0 ⫾ 5.7 47.7 ⫾ 1.4 40.4 ⫾ 1.7
62.4 ⫾ 8.4* 53.9 ⫾ 1.5* 51.5 ⫾ 1.1 42.0 ⫾ 0.8
After culture in serum free media for 60 hours, the cells were suspended in culture media either without exogenous cytokine or with rIL-6, IFN-␣ (1000 U/ mL), rIL-6 and IFN-␣. Cell proliferation was estimated as follows: the number of cells cultured in media with rIL-6 for 48 hours/the number of cells cultured in media alone for 48 hours. After 24-hour (PCM6), 48-hour (NOP-2), or 72-hour (U266, RPMI 8226) culture, cells were harvested and apoptotic cells were detected by analysis of FITC-annexin V-stained cells on FACScan analysis. Results are the mean ⫾ SEM of three separate experiments. Percent apoptosis in each group was compared using a paired t-test. * Significantly different (p ⬍ 0.01) from corresponding values in cultures with IFN-␣ alone.
western blot. Cells showed a transition from the hypophosphorylated to the hyperphosphorylated form of Rb when cells were treated without IFN-␣, and rIL-6 slightly upregulated the protein level of hyperphosphorylated Rb (Fig. 4). There was no remarkable difference in the protein level of cyclinD1 and cyclinE between cells treated without any cytokines or with IFN-␣, rIL-6, or IFN-␣⫹rIL-6 (data not shown). c-Myc was increased in the presence of IFN-␣ To investigate the molecular mechanism by which rIL-6 and IFN-␣ influence the apoptosis of PCM6, we examined expression of apoptosis-related proteins by western blotting (Fig. 4). Under serum-free conditions, c-Myc was not detected by western blotting (data not shown) but was induced within 12 hours after exposure to FCS, with or without exogenous cytokines. Addition of IFN-␣ remarkably increased the amount of c-Myc, which was most evident after a 24-hour culture. Addition of rIL-6 did not influence c-Myc expression. Neither IFN-␣ nor rIL-6 influenced the expression of p53, Bcl-2, and Bcl-x protein. Fas-related apoptosis pathway was enhanced by IFN-␣ and IL-6 PCM6 expressed Fas under regular culture conditions with FCS ⫹ rIL-6 (data not shown). When IFN-␣ was added to the culture, expression of Fas was significantly increased, compared to findings in cultures without IFN-␣ (Fig. 5A[a] and [c], and 5B). Addition of rIL-6 to the culture with IFN-␣ significantly increased the expression of Fas, compared with observations of cultures with IFN-␣ alone (Fig. 5A[c] and [d], and 5B). In the absence of IFN-␣, expression of Fas was not significantly increased by rIL-6 (Fig. 5A[a] and [b], and 5B). Next, we examined the activity of caspase3, which is activated by the stimulation of Fas and plays critical roles as a downstream mediator in Fas mediated apoptosis [10,12]. We preformed western blot analysis for caspase3 (Fig. 5D), and for the p17 fragment of caspase3, that is the activated form of caspase3, obtained from cells treated with
IFN-␣, and those treated with IFN-␣ plus IL-6. We wanted to determine if caspase3 would be activated in intact cells treated with exogenous cytokines by a cell-permeable fluorigenic caspase substrate, PhiPhilux G1D2. Addition of IFN-␣ significantly increased the activity of caspase3, as compared with findings without IFN-␣ (Fig. 5C[a] and [c], and 5D). When rIL-6 was added together with IFN-␣, activity of caspase3 was further increased over that seen with IFN-␣ alone (p ⬍ 0.05, Fig. 5C[c] and [d], and 5D). To confirm that caspases are involved in the apoptosis induced by IFN-␣, with or without IL-6, we examined the effect of the caspase inhibitor, Z-VAD-fmk, on apoptosis of PCM6 (Table 3). In cells treated with Z-VAD-fmk, apoptosis induced by IFN-␣ was inhibited and the apoptosis sensitizing effect of IL-6 was not observed. These results indicate that IFN-␣ induces a caspase dependent death and the apoptosis sensitizing effect of IL-6 also depends on caspases. To determining IL-6 influences the Fas-mediated signal to enhance apoptosis, we examined the effect of rIL-6 on apoptosis and caspase3 activity in the presence of anti-Fas (Fig. 6A and 6B) instead of IFN-␣. Addition of anti-Fas Ab increased the percentage of apoptotic cells, compared with findings without Ab, and the addition of rIL-6 plus anti-Fas Ab further increased the percentage of apoptotic cells, compared to findings with Ab alone (Fig. 6A). When the activity of caspase3 was measured, addition of rIL-6 plus anti-Fas Ab resulted in greater activity than that seen with anti-Fas Ab alone (Fig. 6B). These results suggest that IFN-␣ may induce apoptosis of PCM6 partially via a Fas-related pathway, and that IL-6 interacts and modulates the members of Fas signaling pathway such as Fas and caspase3. Apoptosis induced by serum deprivation was inhibited by IL-6 While IL-6 enhanced the apoptosis induced by IFN-␣, IL-6 is also a factor in survival of multiple myeloma cells. To investigate whether IL-6 has an anti-apoptotic function on PCM6 cells, we studied the effect of IL-6 on apoptosis in-
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Figure 4. Western blot analysis of Rb, c-Myc, Bcl-2, Bcl-x, p53 of PCM6. After culture in serum-free media for 60 hours, the cells were incubated in media containing 20% FCS either without exogenous cytokine (lane 1) or with rIL-6 (lane 2), IFN-␣ (1000 U/mL) (lane 3), rIL-6 and IFN-␣ (lane 4) for 12 hours (Rb), or 24 hours (c-Myc, Bcl-2, Bcl-x, p53). Lysates of cells of each group were subjected to SDS-PAGE electrophoresis and immunoblotted with Rb, c-Myc, Bcl-2, Bcl-x, p53 Abs. Blots were then stripped and reprobed with anti-actin Ab to ensure that equivalent levels of proteins were present in each lane.
Figure 3. Effect of rIL-6 and IFN-␣ on cell cycle status and DNA synthesis. After culture in serum-free media for 60 hours, PCM6 were suspended in media containing 20% FCS either without exogenous cytokine (䊊) or with rIL-6 (䊉), IFN-␣ (1000 U/mL) (䊐), rIL-6 and IFN-␣ (䊏). The percentages of cells in different phases of the cell cycle were determined after PI staining using ModFit LT software. (B) PCM6 (105 cells) were incubated in serumfree medium with or without a cytokine (rIL-6 or IFN-␣). Cells were pulsed with [3H] thymidine (1.25 Ci) for 1 hour, and the incorporated radioactivity was measured, as described in the materials and methods section. Results are the mean ⫾ SEM of three separate experiments. The p values were determined by using paired t-test. Percent apoptosis in each group was compared using paired t-test. *Significantly different (p ⬍ 0.01) from corresponding values in cultures without exogenous cytokines; †significantly different (p ⬍ 0.05) from corresponding values in cultures without exogenous cytokine.
duced by serum deprivation (Fig. 7A). When PCM6 cells cultured in media with FCS were transferred to serum-free media, apoptotic cells gradually increased and the percentage of apoptotic cells was smaller in the culture with than
without rIL-6 after culture for 72 hours (Fig. 7A; 21.9% ⫾ 0.3% with rIL-6 vs 42.3% ⫾ 2.4% without rIL-6). Similar results were obtained when the viability of the cells was determined by trypan blue exclusion (data not shown). Therefore, rIL-6 apparently protects PCM6 cells from the apoptosis induced by serum deprivation.
Effects of IL-6 on apoptosis as influenced by a PI-3-K inhibitor (LY294002) To examine the role of PI-3-K in the regulation of apoptosis by rIL-6 and IFN-␣, the effect of the PI-3-K inhibitor, LY294002, on apoptosis of PCM6 was determined. When LY294002 was added to the culture with IFN-␣ and/or rIL-6, PCM6 were protected from the apoptosis induced by IFN-␣, and the apoptosis-promoting effect of rIL-6 was abolished (Fig. 7B). In addition, LY294002 blocked cell cycle progression after exposure to FCS ⫹ rIL-6 at G0/G1 phase (data not shown). When the effect of LY294002 on apopto-
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Figure 5. Effect of IFN-␣ and rIL-6 on Fas expression and activity of caspase 3. After culture in serum-free media for 60 hours, the PCM6 cells were suspended in media containing 20% FCS either without exogenous cytokine [(a) and 䊊] or with rIL-6 [(b) and 䊉]; IFN-␣ (1000 U/mL) [(c) and 䊐]; rIL-6 and IFN-␣ [(d) and 䊏]. (A) Cells were stained with FITC-conjugated anti-Fas mAb after 0-hour (—) and 48-hour (—) culture. The population of Fas-positive cells was gated using mouse anti-IgG1 antibody (· · ·). The cells were analyzed by FACScan. The fluorescence intensity is given in logarithmic scale. Data are representative of one of three separate experiments. (B) Time course of Fas expression. Results are the mean ⫾ SEM of three separate experiments; p values were determined by using paired t-test. *Significantly different (p ⬍ 0.01) from corresponding values in cultures with IFN-␣ alone; †significantly different (p ⬍ 0.05) from corresponding values in cultures with IFN-␣ alone. (C) After 48 hours, cells were incubated with a fluorigenic caspase3 substrate PhiPhilux-G1D2 for 1 hour and then analyzed by FACScan (—). The fluorescence intensity is given in logarithmic scale. The percentages of cells with caspase3 activation are indicated. Nonapoptotic cells prestimulated by any cytokine we used served as negative controls, the absence of caspase3 activation (—). (D) Time course of caspase3 activation. Results are the mean ⫾ SEM of three separate experiments; p values were determined by using paired t-test. *Significantly different (p ⬍ 0.01) from corresponding values in cultures with IFN-␣ alone; †significantly different (p ⬍ 0.05) from corresponding values in cultures with IFN-␣ alone. The inset shows western blot analysis of caspase3. Cell lysates were obtained after 48-hour culture without exogenous cytokine (lane 1) or with rIL-6 (lane 2), IFN-␣ (1000 U/mL) (lane 3), and rIL-6 and IFN-␣ (lane 4).
Table 3. Effect of Z-VAD-fmk on apoptosis by IFN-␣ Apoptotic cell (%)
None IFN-␣ IFN-␣ ⫹ rIL-6
Z-VAD-fmk (⫺)
Z-VAD-fmk (⫹)
5.51 28.2 45.8
5.06 5.25 5.67
After culture is serum free media for 60 hours, PCM6 were incubated for 24 hours in media containing 20% FCS in the absence or presence of Z-VAD-fmk (20 M), without any cytokines, or with IFN-␣ (1000 U/mL), rIL-6 (3 ng/mL) plus IFN-␣. Apoptosis was detected by analysis of FITCannexin V-stained cells on FACScan.
sis induced by serum starvation was examined, addition of LY294002 led to an increase in the percentage of apoptotic cells, with or without rIL-6 (Fig. 7A). Because the absence of rIL-6 augmented the apoptosis, the anti-apoptotic effect of rIL-6 was not affected by LY294002. These data suggest that the function of IL-6, as a growth and apoptosis-promoting factor, is mediated by the activity of PI-3-K, however, the function as a survival factor is not related to PI-3-K.
Discussion We obtained evidence that IL-6 sensitized IL-6-responsive myeloma cells for apoptosis and stimulated cellular proliferation. We used four IFN-␣ sensitive myeloma cell lines,
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Figure 6. Effect of rIL-6 on Fas-induced apoptosis. After culture in serum-free media for 60 hours, PCM6 were suspended in media containing 20% FCS with control mouse IgM [(a)]; rIL-6 and control mouse IgM, anti-Fas (50 ng/mL) [(b)]; and rIL-6 and anti-Fas (50 ng/mL) [(c)]. (A) After 24 and 48 hours, the cells were harvested. Apoptotic cells were detected by analysis of FITC-annexin V-stained cells on FACScan analysis. Results are the mean ⫾ SEM of three separate experiments. Percent apoptosis in each group was compared using paired t-test. *Significantly different (p ⬍ 0.01) from corresponding values in cultures with IFN-␣ alone. (B) After a 24-hour culture, PCM6 were incubated with a fluorigenic caspase3 substrate PhiPhilux-G1D2 for 1 hour and then analyzed using FACScan. The fluorescence intensity is given in logarithmic scale. The percentages of cells with caspase3 activation are indicated. Nonapoptotic cells prestimulated by any cytokine we used served as negative controls, the absence of caspase3 activation (—). The percentages of cells with caspase3 activation are indicated. Nonapoptotic cells used served as negative controls, the absence of caspase3 activation. Data are representative of three separate experiments.
all of which respond to IL-6 to a different degree. Apoptosis was more strongly enhanced by rIL-6 when the cell line used was the more sensitive to rIL-6 in proliferative response. This suggests that signaling pathways to initiate apoptosis and cellular proliferation are simultaneously activated through the IL-6 receptor. It was recently reported that hematopoietic growth factors such as IL-3, G-CSF, or GM-CSF increased Fas-induced apoptosis of bone marrow precursor cells [25]. Together with our data, this suggests that cytokines that promote cellular proliferation also possess the “death signal,” related to the Fas/FasL system, as a intracellular negative-feed back mechanism. The role of the Fas system has been demonstrated primarily in cases of activation-induced cell death (AICD), which closely relates to the cellular proliferation of T lymphocytes. Expression of Fas and c-myc is required for T cells to
undergo AICD [33]. In the myeloma cell line PCM6, the addition of IFN-␣ increased expression of Fas and c-Myc prior to the initiation of apoptosis, as well as AICD. The activity of caspase3, one of the downstream components in Fas/Fas ligand (FasL) signaling pathway, also increased in the presence of IFN-␣. Addition of rIL-6 influenced this cascade by further enhancing Fas expression and caspase3 activity. Our data obtained using anti-Fas confirmed that IL-6 influences the apoptotic signal related to the Fas/FasL system, therefore, this system seems to play a key role in the signaling pathway of apoptosis induced by “growth factors.” Our finding that IL-6 enhanced Fas related apoptosis differs from the data of Chauhan et al. [34], which IL-6 inhibits Fas-induced apoptosis in the myeloma cell line, RPMI 8226. This discrepancy might relate to differences in experimental systems. The most obvious difference is that
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Figure 7. Effect of LY294002 on apoptosis induced by serum starvation and IFN-␣. (A) After culture in media containing 20% FCS without exogenous cytokine for 60 hours, PCM6 were incubated in serum-free media in the absence or presence of LY294002 (50 ng/mL) with or without rIL-6 (3 ng/mL). At the indicated times cells were harvested and apoptotic cells were detected by analysis of FITC-annexin V-stained cells on FACScan. (B) After culture in serumfree media for 60 hours, PCM6 were incubated in media containing 20% FCS in the absence or presence of LY294002 (50 ng/mL) without any cytokine we used or with rIL-6 (3 ng/mL), IFN-␣ (1000 U/mL), rIL-6 (3 ng/mL), and IFN-␣. Results are the mean ⫾ SEM of three separate experiments. Percent apoptosis in each group was compared using paired t-test. *Significantly different (p ⬍ 0.05) from corresponding values in cultures without exogenous cytokines; † significantly different (p ⬍ 0.05) from corresponding values in cultures with LY294002 alone; †significantly different (p ⬍ 0.05) from corresponding values in cultures with IFN-␣ alone.
an IL-6 independent myeloma cell line was used in the previous study [34]. Furthermore, unlike our culture condition, the cells were not synchronized in G0/G1 phase and were preincubated with rIL-6 prior to stimulation with anti-Fas. In IL-6 responsive PCM6 cells, IL-6 has a multifunction, such as stimulating proliferation, reducing apoptosis, and, as described here, enhancing apoptosis. It has been reported that different signals induced through gp130 may have distinct biologic functions; growth stimulation via the MAPK cascade and anti-apoptosis via other cascades including the STAT 1/3 pathway [35]. IL-6 may trigger proliferation, survival, and apoptosis-enhancing effects through different sig-
naling pathways. We examined the association of the effects of IL-6 with PI-3-K, which plays a critical role in signaling from gp130 subunit of IL-6 receptor [36]. PI-3-K is a downstream target of Ras [37] and seems to be important for differentiation or proliferation of various cells. On the other hand, it has been reported that Fas induces apoptosis by Ras-regulated PI-3-K activation in Jurkat cells [38], and Uddin et al. [39] reported the activation of PI-3-K serine kinase by IFN-␣ in U266 and Daudi cells. Our experiments clearly indicated that PI-3-K mediated the apoptosisenhancing effects of IL-6. All these observations taken together suggest that PI-3-K might play an essential role in
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the signaling pathway for the proliferation and apoptosis induced by cytokine stimuli, or activation of Fas. In conclusion, IL-6 is multifunctional and has distinct effects on both apoptosis and proliferation of myeloma cells. PI-3-K is likely to be involved in the function of IL-6. Further studies on precise mechanisms determining the direction of IL-6 action are warranted and may yield pertinent findings, which may aid in developing protocols for treating subjects with multiple myeloma.
Acknowledgments We are grateful to Michio Kawano, M.D., for critical comments on the manuscript. We also thank Seiichi Motomura, M.D., for helpful advice; Ms. Shizuko Aoki and Ms. Shiho Isewaki for technical support; Sumitomo Pharmaceuticals for human IFN-␣; and Ms. M. Ohara provided language assistance.
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