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Bacterial Lipopolysaccharide Induces Proliferation of IL-6- Dependent Plasmacytoma Cells By MAPK Pathway Activation

Posted on: Friday, 2 July 2004, 06:00 CDT

Abstract

Mouse plasmacytomas are appropriate models to study the biology of human multiple myeloma (MM). Growth of murine interleukin-6 (IL- 6)-dependent hybridoma/plasmacytoma lines can be stimulated by bacterial lipopolysaccharides (LPS). However, the molecular mechanisms of this phenomenon are still not elucidated. In this study the in vitro action of bacterial LPS on the mouse IL-6- dependent B9 hybridoma/plasmacytoma cell line and two IL-6- dependent hybridomas was investigated. The involvement of different signal transduction pathways was established using specific kinase inhibitors in proliferation assays and immunoblotting analysis of the kinase activity. Selective mitogen-activated protein kinase (MAPK) kinase inhibitor PD989059 inhibited both IL-6- and LPS- induced B9 cell proliferation. In contrast, in H187 and H188 cells, PD98059 inhibited only LPS-, but not IL-6-stimulated cell growth. The kinetics of MAPK activation in all cell lines showed that phosphorylation of p42 MAPK (encoded as ERK2) but not of p44 MAPK (ERK1), was considerably increased after treatment with LPS. We found that in H187 and H188 hybridomas IL-6 induced proliferation by a different STAT3-dependent mechanism. This study demonstrates the key role of the MAPK pathway in LPS-stimulated growth of mouse IL-6- dependent plasmacytoma cells. These findings suggest the presence of signaling mechanism in MM cells inducible by bacterial mitogens and possibly mediated by Tolllike receptors (TLR) - evoludonarily conserved molecules playing a central role in the microbial recognition and initiation of the cellular innate immune response.

Abbreviations: ERK = extracellular signal-regulated kinases 1 and 2; FBS = fetal bovine serum; IL-6 = interleukin-6; JNK = c-Jun N- terminal kinase; LPS = lipopolysaccharide; MAPK = mitogen-activated protein kinase; MM = multiple myeloma; PI3-K = phosphatidylinositol 3-kinase; PKC = protein kinase C; PxB = polymyxin B; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis; STAT1/ STAT3 = signal transducers and activators of transcription 1/3; TLR = toll-like receptor

Introduction

Lipopolysaccharide (endotoxin of Gram-negative bacteria) induces a broad spectrum of pathophysiological reactions in the host upon generalized Gramnegative infection (Freudenberg & Galanos, 1990). The biological effects of LPS are mediated by direct or indirect mechanisms stimulating cytokine release. LPS-induced activation of signal transduction pathways in cells has been studied in detail in isolated macrophages and macrophage cell lines. LPS binds to CD 14 and triggers the activation of several protein kinases including protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3-K) and the three MAPK pathways: p38, c-Jun N-terminal kinase (JNK) and p42/ p44 MAPK also known as extracellular signal-regulated kinases 1 and 2 (ERK) (Guha & Mackman, 2001; Monick et al., 2000). These signaling pathways activate a variety of transcription factors including NF- [Hamiltonian (script capital H)]B and AP-1, which lead to the expression of many genes encoding inflammatory mediators and immunomodulatory molecules such as proinflammatory cytokines (Guha et al., 2001; Zhang et al., 2000). Recent data illustrate the important role of the Toll-like receptor family in the inflammatory response and innate immunity to microorganisms (reviewed in Means et al., 2000; Vasselon & Detmers, 2002). TLRs are composed of an extracellular ligand-binding domain and an intracellular domain. The intracellular domain structure is similar to that of the IL-1 receptor. Family members TLR2 and TLR4 can be activated by a variety of bacterial and yeast cell wall components. Interaction of microbial components with the extracellular domain of TLRs can trigger multiple intracellular signaling mechanisms, activation of transcription factors and cytokine release. TLR4 is strictly involved in the response to bacterial LPS in different cell types. It has been established that LPS can induce activation of NF- [Hamiltonian (script capital H)]B and p44/ p42 MAPK pathways through a TLR4-dependent mechanism (Chow et al., 1999; Yang et al., 2000). These signaling pathways can play a central role in cell proliferation. For example, exposure to Chlamydia pneumoniae results in a rapid TLR4-mediated MAPK activation and subsequent stimulation of proliferation in human vascular smooth muscle cells (Sasu et al., 2001). LPS stimulates the proliferation of murine B lymphocytes and promotes their differentiation into antibody-secreting plasma cells. Both TLR4 and structurally related RP105 protein play a key role in the response of mature B cells to bacterial endotoxins (Vasselon & Detmers, 2002). However, the signaling mechanisms involved in LPS- induced B cell activation arc not well understood. In previous studies it has been demonstrated that LPS directly activates PKC by mimicking diacylglycerol (Bosca & Diaz-Guerra, 1988). The role of PKC in the mitogenic response of B cells to Salmonella typhimurium LPS has been reported recently (Mey & Revillard, 1998). LPS stimulation of B cells involves not only a PKC-dependent pathway, but also one dependent upon protein tyrosine kinases. PI3-K is also involved in the mechanisms of B cell development, differentiation and function upon mitogenic stimulation (Bone & Williams, 2001). Recently it has been reported that both LPS-mediated B cell proliferation and IL-6 secretion are dependent on the PI3-K signaling pathway (Venkataraman et al., 1999).

Mouse models are widely used and accepted in the elucidation of the role of growth factors and signal transduction pathways involved in the mechanisms of cell transformation and cell survival in human plasmacytomas (reviewed in Gado et al., 2001). B9 murine hybridoma/ plasmacytoma cell proliferation is strictly dependent on the presence of IL-6 in culture medium and a commonly used sensitive bioassay for quantification of IL-6 is based on the B9 cell line (Aarden et al., 1987). On the other hand, B9 cells are a suitable model for the examination of signal transduction mechanisms and growth regulation in human IL-6-dependent multiple myeloma cells (Ogata et al., 1997a; Iankov et al. 2002a). It is reported that bacterial LPS can induce B9 cell proliferation in a dose-dependent way (Pedersen et al., 1995). However, there are no detailed investigations concerning the molecular mechanisms of that phenomenon. On the other hand, the effect of LPS and other bacterial mitogens on human MM cells is also not well elucidated. In our study we investigated the role of p44/p42 MAPK (ERK), PKC and transcription factors STAT1 and STAT3 in LPS-induced proliferation of B9 cells and two IL-6dependent hybridomas.

Materials and Methods

Cell culture and growth factors

H187 and H188 are IL-6-dependent clones of hybridomas 187g3 and 188ND9, that produce monoclonal antibodies of IgA isotype directed against Salmonella enteritidis flagellar antigen (Iankov et al., 2002b). They are selected in our laboratory after cloning by limiting dilution in the presence of IL-6 in hybridoma culture medium.

IL-6-dependent hybridomas and B9 cells were grown in RPMI1640 medium (Sigma Chemical Co.) supplemented with 10% fetal bovine serum (FBS) CELLectTM Gold with low (< 1 ng/ml) endotoxin level (ICN Pharmaceuticals, Inc.). Proliferation was stimulated by adding different concentrations (in U/ ml) of recombinant human IL-6. The specific activity of IL-6 was defined in Units by the amount needed to induce growth in B9 cells as described by the manufacturer (cat. no. 152352, ICN Pharmaceuticals, Inc.). Polymyxin B (Sigma Chemical Co.) was used as a specific inhibitor of LPS-induced growth response in B9 cells (Pedersen et al., 1995).

Reagents

Purified LPS of Salmonella abortus equi was kindly given by C. Galanos, Max-Planck-Institute of Immunobiology, Freiburg, Germany. Selective MAPK kinase inhibitor PD98059 and PKC inhibitors Go976 and Go983 were kindly provided by Debiopharm S.A., Lousanne, Switzerland. Mouse monoclonal antibodies p-ERK (E-4) - specific for Tyr-204 phosphorylated ERKl and ERK2 activated forms (p44/p42 MAPK), p-STATl (A-2) - specific for Tyr-701 phosphorylated active STATl and pSTAT3 (B-7) - specific for Tyr-705 phosphorylated active STAT3, rabbit polyclonal antibody ERKl (K23) - reactive with both ERK1and ERK2 and rabbit polyclonal antibodies specific for TLR2 (H-175) and TLR4 (H-80) were purchased from Santa Cruz Biotechnology, Inc., USA.

DNA synthesis assay

Proliferation was determined by measurement of [3H]-thymidine incorporation into DNA. Briefly, 10^sup 5^ cells in 1 ml RPMI1640 medium (supplemented with 10% FBS for B9 and without FBS for Hl 87 and H18 cells) were cultured in 24-well plates in the presence or absence of IL-6 and LPS. To establish the importance of the MAPK pathway cells were prctrcatcd with 25 M PD98059, a potent MAPK kinase inhibitor, for 72 hours and 5 Ci per well of [3H]-thymidine (Amersham) were added for the last 12 hours of incubation. Portions of 200 ?? cell suspension of each well were pipetted on glass microfibre filters GF/B (Whatman) and counted in a Beckman Liquid Scintillation Counter. Samples were run in six wells and each experiment was repeated four times.

lmmunoblotting analysis of phosphorylated p44/p42 MARK proteins

\Cells growing exponentially in the presence of IL-6 were washed three times in RPMI1640 and were left to starve for 5 hours in RPMI1640 medium without serum and growth factors. Purified LPS (10 ^ig/ml) and IL-6 (100 U/ml) were added and cells were incubated for different time periods. After incubation cells were washed once in phosphate buffered saline (PBS) and lysed in 1 ml ice-cold lysis buffer (PBS with 0.5% sodium deoxycholate, 1% Nomdet P-40 and 0.1% SDS), containing freshly added protease inhibitors (5 g/ml aprotinin and 1 mM PMSF) and 1 mM sodium orthovanadate. After incubation on ice for 30 minutes cell lysates were centrifuged at 10000 g for 10 minutes at 40C and supernatant total cell lysates were collected. Equal volumes (containing equal protein amounts, measured by the Bradford method) of the cell lysates were mixed with sample buffer and were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then proteins were transferred to PVDF membranes (Millipore) and incubated with the phospho-specific antibodies according to the manufacturer's recommendations. After incubation with a secondary peroxidase-conjugated antibody (Santa Cruz Biotechnology), proteins were visualized by Chemiluminescence Luminol Reagent (Santa Cruz Biotechnology).

To investigate the effect of selective kinase inhibitors on the MAPK pathway and the possible involvement of PKC in MAPK activation, Hl 87 and Hl 88 cells were pretreated with 25 ?? PD98059, 1 M Go6976 or 1 M Go6983 for 1 hour and then were stimulated with LPS and IL6. Cells were lysed and MAPK activity was analyzed by immunoblotting as described above.

Results

The maximal effect on cell proliferation was obtained after incubation with 100 U/ml IL-6 and 10 [ig/ml LPS, respectively. B9 and hybridoma cells were strongly dependent on the presence of IL-6 in the culture medium. Serial experiments showed that in serum-free conditions 100 U/ml IL-6 stimulated B9 proliferation 6.5 -12 times greater than the control. Under the same conditions IL-6 could increase up to eight (3.8-8.7 in different experiments) times [3H]- thymidine incorporation into DNA in Hl 87 (growth curve in Fig. 1) and more than twenty times (17.3-38.5) in H188 cell (Fig. 2A). LPS stimulated growth of both H187 and H188 hybridorna cells in serum- free conditions. In contrast, LPS induced B9 cell proliferation only in the presence of FBS in the culture medium (growth curves in Fig. 1). The calculated effect of stimulation of 10 g/ml LPS corresponded to the stimulation effect of 10-50 U/ml IL-6 in repeated experiments. The specificity of LPS action on B9 cells in the presence of FBS was demonstrated using Polymyxin B. As shown in Fig. 1C, Polymyxin B in 10 ug/ml final concentration completely inhibited LPS induced growth, but had no effect on IL-6-induced proliferation (not presented).

Fig. 1. Dose response of H187 and B9 cells to recombinant human IL-6 and Salmonella abortus equi LPS stimulation. (A) Both IL-6 and LPS could induce H187 proliferation in serum free conditions. (B) In contrast, only IL-6 stimulated growth of B9 cells in the absence of FBS. The co-stimulatory effect of FBS in the culture medium was required for LPS stimulation of B9 growth. (C) The specificity of LPS action was demonstrated using Polymyxin B (PxB). LPS and PxB (10 g/ml final concentration in medium) were mixed prior to the stimulation of B9 cells. The results shown are from one of five (for H187 hybridoma cells) and one of six (for B9 cells) independent experiments.

Fig. 2. Results of [3H]-thymidine incorporation into DNA of B9, H187 and H188 cells stimulated with 100 U/ml human recombinant IL-6 and 10ng/ml purified S, abortus equi LPS. (A) IL-6 and LPS induced DNA synthesis compared to the non-stimulated control cells. LPS induced B9 proliferation only in the presence of FBS. The effect of LPS on B9 proliferation was calculated in comparison with the control cells grown in the presence of FBS. (B) The result of selective MAPK pathway inhibition on proliferation was examined in a parallel series. The effect of 25 M PD98059 on [3H]-thymidine incorporation into DNA of IL-6- and LPS-stimulated cells was calculated as % of the controls without inhibitor. The results shown are from one of four experiments.

To determine the possible involvement of the PKC and MAPK cascade in IL-6- and LPS-induced proliferation, cells were pretreated with selective kinase inhibitors. The selective blockade of MAPK kinase with 25 M PD98059 led to inhibition of both IL-6- and LPS-induced proliferation in B9 cells (Fig. 2B). The effect of the inhibitor was calculated in comparison with non-stimulated control cells cultivated in the presence of FBS, because LPS-stimulated B9 cell growth required FBS. PD98059 decreased to 50% DNA synthesis in cells incubated for 72 hours in the presence of 10 ng/ml LPS. In contrast, PD98059 did not affect IL-6-induced proliferation of H187 and H188 hybridorna cells. LPS-stimulated DNA synthesis in serum-free conditions was decreased approximately to 50% as it was in the case of B9 cells. Thus chemical blockade of p44/p42 MAPK activation in all three cell lines led to inhibition of LPS-induced proliferation indicating the important role of the MAPK cascade.

Phosphorylation of p44/p42 MAPK proteins was detected by immunoblotting in LPS-stimulated cells for different time intervals. LPS led to MAPK phosphorylation in B9 cells with a peak at 30 minutes (Fig. 3A), as similar to the kinetics of MAPK activation after IL-6 stimulation (lankov et al. 2002a; Ogata etal., 1997a). The effect was seen mainly in p42 MAPK and LPS did not require the presence of FBS in order to activate the MAPK pathway. FBS alone could not stimulate MAPK and had no effect on the LPS-induced p44/ p42 MAPK phosphorylation.

LPS also induced MAPK activation and led to considerable p42 MAPK phosphorylation with a similar kinetics in Hl87 hybridoma cells (Fig. 3B). MAPK activation was detected at 5 minutes, reaching the maximum at 30 minutes and markedly decreased 90 minutes after stimulation with 10 ug/ ml LPS. Phosphorylation of p44 MAPK protein was not detected. In contrast to B9 cells, IL-6 at a concentration of 100 U/ml did not activate the MAPK pathway in H187 cells.

The kinetics of MAPK activation in LPS-stimulated H188 cells was absolutely distinct from that observed in B9 and H187 cells (Fig. 3C). Phosphorylation of p42 MAPK (and to a smaller extent of p44 MAPK) rapidly increased within 5 minutes, maintained at the same level for 30-45 minutes and reached the basic p42 MAPK activity after 90 minutes of incubation with 10 g/ml LPS. No change of the p44 MAPK phosphorylation was detected and the MAPK pathway was not activated in the presence of 100 U/ml IL-6.

LPS-induced p42 MAPK activation was completely abolished in the presence of the MAPK kinase inhibitor PD98059 (Figs. 3A, 4). In our previous work we observed that pretreatment of B9 cells with selective PKC inhibitors GO6976 and GO6983 led to increased ERK1/2 activity (lankov et al., 2002a). These data suggested a possible PKC- mediated mechanism of negative control on the MAPK pathway in B9 cells. This negative regulation of MAPK activity by PKC was not found in the two hybridoma lines, even Go6976 weakly decreased ERK1/ 2 phosphorylation in H188 cells (Fig. 3B). IL-6 did not induce MAPK activation in H187 and H188 cells. The immunoblotting analysis of STATl and STAT3 proteins showed that STAT3 was phosphoryIated30 minutes after stimulation with IL-6 (Fig. 3). No change of STATl phosphorylation was detected (not presented).

The presence of TLRs that could mediate the effect of LPS was evaluated by immunoblotting. Both TLR2 and TLR4 were expressed in the three cell lines (Fig. 5A). The differences observed in the mechanisms of IL-6- and LPS-induced cell proliferation and the involvement of the MAPK pathway are summarized in Fig. 5B.

Discussion

The aim of the present study is to assess the mitogenic potential of bacterial LPS on IL-6-dependent hybridoma/plasmacytoma cells. Our results demonstrate that LPS-induced cell growth involves the MAPK pathway and show the key role of p42 MAPK activation.

The MAPK cascade plays a crucial role in the regulation of gene expression in response to extracellular stimulation signals and subsequent activation of cell proliferation (Chang & Karin, 2001). The activation of signal transduction mechanisms by growth factors is mediated by p44/p42 MAPK (encoded by ERKl and ERK2) connecting cellsurface receptors to critical regulatory targets within cells. ERK1/2 can induce proliferation by different mechanisms. TLRs could also be involved in the MAPK-mediated signaling mechanisms. Chlamydia pneumoniae and chlamydial heat shock protein 60 were demonstrated to activate p44/p42 MAPK and promote proliferation of vascular smooth muscle cells via TLR4 (Sasu et al., 2001). A similar effect of MAPK activation was observed in murine L-929 fibroblast cells treated with the same pathogen (Haralambieva et al., 2002).

Fig. 3. (A) Kinetics of MAPK activation in B9 cells after stimulation with LPS and FBS. The effect of 10 g/ml LPS on the MAPK pathway is demonstrated by immunoblotting with monoclonal antibody specific for Tyr-204 phosphorylated active ERK1/2 forms (p-ERKl/2). lmmunoblotting for detection of ERK1/2 expression was performed by rabbit antibody K-23 to show the equal protein amount in the samples (ERK1/2). Arrowheads indicate the position of 43 kDa protein standard (prestained molecular weight standards, Gibco). (B, C - top panels) ERK1/2 phosphorylation in H187 cells (B) and H188 cells (C) after stimulation with LPS and IL-6. (B, C - bottom panels) Immunoblotting for detection of Tyr-705 phosphorylated active forms of STAT3 protein in the same samples stimulated with LPS and IL-6 for Hl 87 (B) and H188 (C) cells.

Previous reports had demonstrated that LPS in concentrations above 40 ng/ml could c\ompromise the sensitive bioassay for IL-6 measurement based on IL-6-dependent B9 cells (Pedersen et al., 1995). The authors concluded that it was reasonable because B9 was a hybridoma derived from B cells and LPS was known to stimulate proliferation of B cells directly in a way independent of T cells. IL-6 was shown to trigger Ras-dependent MAPK cascade activation in B9 cells, which had been established also for human IL-6-dependent MM cells (Ogata et al., 1997a). Thus, the investigation of signal transduction pathways in B9 cells may contribute to the clarification of the mechanisms of growth and cell survival in human myelomas. IL-6 is a major myeloma growth factor, which mediates proliferation by autocrine and paracrine mechanisms (Kawano et al., 1988; Klein et al., 1989). In addition, IL-6 may also act as a survival factor for MM by inhibition of apoptosis (Chauhan et al., 1997). The blockade of the MAPK cascade is associated with loss of responsiveness to IL-6 in MM cells indicating the important role of this pathway (Ogata et al., 1997b). However, the response and the mechanisms triggered by bacterial mitogen stimulation in IL-6- dependent MM cells are not well investigated.

Fig. 4. The effect of selective kinase inhibitors on MAPK activity in H187 cells (A) stimulated with LPS and IL-6 for 30 minutes and in H188 cells (B) stimulated for 5 minutes (p-ERK1/2). The samples were controlled for equal protein concentration by immunoblotting for ERK1/2 expression (ERK1/2). Arrowheads indicate the position of 43 kDa standard protein.

Fig. 5. (A) The expression of TLR2 and TLR4 in B9 (lane 1), H187 (lane 2) and H188 (lane 3) cells established by immunoblotting. (B) Involvement of the MAPK pathway in LPS- and IL-6-induced proliferation of B9, H187 and H188 cells. In all IL-6-dependent hybridoma/plasmacytoma cells LPS induces MAPK activation and subsequent cell proliferation possibly by a TLR-mediated pathway. Only in B9 cells the MAPK cascade is involved in the mechanisms of IL-6-induced cell proliferation.

In our study we observed that LPS induced proliferation of B9 cells and two hybridomas H187 and H188. All cells were strictly dependent on IL-6 for growth. The role of the MAPK pathway in LPS- stimulated proliferation was proved in two distinct ways. First, the specific MAPK inhibitor PD98059 markedly decreased DNA synthesis in LPS-treated cells and second, LPS-triggered ERK2 activation was confirmed by the results of the ERK1/2 phosphorylation kinetics. Although MAPK cascade activation is required for proliferation, the mechanism of activation and its role are distinct in the different hybridoma/plasmacytoma cells used in this investigation. In B9 cells, like IL-6, LPS alone led to increasing ERK2 activity with a peak of phosphorylation within 30 minutes. However, FBS was required for LPS- but not for IL-6-induced B9 cell proliferation. On the other hand, FBS alone did not activate ERK1/2 indicating that the FBS effect on B9 cell proliferation was MAPK cascade independent. These observations show that ERK2 activation has a crucial role after LPS stimulation. However, in contrast to IL-6 stimulation, LPS stimulation alone is not sufficient and necessarily requires the presence of serum for triggering proliferation. LPS stimulation led to ERK1/2 activation with similar kinetics in H187 cells and that was sufficient to promote proliferation. In contrast to B9 and H187 cells, in H188 cells LPS led to rapid ERK2 phosphorylation within 3- 5 minutes, which corresponded to the typical MAPK cascade activation. As in H187 cells, LPS induced H188 cell growth without the necessity of additional signals such as FBS. In both hybridoma clones the MAPK pathway was not activated after IL-6 stimulation. However, the MAPK cascade is not the only pathway triggering cell proliferation after IL-6 stimulation. The JAKSTAT signaling mechanism and PI3-K could also be possible pathways for IL-6- induced growth and cell survival (Hirano, 1998; Hirano et al., 1997, 2000). Obviously, the mechanism of signal transduction pathways activated in H187 and H188 hybridomas is different for LPS and IL- 6. The MAPK pathway is involved in LPS-induced proliferation, while STAT3 possibly mediates IL-6-induced proliferation in these cells. In IL-6-stimulated B9 cells we observed the mechanism of negative control of MAPK phosphorylation by PKC (lankov et al., 2002a). In contrast pretreatment of H187 and H188 hybridomas with selective PKC inhibitors did not increase MAPK activity both in LPS-stimulated and in control cells. In H188 cells selective blockade of PKD (formerly PKC isoform) by Go6976 led to weak inhibition of ERK1/2 phosphorylation. All three cell lines expressed TLR2 and TLR4. TLR4 is now known as a major surface transducer molecule of the cellular signals in response to bacterial LPS (Vasselon & Detmers, 2002). In addition, TLR2 could also be involved in cell activation after stimulation with leptospiral LPS. Thus, the mitogenic effect of LPS on IL-6-dependent plasmacytomas could be possibly explained by a universal TLR-mediated mechanism of MAPK activation. In contrast to bacterial LPS, Streptococcus pyogenes lipoteichoic acid did not induce cell proliferation of IL-6-dependent cells under the same conditions (unpublished results). Lipoteichoic acids of Gram- positive bacteria can also activate cellular signaling through TLR2 in human or by TLR4 in mouse cells (Vasselon & Detmers, 2002). All three cell lines show differences in kinetics of IL-6- and LPS- induced MAPK activation and response to selective PKC inhibitors. Although the kinetics of MAPK activation are distinct for B9 and H187 cells on one hand and for H188 cells on the other hand, ERK2 phosphorylation seems to play a key role for cell cycle development. PKC and PKD (former member of the PKC family - PKC) could participate in the regulation of MAPK activity and thus could be involved in regulation of proliferation. Cellular signaling in human MM also is too complex and may vary in cells isolated from different patients (Ogata et al., 1997a). Recently, chemical blockade of signaling pathways such as PKC has been used in the treatment of hematological malignancies (Ganeshaguru et al., 2002). Thus murine IL-6-dependent plasmacytoma models may contribute to the development of a new strategy for therapy of human MM.

In conclusion our results suggest the presence of a unique mechanism of LPS-induced cell proliferation through the MAPK pathway in mouse IL-6-dependent plasmacytoma cells. LPS-triggered signaling is different from that of IL-6 and possibly involves Toll-like receptors as evolutionarily conserved surface molecules that play a key role in host response to bacterial mitogens.

Acknowledgements. This work is supported by Medical University of Sofia Grant no. 5-2001. We thank Vania Paskova from Department of Microbiology, Medical University - Sofia for excellent technical assistance. We also wish to thank to V. Nickolchev and K. Chokoisky from Preclinical University Center, Medical University -Sofia.

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Ianko Iankov(a), Ganka Atanasova(b), Maria Praskova(b), Silvia Kalenderova(b), Dragomir Petrov(a), Vanio Mitev(b), Ivan Mitov(a)

(a) Department of Microbiology, Medical University, Zdrave 2 str., 1431 Sofia, Bulgaria

(b) Department of Chemistry and Biochemistry, Medical University, Zdrave 2 str., 1431 Sofia, Bulgaria

Received: March 3, 2003 * Accepted: January 15, 2004

Corresponding author: Dr. Ianko Iankov, Department of Microbiology, Preclinical University Center, Medical University, Zdrave 2 str., 1431 Sofia, Bulgaria, Tel./Fax: +35929515317, e- mail: ianko@medfac.acad.bg

Copyright Urban & Fischer Verlag 2004

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