In this study we demonstrate that tumor necrosis factor α (TNFα) triggers only modest proliferation, as well as p44/p42 mitogen-activated protein kinase (MAPK) and NF-κB activation, in MM.1S multiple myeloma (MM) cells. TNFα also activates NF-κB and markedly upregulates (fivefold) secretion of interleukin-6 (IL-6), a myeloma growth and survival factor, in bone marrow stromal cells (BMSCs). TNFα in both a dose and time dependent fashion induced expression of CD11a (LFA-1), CD54 (intercellular adhesion molecule-1, ICAM-1), CD106 (vascular cell adhesion molecule-1, VCAM-1), CD49d (very late activating antigen-4, VLA-4), and/or MUC-1 on MM cell lines; as well as CD106 (VCAM-1) and CD54 (ICAM-1) expression on BMSCs. This resulted in increased (2–4-fold) per cent specific binding of MM cells to BMSCs, with related IL-6 secretion. Importantly, the proteasome inhibitor PS-341 abrogated TNFα-induced NF-κB activation, induction of ICAM-1 or VCAM-1, and increased adhesion of MM cells to BMSCs. Agents which act to inhibit TNFα may therefore abrogate the paracrine growth and survival advantage conferred by MM cell adhesion in the BM microenvironment.
TNFα is known to be a potent mediator of inflammation and bone resorption. Bone barrow mononuclear cells (BMMCs), as well as plasma cells isolated from multiple myeloma (MM) patients, express TNFα mRNA and protein (Garrett et al., 1987; Lichtenstein et al., 1989; Filella et al., 1996; Sati et al., 1999). Moreover, TNFα secretion in BM culture supernatants was significantly higher in patients with bone disease than in patients without bone disease (Davies et al., 2000). To date, however, the direct effects of TNFα on MM cell growth and survival are not fully characterized. This is of particular interest due to the recent remarkable clinical responses in MM patients treated with thalidomide (Singhal et al., 1999), which is known to inhibit TNFα, and our recent demonstration of the direct action of thalidomide on MM cells (Hideshima et al., 2000).
Additional interest in studying TNFα stems from its potential impact on paracrine MM cell growth and survival in the BM millieu. We (Ogata et al., 1997; Urashima et al., 1997; Chauhan et al., 1997, 2000) and others (Akira et al., 1990; Lichtenstein et al., 1995; Catlett-Falcone et al., 1999) have reported that IL-6 both induces proliferation of MM cells, mediated via the mitogen activated protein kinase (MAPK) cascade, and confers protection against dexamethasone (Dex)-induced apoptosis of MM cells, via specific activation of protein tyrosine phosphatase (SHP2). Others have shown that adhesion of MM cells to bone marrow stromal cells (BMSCs) confers protection against drug-induced apoptosis (Damiano et al., 1999), and we have shown that adherence of MM cells to BMSCs induces NF-κB dependent IL-6 transcription and secretion by BMSCs (Chauhan et al., 1996). To date, however, regulation of TNFα secretion by either MM cells or BMSCs alone, or by tumor cells bound to BMSCs, has not been characterized. Since there is a TNFα response element in the IL-6 promoter (Ray et al., 1989), the indirect effects of TNFα mediated via IL-6 are also of great interest. Finally, TNFα is known to induce NF-κB-dependent expression of adhesion molecules (Palombella et al., 1998; Li et al., 2000), further supporting its potential role in altering binding of MM cells to BMSCs within the BM millieu.
In this study, we show that TNFα induces a modest increase in proliferation, as well as MAPK/ERK activation, in MM cells. It induces IL-6 secretion, as well as NF-κB activation, in BMSCs. Importantly TNFα induces adhesion molecules on MM cells and BMSCs, with resultant increased MM cell to BMSC binding and IL-6 secretion; conversely, blockade of TNFα-induced NF-κB activation inhibits these sequelae. These studies confirm a central role for TNFα in the growth and survival of MM cells in the BM millieu and suggest the utility of novel therapeutics targeting TNFα in MM.
TNFα secretion from IM-9 MM cells stimulates IL-6 secretion from BMSCs
We first measured TNFα in culture supernatants of IM-9 MM cells alone, BMSCs alone, and IM-9 MM cells adherent to BMSCs. As can be seen Figure 1a, IM-9 cells secreted 30 and 72 pg/ml of TNFα in 24 and 48 h cultures, respectively, whereas BMSCs alone did not produce TNFα. Adhesion of IM-9 MM cells to BMSCs did not significantly augment TNFα secretion above that noted by IM-9 cells alone. Low levels of IL-6 are secreted by BMSCs, but not IM-9 cells; as in our prior studies (Uchiyama et al., 1993; Chauhan et al., 1996), adhesion of IM-9 cells to BMSCs significantly increased IL-6 secretion (Figure 1b). Similar results were observed in MM.1S cells (data not shown).
To determine whether TNFα stimulates IL-6 secretion from BMSCs, we cultured BMSCs with TNFα for 48 h; VEGF and TGFβ1 were used as positive controls for induction of IL-6 (Dankar et al., 2000; Junn et al., 2000). As can be seen in Figure 1c, TNFα induced significant (16-fold, P<0.001) increases in IL-6 secretion in BMSCs, whereas VEGF and TGFβ1 triggered only 2–3-fold increases in IL-6 secretion. Finally, a dose-dependent effect of TNFα on IL-6 secretion was demonstrated when BMSCs from two MM patients were incubated with TNFα (0.0001–10 ng/ml) (Figure 1d).
TNFα induces proliferation, NF-κB and p44/42 MAPK activation in MM.1S cells
We next examined the direct effect of TNFα on proliferation and signal transduction in MM.1S cells. Like IL-6 (Hideshima et al., 2000), TNFα (0.0016–10 ng/ml) induced modest (<twofold) increase in [3H]TdR incorporation (n=3, P=0.017–0.035, respectively) and viable cell number, which was blocked by the MEK1 inhibitor PD098059 (Figure 2a). Degradation of IκBα was also triggered by treatment of MM.1S cells with TNFα (Figure 2b), and TNFα-induced NF-κB activation in MM.1S cells was further confirmed using electrophoretic mobility shift analysis (EMSA) (Figure 2c).
Since we (Chauhan et al., 1997; Ogata et al., 1997; Urashima et al., 1997) and others (Borset et al., 1994; Catlett-Falcone et al., 1999) have shown that activation of p44/42 MAPK and STAT3 play role in cell proliferation and survival, respectively, in MM cells, we next examined whether TNFα triggered activation of p44/42 MAPK or STAT3 in MM.1S cells. As can be seen in Figure 2d, TNFα triggered phosphorylation of p44/42 MAPK, but not STAT3, in MM.1S cells. IL-6, which is known to activate MAPK and STAT3 in MM cells, was used as a positive control for activation of these kinases (Ogata et al., 1997; Catlett-Falcone et al., 1999).
TNFα induces NF-κB and p44/42 MAPK activation in BMSCs
We similarly examined the effect of TNFα on proliferation and signal transduction in BMSCs. As can be seen in Figure 3a, TNFα (0.1–10 ng/ml) for 48 h did not augment proliferation in BMSCs, assessed by MTT assay. However, EMSA demonstrated that TNFα treatment of BMSCs for 10 and 20 min activated NF-κB (Figure 3b). Importantly, this activation of NF-κB triggered by TNFα was inhibited by pre-treatment with proteasome inhibitor PS-341 (5 μM for 1 h). Finally, TNFα induced phosphorylation of p44/42 MAPK (Figure 3c), but not of STAT3 (data not shown) in MM.1S cells.
TNFα induces adhesion molecules on MM cell lines
We next determined whether TNFα induces adhesion molecules on MM cells. MM.1S, RPMI-8226 and IM-9 MM cells were examined for CD11a (LFA-1), CD49d (VLA-4), CD54 (ICAM-1), and CD106 (VCAM-1) expression using immunofluorescence flow cytometry before and after treatment with TNFα. As can be seen in Table 1, TNFα (10 ng/ml for 24 h) enhanced expression of CD11a, CD49d, and CD54, but not CD106, on both MM.1S cells and RPMI-8226 cells. Similar results were obtained in IM-9 cells (data not shown). This induction of CD11a, CD49d and CD54 expression on MM.1S cells occurred in a time-dependent fashion (Table 2). Culture with proteasome inhibitor PS-341 (Palombella et al., 1998) blocked TNFα-induced upregulation of CD54 (ICAM-1) expression on RPMI-8226 cells (Figure 4), as well as on MM.1S cells (data not shown).
TNFα induces CD54 (ICAM-1) and (CD106) VCAM-1 in BMSCs
We similarly determined whether TNFα induces adhesion molecules on BMSCs. Specifically, BMSCs were incubated for 2, 4 and 8 h in the presence TNFα (10 ng/ml), and the expression of ICAM-1 and VCAM-1 assessed by Western blotting. As can be seen in Figure 5a, expression of ICAM-1 and VCAM-1 was maximally induced after culture with TNFα for 2 h. Incubation of BMSCs with TNFα (0.4–50 ng/ml) for 2 h demonstrated that induction of both ICAM-1 and VCAM-1 on BMSCs was TNFα dose-dependent (Figure 5b).
TNFα augments of adherence of MM.1S cells to BMSCs
Having shown that TNFα induces adhesion molecules on both MM cells and BMSCs, we next examined whether MM cell to BMSC binding was also increased. BMSCs from three MM patients were pre-treated with DMSO or TNFα to induce ICAM-1 and VCAM-1 expression, and MM.1S binding to these BMSCs was assessed as in prior studies (Uchiyama et al., 1993). As can be seen in Figure 6, TNFα triggered a dose-dependent significant increase (2.2–4.5-fold, P<0.01) in per cent specific binding of MM.1S cells to BMSCs.
Adhesion of MM.1S cells induces p44/42 MAPK activation in BMSCs
We next examined whether adhesion of MM.1S cells could activate p44/42 MAPK in BMSCs. Although constitutive activation of both MEK1/2 and p44/42 MAPK in BMSCs was observed, maximal induction of MEK1/2 and p44/42 MAPK phosphorylation occurred after adhesion of 1% formaldehyde-fixed MM.1S cells for 10 min (Figure 7). IL-6 did not activate MAPK activity in formaldehyde-fixed MM.1S cells, confirming that the adhesion-induced MAPK activation was in BMSCs.
Mechanism of TNFα-induced increased binding of MM.1S cells to BMSCs
To determine whether TNFα induced ICAM-1 and VCAM-1 expression on BMSCs via activation of NF-κB, we next examined the effect of the proteasome inhibitor PS-341 on induction of these adhesion molecules. As seen in Figure 8a, pre-treatment of BMSCs with PS-341 (5 μM for 1 h), but not DMSO, abrogated induction of ICAM-1 or VCAM-1 by TNFα.
To confirm the mechanism of increased MM.1S binding to BMSCs induced by TNFα, we next examined whether anti-ICAM-1 Ab, anti-VCAM-1 Ab, or PS-341 could block the augmentation of MM.1S cell to BMSC adhesion triggered by TNFα. PS-341 completely, whereas anti-ICAM-1 and/or anti-VCAM-1 Abs partially, abrogated the increased adhesion of MM.1S cells to BMSCs triggered by TNFα (Figure 8b).
TNFα is an inflammatory and bone resorbing cytokine with both direct and indirect effects mediated by induction of other cytokines. Previous reports of low levels of TNFα mRNA and protein in MM patient samples (Sati et al., 1999; Davies et al., 2000), coupled with responses after treatment of MM patients with thalidomide (Singhal et al., 1999) and its known TNFα inhibitory activity (Corral et al., 1999), provided the impetus for the current study to delineate the role of TNFα in the pathogenesis of MM. Although the direct effect of TNFα on MM cell proliferation is modest, it mediates two major effects in the BM to promote growth and survival of MM cells: direct stimulation of IL-6 production in BMSCs; and upregulation of adhesion molecules on MM cells and on BMSCs, thereby enhancing binding of MM cells to BMSCs and related IL-6 secretion. These studies highlight the importance of studying the MM cell within the BM millieu, and suggest TNFα as a novel therapeutic target in MM.
Previous reports have detected TNFα mRNA and protein in six of 10 MM patients (Sati et al., 1999), and demonstrated that TNFα induces in vitro proliferation of a MM cell line (Jourdan et al., 1999). Our findings also suggest that TNFα is secreted by MM cells, but that it is not a major MM growth factor. Our (Urashima et al., 1997; Chauhan et al., 1997, 2000; Hideshima et al., 2000) and other (Borset et al., 1994; Lichtenstein et al., 1995; Catlett-Falcone et al., 1999) studies have demonstrated that IL-6 is the major growth and survival factor for human MM cells, and specifically shown that proliferation induced by IL-6 is mediated via the MAPK signaling pathway (Ogata et al., 1997). The current study shows that TNFα-induced MM cell proliferation is also mediated via this cascade. There appear to be differences, however, since IL-6 triggers STAT3 activation whereas TNFα does not. Importantly, there is known to be a TNFα response element in the IL-6 promoter (Ray et al., 1989), and we show that TNFα markedly up-regulates IL-6 secretion in BMSCs. We have previously shown that TGFβ secreted by MM cells augments IL-6 secretion in BMSCs (Urashima et al., 1996), but the induction of IL-6 in BMSCs by TNFα observed in this study is much greater. Therefore one key role for TNFα in MM is to augment paracrine IL-6 mediated MM cell growth and survival.
We have shown that adherence of MM cells to MM patient BMSCs triggers IL-6 secretion from BMSCs via activation of NF-κB (Chauhan et al., 1996). Moreover, adherence of MM cells to BMSCs protects MM cells against drug-induced apoptosis (Damiano et al., 1999). These reports confirm the critical role of interactions between MM cells and BMSCs in the BM microenvironment. In our previous studies, we have delineated those adhesion molecules on MM cells (CD49d, VLA-4; CD11a, LFA-1; Muc-1) mediating adherence to extracellular matrix proteins (fibronectin) and to BMSCs (CD54, ICAM-1; CD106, VCAM-1) (Uchiyama et al., 1992, 1993; Kim et al., 1994; Teoh and Anderson, 1997). Moreover, TNFα induces VCAM-1 and ICAM-1 expression in a NF-κB dependent manner in human umbilical vein endothelial (HUVE) cells (Palombella et al., 1998). In the present study, we demonstrate that TNFα induces adhesion molecules on both MM cells (CD49d, VLA4; CD11a, LFA-1; Muc-1) and BMSCs (CD54, ICAM-1; CD106, VCAM-1), which results in increased MM cell to BMSC adhesion (Figure 9). Increased tumor cell binding in turn induces IL-6 secretion from MM patient BMSCs. Our recent studies further suggest that vascular endothelial growth factor (VEGF) is secreted by MM cells, and that this secretion is also markedly upregulated by MM cell adhesion to BMSCs (Gupta et al., 2001). VEGF may not only account for the increase in MM BM angiogenesis, but our studies further demonstrate that VEGF directly acts on MM cells to trigger migration (Podar et al., 2001). Therefore inhibition of increased adhesion triggered by TNFα will likely inhibit not only growth and survival effects of adhesion, but also tumor cell migration.
We have previously shown that the proteasome inhibitor PS-341 directly induces apoptosis even of MM cells resistant to conventional therapy, and downregulates the NF-κB dependent induction of IL-6 in BMSCs triggered by MM cell binding (Chauhan et al., 1996). Since PS-341 has previously been reported to inhibit TNFα-induced ICAM-1, VCAM-1, or E-Selectin expression on HUVE cells (Palombella et al., 1998), we assessed whether it acted similarly in MM. Specifically, we show that PS-341 abrogates TNFα-induced upregulation of adhesion molecules on both MM cells and BMSCs and, most importantly, inhibits MM cell to BMSC binding. This blockade both further validates strategies to target TNFα in MM and has important implications for other novel treatment approaches in MM. For example, thalidomide has recently been used to achieve responses in MM refractory to conventional therapy (Singhal et al., 1999), based upon its anti-angiogenic activity (D'Amato et al., 1994) and the increased angiogenesis observed in MM BM (Vacca et al., 1999). However, we have recently shown that thalidomide and its immunomodulatory derivatives have multiple anti-MM activities: direct action on MM cells to induce apoptosis or G1 growth arrest; blockade of the increased secretion of MM cell growth, survival, and migration factors (IL-6, VEGF) triggered by binding of MM cells to BMSCs; expansion of patients' natural killer cell number and function against MM cells; and downregulation of protectin expression in MM cells (Raje and Anderson, 1999; Hideshima et al., 2000; Treon et al., 2001; Davies et al., 2001). Although both thalidomide and the immunomodulatory derivatives inhibit TNFα production from LPS-stimulated monocytes (Marriott et al., 1998), they also inhibit IL-1β and IL-6 and increase IL-10 production in these cells (Corral et al., 1999); stimulate T-cell proliferation, IL-2, and interferon-γ production (Haslett et al., 1998); and do not inhibit phosphodiesterase-4. Characterization of these independent bioactivities of TNFα on MM cells in the BM millieu will not only aid in determining which of these multiple activities of thalidomide and the immunomodulatory drugs is clinically relevant, but also may allow for the development of novel therapeutics with increased efficacy and decreased toxicity.
The current study therefore demonstrates important effects mediated by TNFα on MM cell growth, survival, and migration which would not have been appreciated if MM cells were examined alone and only became apparent when studying MM cells in the context of the BM millieu. These studies therefore confirm the importance of evaluating new agents not only for their direct anti-tumor effects, but also for their impact on intercellular interactions and cytokine production within the BM. Importantly, these studies provide a strong rationale for targeting TNFα in novel therapeutics in MM.
Materials and methods
MM.1S cells were kindly provided by Dr Steven Rosen (Northwestern University, Chicago, IL, USA). The cells were cultured in RPMI-1640 media containing 10% fetal bovine serum (FBS, Sigma Chemical Co., St. Louis, MO, USA), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (GIBCO, Grand Island, NY, USA). BM specimens were obtained from patients with MM with appropriate Institutional Review Board approval. Mononuclear cells (MNCs) separated by Ficoll-Hypaque density sedimentation were used to establish long-term BMSC cultures, as previously described (Uchiyama et al., 1993). When an adherent cell monolayer had developed, BMSCs were harvested in Hank's Buffered Saline Solution containing 0.25% trypsin and 0.02% EDTA, washed, and collected by centrifugation.
Measurement of TNFα and IL-6
Both TNFα and IL-6 were measured in cell supernatants using Duoset ELISA (R&D System, Minneapolis, MN, USA). To examine whether adherence of IM-9 MM cells to BMSCs induces TNFα and IL-6 secretion from BMSCs, BMSCs were incubated with or without IM-9 cells and culture supernatants harvested at 24 or 48 h. BMSCs were also incubated in the presence of VEGF (10 ng/ml), TNFα (10 ng/ml) or TGFβ1 (10 ng/ml) for 24 h to similarly assay for induction of TNFα and IL-6 secretion.
Colorometric assays were performed to assess the effect of TNFα on proliferation of BMSCs, as in prior studies (Hideshima et al., 2000). Cells from 48 h cultures of BMSCs were pulsed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT, Chemicon International Inc., Temecula, CA, USA) for 4 h (10 μl of 5 mg/ml per well), followed by 100 μl isopropanol containing 0.04N HCl. Absorbance readings at 570 nm were measured on a spectrophotometer (Molecular Devices Corp., Sunnyvale, CA, USA). Tritiated thymidine incorporation assays ([3H]TdR) were also performed to assess cell proliferation, as previously described (Hideshima et al., 2000). Viable cells were enumerated using Trypan blue exclusion.
ICAM-1 or VCAM-1 expression in BMSCs was assayed by Western blotting. BMSCs were incubated in the presence of 10 ng/ml human recombinant TNFα (R&D System) at 37°C for 2, 4 and 8 h. To assay for dose dependency, BMSCs were incubated for 4 h in the presence of TNFα (0.2 to 50 ng/ml). The cells were lysed in lysis buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 5 mM EDTA, 5 mM NaF, 2 mM Na3VO4, 1 mM PMSF, 5 μg/ml leupeptine, 5 μg/ml aprotinin. For detection of ICAM-1, VCAM-1, and phosphorylated MAPK, cell lysates were subjected to SDS–PAGE, transferred to PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA), and immunoblotted with anti-ICAM-1, anti-VCAM-1 (Santa Cruz Biotech., Santa Cruz, CA, USA), and anti-phospho MAPK (New England Biolabs, Beverly, MA, USA) Abs, respectively. Immunoblotting with anti-alpha-tubulin Ab (Sigma) confirmed equivalent protein loading. Similar immunoblotting was performed on BMSCs pre-treated with proteasome inhibitor PS-341 (10 μM, Millennium Pharmaceuticals Inc., Cambridge, MA, USA) for 1 h prior to incubation with TNFα (10 ng/ml) for 2 h.
Flow cytometric analysis
MM.1S, RPMI-8226, and IM-9 cells were cultured for 24 h in the presence or absence of TNFα (10 ng/ml), washed, and stained with specific Abs directed against CD11a (LFA-1), CD49d (VLA-4), CD54 (ICAM) (Immunotech, Marseille, France), CD106 (VCAM) (R&D System, Minneapolis, MN, USA) in either direct (CD11a, CD49d and CD54) or indirect (CD106) immunofluorescence assays and flow cytometric analysis, as previously described (Uchiyama et al., 1992).
Assays of adherence of MM cells to BMSCs were performed as previously described (Uchiyama et al., 1993). MM.1S cells labeled with Na2CrO4 (NEN, Boston, MA, USA), were added to either TNFα pre-treated (10 ng/ml for 2 h) or non-treated BMSC-coated 96-well plates and incubated for 1 h. After incubation, each well was washed twice with media and lysed with 0.5% NP-40; lysate radioactivity was counted on a gamma counter. The proteasome inhibitor PS-341, anti-ICAM-1 Ab, and/or anti-VCAM-1 Ab (R&D System) were used to block TNFα-induced augmentation of adhesion. Specifically, BMSCs were pre-treated with either DMSO (0.1%) or PS-341 (10 μM) before stimulation with TNFα (10 ng/ml) for 2 h and MM cell adhesion. TNFα-stimulated BMSCs were incubated with anti-ICAM-1 Ab and/or anti-VCAM-1 Ab (R&D System) prior to addition of MM.1S cells and adhesion assays.
Assays for activation of NF-κB
To assay for NF-κB activation in BMSCs, BMSCs were pre-incubated with PS-341 (10 μM for 1 h) before stimulation with TNFα (10 ng/ml for 10, 20 or 30 min). Degradation of IκBα was used to assay for activation of NF-κB in MM cells (Palombella et al., 1998). To analyse the effect of TNFα on degradation of IκBα in MM.1S cells, the cells were incubated with TNFα (5 ng/ml), washed, and lysed. Whole cell extracts were prepared and analysed by Western blotting with anti-IκBα Ab (Santa Cruz Biotech.)
Electrophoretic mobility shift analysis
Nuclear extracts for electrophoretic mobility shift analyses (EMSA) were carried out as in our previous studies (Hideshima et al., 2001). Briefly, double-stranded NF-κB consensus oligonucleotide probe (5′ -GGGGACTTTCCC-3′, Santa Cruz Biotech.) was end-labeled with γ32P-ATP (50 μCi at 222 TBq/mM; NEN, Boston, MA, USA). Binding reactions containing 1 ng of oligonucleotide and 3 μg of nuclear protein were conducted at room temperature for 20 min in total volume of 10 μl of binding buffer [(10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 4% glycerol (v/v), and 0.5 μg poly (dI-dC) (Pharmacia, Peapack, NJ, USA)]. For supershift analysis, 1 μg of anti-p65 NF-κB Ab (Santa Cruz Biotech.) was added 5 min before the reaction mixtures, immediately after addition of radiolabeled probe. The samples were loaded onto a 4% polyacrylamide gel, transferred to Whatman paper (Whatman International, Maidstone, UK), and visualized by autoradiography.
Assay for MAPK signaling in BMSCs triggered by MM cell adhesion
BMSCs were cultured overnight in ISCOVE's MEM supplemented with 2% FBS (2%–ISCOVE), washed twice, and re-suspended in 2%–ISCOVE. BMSCs were subsequently incubated with TNFα (10 ng/ml) for 2 h and washed two times with 2%–ISCOVE. MM.1S cells were fixed in 1% paraformaldehyde (4°C for 40 min), washed three times with PBS, resuspended in 2%–ISCOVE, and then added to TNFα pre-pretreated BMSCs. After incubation for 10, 20 or 30 min, the cells were scraped, washed, lysed, and immunoblotted to detect tyrosine phosphorylated P44/42 MAPK and serine phosphorylated MEK1/2 with anti-phospho MAPK and anti-phospho MEK1/2 Abs, respectively (New England Biolabs).
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Supported by National Institutes of Health Grant PO-1 78378 and the Doris Duke Distinguished Clinical Research Scientist Award to KC Anderson.
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Hideshima, T., Chauhan, D., Schlossman, R. et al. The role of tumor necrosis factor α in the pathophysiology of human multiple myeloma: therapeutic applications. Oncogene 20, 4519–4527 (2001) doi:10.1038/sj.onc.1204623
- multiple myeloma
- adhesion molecule
Peripheral neuropathy following bortezomib therapy in multiple myeloma patients: association with cumulative dose, heparanase, and TNF-α
Annals of Hematology (2019)
Targeting the β2-integrin LFA-1, reduces adverse neuroimmune actions in neuropathic susceptibility caused by prenatal alcohol exposure
Acta Neuropathologica Communications (2019)
Drug Resistance Updates (2019)
The NEDD8-activating enzyme inhibitor MLN4924 sensitizes a TNFR1+ subgroup of multiple myeloma cells for TNF-induced cell death
Cell Death & Disease (2019)
Clinical Immunology (2019)