Myeloma cell growth arrest, apoptosis, and interleukin-6 receptor modulation induced by EB1089, a vitamin D3 derivative, alone or in association with dexamethasone

We have previously shown that malignant plasma cells expressed the specific receptor for 1,25-dihydroxyvitamin D3 and that this derivative could significantly inhibit the proliferation of such malignant cells. More recently, new vitamin D3 derivatives have been generated with extraordinarily potent inhibitory effects on leukemic cell growth in vitro. These new data prompted us to (re)investigate the capacity of such new vitamin D3 derivatives to inhibit myeloma cell growth in comparison with that of dexamethasone, a potent antitumoral agent in multiple myeloma. In the current study, we show that EB1089, a new vitamin D3 derivative, (1) induces G1 growth arrest of human myeloma cells, which is only partially reversed by interleukin-6 (IL-6); (2) induces apoptosis in synergy with dexamethasone, IL-6, leukemia-inhibitory factor, and Oncostatin M, with an agonistic anti-gp130 monoclonal antibody being unable to prevent this apoptosis; (3) downregulates both the gp80 (ie, the alpha chain of the IL-6 receptor [IL-6Ralpha]) expression on malignant plasma cells and the production of soluble IL-6Ralpha, and finally (4) inhibits the deleterious upregulation of gp80 expression induced by dexamethasone while limiting the dexamethasone-induced upregulation of gp130 expression. Considering that these in vitro effects of EB1089 have been observed at doses obtainable in vivo (without hypercalcemic effects), our present data strongly suggest that EB1089 could have a true interest in the treatment of multiple myeloma, especially in association with dexamethasone.     

Immunoreactivation of Epstein-Barr virus due to cytomegalovirus primary infection

Interleukin (IL)-6 plays a major role in the control of the survival and proliferation of myeloma cell lines and primary myeloma cells. The genes of the receptors of IL-6 have been cloned, and the major signaling pathways involved in gp130 IL-6 transducer activation have been identified. In addition, another five cytokines that activate the gp130 IL-6 transducer have been identified. We review the recent data on gp130 cytokines and gp130-mediated signal transduction, their involvement in myeloma cell biology, and we discuss the possible therapeutic applications of this knowledge.     

Dimerization and activation of the common transducing chain (gp130) of the cytokines of the IL-6 family by mAb

The objective of our research was to study the mechanisms of activation of mAb against the gp130 transducer chain common to the IL-6 cytokine family. It has been found that among the 56 anti-gp130 available worldwide, none was able to activate the growth of IL-6-dependent myeloma cell lines. When certain of them were associated in pairs they allowed the cells to grow; alone, they were inhibitory. The same activation was also obtained by cross-linking certain anti-gp130 mAb on the cell membrane with a goat anti-mouse Ig antiserum. A bispecific mAb was prepared by the somatic fusion of two hybridomas secreting two mAb whose association was able to activate gp130 signaling; the bispecific mAb was inactive. The activating mAb were able to support long-term proliferation of the IL-6-dependent myeloma cell lines, which indicates that they are potential valuable growth factors of tumor cells and hematopoietic stem cells. When they were injected into SCID mice, they allowed human IL-6-dependent myeloma cell lines to grow, develop tumors and metastasize. By studying the functional epitopes of the cell membrane gp130 receptors, it was shown that the activating mAb induced gp130 dimerization and STAT3 activation, as did IL-6.     

Human herpesvirus type 8 interleukin-6 homologue is functionally active on human myeloma cells

Seroepidemiology and polymerase chain reaction studies have strongly suggested that human herpesvirus type 8 (HHV-8) is associated with Kaposi's sarcoma, Castleman's disease, and body cavity-based lymphoma. The genome of HHV-8 harbors a viral analogue of the interleukin-6 (IL-6) gene. The amino acid sequence of the viral IL-6 (vIL-6) protein is 24.7% identical to human IL-6 (hIL-6). IL-6 as a B-cell growth and differentiation factor is known to play an essential role in the pathophysiology of B-cell tumors. Thus, it seems possible that virus-encoded IL-6 contributes to malignant growth of HHV-8-positive B-cell lymphatic tumors. We have tested a preparation of HHV-8-derived IL-6 for the ability to promote the proliferation of the human myeloma cell line INA-6, which is strictly dependent on exogenous IL-6 for growth and survival. Viral IL-6 significantly induced DNA synthesis of INA-6 cells, but required much more protein on a weight basis when compared with hIL-6 for maximal proliferation. The proliferative effect of vIL-6 was almost completely inhibited by a combination of anti-IL-6 receptor (IL-6R) and anti-gp130 antibodies or IL-6R superantagonist Sant7 and anti-gp130 antibodies. This report demonstrates that vIL-6 has proliferative activity on human cells and that the IL-6R and gp130 are involved in vIL-6 signaling in the myeloma cell line INA-6.     

Differential human multiple myeloma cell line responsiveness to interferon-alpha. Analysis of transcription factor activation and interleukin 6 receptor expression

Although IFN-alpha is commonly used as maintenance treatment for multiple myeloma patients, its effectiveness is varied. In this study, we have used a panel of IL-6 responsive myeloma cell lines that vary remarkably in responsiveness to IFN-alpha. Three cell lines were growth arrested by IFN-alpha; however, IFN-alpha significantly stimulated growth of the fourth cell line, KAS-6/1. Our studies have focused on elucidating the mechanism of differential IFN-alpha responsiveness. First, we have shown that IFN-alpha-stimulated growth of the KAS-6/1 cells did not result from induction of autocrine IL-6 expression. Second, analysis of Stats 1, 2, and 3 and IFN regulatory factor-1 (IRF-1) and IRF-2 activation failed to reveal differences between the IFN-alpha growth-arrested or growth-stimulated cells. Third, although IFN-alpha treatment of the IFN-alpha growth-inhibited cell lines reduced IL-6 receptor (IL-6R) expression, IFN-alpha also reduced KAS-6/1 IL-6R expression. Finally, although IFN-alpha treatment reduced IL-6R numbers on each cell line, analysis of Stat protein activation revealed that the receptors were still functional. We conclude that myeloma cell responsiveness to IFN-alpha is heterogeneous and that mechanisms of IFN-alpha-mediated growth inhibition other than IL-6R downregulation must exist in myeloma. Identification of these mechanisms may allow development of agents that are more universally effective than IFN-alpha.     

gp130 transducing receptor cross-linking is sufficient to induce interleukin-6 type responses

gP130 transducing receptor is involved in the formation of high affinity receptors for the cytokines of the interleukin-6 (IL-6) family. Recruitment of gp130 by IL-6 associated to its receptor leads to the dimerization of the transducing component. In the present study we did characterize the B-S12 monoclonal antibody raised against gp130 and able to elicit IL-6 type biological activities. B-S12 antibody triggered strongly the proliferation of TF1 and XGI hematopoietic cell lines and was able to increase the synthesis of acute phase proteins in HepG2 hepatoma cell line. B-S12 also behaved as a synergistic factor with granulocyte-macrophage colony-stimulating factor for both proliferation and differentiation of CD34-positive hematopoietic cell progenitors. By using a symmetric enzyme-linked immunosorbent assay, allowing the detection of dimeric forms of soluble gp130, we found that addition of B-S12 to gp130 led to its dimerization. Analysis of the tyrosine phosphorylation events in gp130 and Jak kinase family members revealed that B-S12 quickly induced the phosphorylation of gp130 in a neural derived cell line, and that Jak1 and Jak2 were also recruited. In conclusion, we show that gp130 cross-linking with the B-S12 monoclonal antibody was sufficient to generate functional IL-6 type responses in hematopoietic, neural, and hepatic cells.     

Fas/APO-1 (CD95)-mediated apoptosis is activated by interferon-gamma and interferon- in interleukin-6 (IL-6)-dependent and IL-6-independent multiple myeloma cell lines

A poor response to Fas-induced apoptosis is evident in some multiple myeloma (MM) cell lines and primary cells. In this study, we have examined the possibility to increase the sensitivity to Fas-induced apoptosis by pretreatment of MM cells with interferon-gamma (IFN-gamma) or interferon-alpha (IFN-alpha). Both IFN-gamma and IFN-alpha markedly increased the Fas-induced apoptosis in all cell lines tested (U-266-1970, U-266-1984, and U-1958). In the U-266-1970 and U-1958 cell lines, pretreatment with either IFN-gamma or IFN-alpha also inhibited proliferation in a dose-dependent manner. In contrast, IFN-gamma activation of the Fas death pathway in the U-266-1984 cells was not accompanied by growth inhibition. Incubation with the IFNs increased the Fas antigen expression in one of three cell lines but did not alter the expression of Bcl-2 or Bax. The IFNs are important regulators of growth and survival in MM cells. Our results suggest that activation of Fas-mediated apoptosis is a novel mechanism by which the IFNs exert inhibitory effects on MM cells.     

IL-6 triggers cell growth via the Ras-dependent mitogen-activated protein kinase cascade

IL-6 mediates growth of some human multiple myeloma (MM) cells and IL-6-dependent cell lines. Although three IL-6 signaling pathways (STAT1, STAT3, and Ras-dependent MAPK cascade) have been reported, cascades mediating IL-6-triggered growth of MM cells and cell lines are not defined. In this study, we therefore characterized IL-6 signaling cascades in MM cell lines, MM patient cells, and IL-6-dependent B9 cells to determine which pathway mediates IL-6-dependent growth. IL-6 induced phosphorylation of JAK kinases and gp130, regardless of the proliferative response of MM cells to this growth factor. Accordingly, we next examined downstream IL-6 signaling via the STAT3, STAT1, and Ras-dependent mitogen-activated protein kinase (MAPK) cascades. IL-6 triggered phosphorylation of STAT1 and/or STAT3 in MM cells independent of their proliferative response to IL-6. In contrast, IL-6 induced phosphorylation of Shc and its association with Sos1, as well as phosphorylation of MAPK, only in MM cells and B9 cells that proliferated in response to IL-6. Moreover, MAPK antisense, but not sense, oligonucleotide inhibited IL-6-induced proliferation of these cells. These data suggest that STAT1 and/or STAT3 activation may occur independently of the proliferative response to IL-6, and that activation of the MAPK cascade is an important distal pathway for IL-6-mediated growth.     

Characterization of soluble gp130 released by melanoma cell lines: A polyvalent antagonist of cytokines from the interleukin 6 family

gp130 acts as a common transducing signal chain for all receptors belonging to the interleukin (IL)-6 receptor family. The IL-6-related cytokines [IL-6, IL-11, oncostatin M (OSM), leukemia inhibitory factor, ciliary neurotrophic factor, and cardiotrophin] often modulate tumor phenotype and control the proliferation of many tumor cell lines. We demonstrate that melanoma cell lines release, in vitro and in vivo (when transplanted in nude mice), soluble gp130 (sgp130), a potential antagonist of cytokines from the IL-6 family. Biochemical analysis revealed that sgp130 derived from melanoma patients' sera or from culture supernatants of melanoma cell lines is a Mr 104,000 protein that resolved after deglycosylation as a Mr 58,000 protein. PCR and Northern blot analysis only identified one gp130 membrane mRNA, suggesting that the soluble form of gp130 is generated by proteolytic cleavage. OSM reproducibly increases sgp130 released by melanoma cell lines, whereas leukemia inhibitory factor stimulates the production of sgp130 in only one of three cell lines tested. This tumor-derived sgp130 is functional because it binds in solution to the IL-6-soluble IL-6 receptor (gp80) complex. Recombinant sgp130 inhibits the growth inhibitory activity of the IL-6-soluble IL-6 receptor complex and OSM on some melanoma cell lines. Therefore, this soluble gp130 represents a natural antagonist of cytokines from the IL-6 family.     

Mapping genes that underlie ethnic differences in disease risk: methods for detecting linkage in admixed populations, by conditioning on parental admixture

The mechanism by which interleukin-6 (IL-6) protects multiple myeloma (MM) plasma cells from apoptosis induced by anti-fas antibodies and dexamethasone was studied. Anti-apoptotic concentrations of IL-6 had no effect on cell-cycle distribution or activation of RAF-1 or ERK in dexamethasone- or anti-fas-treated 8226 and UCLA #1 MM cell lines. However, IL-6-dependent protection of viability correlated with an inhibition of dexamethasone- and anti-fas-induced activation of jun kinase (JNK) and AP-1 transactivation. To test the hypothesis that cytokine-induced protection was mediated through inhibition of JNK/c-jun, we also inhibited c-jun function in 8226 cells via introduction of a mutant dominant negative c-jun construct. Mutant c-jun-containing MM cells were also resistant to anti-fas-induced apoptosis but were significantly more sensitive to dexamethasone-induced apoptosis. These results support the notion that IL-6 protects MM cells against anti-fas through its inhibitory effects on JNK/c-jun but indicate protection against dexamethasone occurs through separate, yet unknown pathways.     

Expression of IL-6, IL-6 receptor and its signal transducer gp130 mRNAs in megakaryocytic cell lines

IL-6, its receptor and its transducer protein gp130 mRNAs were analyzed in three cell lines with megakaryocytic properties including CMK, K562, and HEL. Their colony proliferation was also assayed in the methylcellulose medium with or without IL-6. CMK had IL-6, IL-6 receptor and gp130 mRNAs, while the other two cell lines lacked IL-6 receptor mRNA expression. K562 responded to IL-6 indicating that it has an alternative receptor to the so called "IL-6 receptor". HEL was not sensitive to IL-6 suggesting that it proliferates with growth factors other than IL-6 via gp130.     

Mechanisms of multiple myeloma cell growth

The mechanism of human multiple myeloma cell growth was studied utilizing eleven myeloma cell lines established in vitro or in vivo (Scid mouse). Four bone marrow derived cell lines grew dependently on IL-6 or bone marrow stromal cells. Seven extramedullary lesion derived cell lines grew spontaneously and additively proliferated in response to IL-6. All cell lines expressed the IL-6 receptor (IL-6R) and IL-6RmRNA, but none expressed IL-6mRNA. No IL-6 activity was detected in the myeloma cell culture supernatant. Both the anti-IL-6 antibody and anti-IL-6R antibody neutralized IL-6-induced proliferation, but did not inhibit spontaneous proliferation of extramedullary lesion derived cell lines. While establishing cell lines, it was found that the proliferating fraction was primarily included in a fraction which was non-adherent to stromal cells and composed of undifferentiated plasmablasts. Undifferentiated plasmablasts proliferated in response to IL-6, in contrast to the adherent, mature form of myeloma cells which did not proliferate in response to IL-6. Innoculation of myeloma cells into Scid mice induced subcutaneous tumor formation. These tumors were composed of undifferentiated plasmablasts, which also proliferated in response to IL-6. These results imply that the growth of bone marrow derived myeloma cell lines are dependent on the IL-6 paracrine mechanism and that the growth of extramedullary lesion derived cell lines primarily autonomous and additively dependent on the IL-6 paracrine mechanism.     

Chloride channels in excised membrane patches from human platelets: effect of intracellular calcium

Interleukin-6 (IL-6) is one of the most important growth factors for myeloma cells. We examined the effect of recombinant IL-6 on the proliferation of five human myeloma cell lines, which were independently established AT Kawasaki Medical School. Only the KMS-11 cell line among these five lines showed growth enhancement induced by IL-6. Based on the results, a possible contribution of Ca(2+)-phospholipid-dependent protein kinase C (PKC) to the signal transduction in KMS-11 cells during growth enhancement was studied, since PKC may play an important role in malignant transformation or cell proliferation induced by some growth factors, such as IL-6. When exogenous IL-6 was added to KMS-11 culture, we observed (1) reduction of total PKC activity, and (2) translocation of PKC activity from its cytosol fraction to the membrane fraction. These findings may indicate that down regulation of PKC occurred during the myeloma cell proliferation induced by IL-6. However, IL-6 does not appear to be involved in cell proliferation and differentiation in the other cell lines studied.     

Inhibition of interleukin-6-induced human immunodeficiency virus type 1 expression by anti-gp130 monoclonal antibody

Interleukin 6 (IL-6) has been reported to upregulate the expression of human immunodeficiency virus type 1 (HIV-1) in infected monocyte/macrophages. Since IL-6 signal is transduced through the membrane glycoprotein gp130, we investigated the inhibitory effect of anti-gp130 monoclonal antibody (mAb) on IL-6-induced viral expression in monocytic cell lines latently infected with HIV-1. IL-6 significantly induced the expression of HIV-1 in U1 cells, whereas it had no effect in OM-10.1 cells. Flow cytometric analysis revealed that U1 but not OM-10.1 cells expressed gp130 on their surface. The anti-gp130 mAb could inhibit IL-6-induced HIV-1 expression in U1 cells in a dose dependent fashion. These results suggest that blocking the gp130 signal transduction may have therapeutic potential for the treatment of HIV-1 infection.     

Differential myeloma cell responsiveness to interferon-alpha correlates with differential induction of p19(INK4d) and cyclin D2 expression

Interferon-alpha (IFN-alpha) has been used as therapy for the treatment of a variety of viral diseases and malignancies including multiple myeloma. The effectiveness of interferon-alpha in treating multiple myeloma, however, has been somewhat variable, and the mechanism(s) accounting for this is not well understood. As a means to examine the basis for the differential effectiveness of this cytokine, we have analyzed IFN-alpha-mediated modulation of the cell cycle in two human myeloma cell lines. These two cell lines, ANBL-6 and KAS-6/1, display dramatically different outcomes in response to this cytokine. Although IFN-alpha inhibited the growth of ANBL-6 cells by blocking cell cycle progression from G0/G1 to S phase, IFN-alpha stimulated cell cycle progression in KAS-6/1 cells. Moreover, the effects of IFN-alpha on cell cycle progression correlated with the phosphorylation status of the retinoblastoma protein. Of interest, IFN-alpha increased cyclin D2 expression and cyclin-dependent kinase activity in the KAS-6/1 cells but not in the ANBL-6 cells. To determine whether the differential effects of IFN-alpha on myeloma cell cycle progression could also result from differences in the expression of cyclin-dependent kinase inhibitors, we examined the effects of IFN-alpha on the induction of cyclin-dependent kinase inhibitors with broad regulatory function (p21 and p27) and those with specificity for G1-associated cyclin-cyclin-dependent kinase complexes (p15, p16, p18, and p19). Although we failed to detect an effect of IFN-alpha on expression levels of p21, p15, p16, or p18, IFN-alpha treatment of the ANBL-6 cell line resulted in induction of p19 expression, whereas it was without effect on the KAS-6/1 cell line. These results suggest that heterogeneity in IFN-alpha-mediated growth effects in myeloma cells correlates with differential induction of cyclin D2 and p19(INK4d) expression.     

Interleukin-1 in multiple myeloma: producer cells and their role in the control of IL-6 production

We studied the role of interleukin (IL)-1beta in patients with multiple myeloma. By in situ hybridization and immunochemistry, myeloid and megakaryocytic cells expressed high levels of the IL-1beta gene and produced IL-1beta. Myeloma cells less potently expressed the IL-1beta gene and IL-1beta protein. IL-1beta gene expression was not constitutive since it was detected in the bone marrow myeloma cells of two patients, unlike circulating tumoural cells. In addition, nine myeloma cell lines failed to express the IL-1beta gene and this expression could not be induced by 12 different cytokines. We demonstrated that IL-1 was mainly responsible for IL-6 production in the tumoural environment through a PGE2 loop. In fact, an IL-1 receptor antagonist (IL-1RA) blocked PGE2 synthesis and IL-6 production by 80%; this blockage could be reversed by adding synthetic PGE2. Similar findings were found with indomethacin, an inhibitor of cyclooxygenase that blocks PGE2 synthesis. Taken together, these data emphasize the possibility of blocking IL-1 by using IL-1RA or other antagonists in order to block IL-6 production, which is a major tumoural survival and proliferation factor.     

Effect of dexamethasone on cell proliferation of neuroepithelial tumor cell lines

The effect of glucocorticoid on cell proliferation, the expression of glucocorticoid receptor, and the relationship between inhibition of cell growth and apoptosis were investigated in four established neuroepithelial tumor cell lines (KNS42, T98G, A172, and U251MG). Glucocorticoid receptor expression was located in the cytoplasm of untreated cells, but translocated into nuclei after treatment with dexamethasone in KNS42, T98G, and A172 cells. U251MG did not express glucocorticoid receptors. Dexamethasone significantly inhibited the growth of KNS42 and T98G cell lines, at high concentrations in contrast to growth stimulation at low concentration. Dexamethasone inhibited proliferation of A172 cell line at all concentrations from 10(-4) M to 10(-7) M. These were prevented by RU38486, a specific glucocorticoid antagonist. Apoptosis did not occur in any cell lines after dexamethasone treatment. There was no response to glucocorticoid by U251MG cells. Dexamethasone treatment of neuroepithelial tumor cells expressing glucocorticoid receptors causes translocation into the nucleus to modulate cell proliferation upon binding of different concentrations of dexamethasone in vitro. Dexamethasone inhibits proliferation of some neuroepithelial cell lines, not by glucocorticoid-induced apoptosis. The bimodal potential of glucocorticoid to stimulate or suppress proliferation of neuroepithelial tumor cells expressing glucocorticoid receptor must be considered in clinical trials.