Inhibitory effect of all-trans retinoic acid on the growth of freshly isolated myeloma cells via interference with interleukin-6 signal transduction [see comments]

We showed the dose-dependent growth inhibition by alltrans retinoic acid (ATRA) of myeloma cells freshly isolated from patients. ATRA downregulated the cell surface expression of interleukin-6 receptor (IL- 6R) and/or glycoprotein (gp) 130. The growth-inhibitory activity of ATRA was well correlated with that of anti-gp 130 antibody in every sample. Furthermore, ATRA inhibited the production of IL-6 from both myeloma cells and marrow stromal cells, and recombinant IL-6 (rIL-6) could partially recover the myeloma cell growth that had been inhibited by ATRA. These data suggest that ATRA may inhibit the proliferation of myeloma cells both by the downregulation of IL-6R and gp130 expression on myeloma cells and by the inhibition of IL-6 production from myeloma and stromal cells. Prednisolone (PSL) and interferon-gamma (IFN-gamma) also inhibited the myeloma growth, while their effects were different from those of ATRA on IL-6 R and gp130 expression, IL-6 production, and morphological change. The inhibitory effect of ATRA on myeloma cell proliferation was observed in 10 of 14 samples obtained from eight patients, which suggests that ATRA may be a potent new therapeutic agent for some myeloma patients.     

Interleukin-21 is a growth and survival factor for human myeloma cells

Interleukin-21 (IL-21) is a recently cloned cytokine with homology to IL-2, IL-4, and IL-15. In this study we examined the effects of IL-21 on human myeloma cells. We found that IL-21 induced proliferation and inhibited apoptosis of the IL-6-dependent human myeloma cell lines ANBL-6, IH-1, and OH-2. The potency of IL-21 was close to that of IL-6 in the OH-2 cell line. Neutralizing antibodies to IL-6 or the IL-6 receptor transducer chain (gp130) did not affect IL-21-induced DNA synthesis, indicating that IL-21-induced proliferation was not mediated through these proteins. Tumor necrosis factor (TNF), another stimulator of myeloma cell growth, up-regulated the expression level of IL-21 receptor (IL-21R), and combinations of TNF and IL-21 gave synergistic effects on myeloma cell proliferation. Furthermore, 4 of 9 purified samples of primary myeloma cells showed a significant increase in DNA synthesis on stimulation of the cells by IL-21. By Western blot analysis, we demonstrated that the intracellular signaling pathways of IL-21 in myeloma cells involved phosphorylation of Jak1, Stat3, and Erk1/2 (p44/42 mitogen-activated protein kinase). IL-21 is a novel growth and survival factor in multiple myeloma and may represent a target for future therapy.     

Involvement of Notch-1 signaling in bone marrow stroma-mediated de novo drug resistance of myeloma and other malignant lymphoid cell lines

The bone marrow (BM) microenvironment plays a critical role in malignant cell growth, patient survival, and response to chemotherapy in hematologic malignancies. However, mechanisms associated with this environmental influence remain unclear. In this study, we investigated the role of Notch family proteins in myeloma and other malignant lymphoid cell line growth and response to chemotherapeutic drugs. All 8 tested cell lines expressed Notch-3 and Notch-4; 7 cell lines expressed Notch-1; and 6 expressed Notch-2 proteins. Interaction with BM stroma (BMS) activated Notch signaling in tumor cells. However, activation of only Notch-1, but not Notch-2, resulted in protection of tumor cells from melphalan- and mitoxantrone-induced apoptosis. This protection was associated with up-regulation of p21(WAF/Cip) and growth inhibition of cells. Overexpression of Notch-1 in Notch-1(-) U266 myeloma cells up-regulated p21 and resulted in protection from drug-induced apoptosis. Thus, this is a first report demonstrating that Notch-1 signaling may be a primary mechanism mediating the BMS influence on hematologic malignant cell growth and survival.     

Histone deacetylase inhibitors increase p21(WAF1) and induce apoptosis of human myeloma cell lines independent of decreased IL-6 receptor expression.

Histone deacetylase (HDAC) inhibitors cause growth arrest and apoptosis of cancer cells by both p21-dependent and independent mechanisms. Decreased expression of growth factor receptors may be a key factor in the p21-independent mechanism, although this has not been directly tested. We have tested the effects of sodium butyrate and trichostatin A on human myeloma cell lines and have observed G1 arrest and apoptosis associated with increased expression of p21(WAF1), Bax, Rb dephosphorylation, and decreased IL-6 receptor (IL-6R) expression. Experiments to determine the role of disruption of IL-6 signaling as a result of decreased IL-6 receptor expression in mediating these effects were conducted using a stable transfectant of the OPM-2 line which constitutively expressed the IL-6 receptor. Our results indicated that decreased IL-6R expression was not required for induction of p21(WAF1) or apoptosis. Thus, HDAC inhibitors appear to activate multiple cellular pathways, leading to growth arrest and apoptosis, and their use in the treatment of myeloma, particularly in combination with other agents, warrants further investigation.     

Interleukin-10 is a proliferation factor but not a differentiation factor for human myeloma cells

Because interleukin-10 (IL-10) is a potent differentiation factor of human B cells into mature plasma cells, we investigated its effect on human malignant plasma cells. IL-10 did not induce any differentiation and increase in Ig synthesis in four human IL-6-dependent malignant plasma cell lines. However, it stimulated the proliferation of two of four cytokine-dependent cell lines in the absence of IL-6 and IL-10- dependent myeloma cell lines have been obtained. The myeloma cell growth activity of IL-10 was unaffected by anti-IL-6 and anti-IL-6R antibodies. Similarly, IL-10 stimulated (P = .001) the proliferation of freshly-explanted myeloma cells in IL-6-deprived cultures of tumor samples from patients with active multiple myeloma (MM) and produced twice as many myeloma cells in these cultures. Again, this cytokine was unable to induce further differentiation (assessed by rate of Ig production) of fresh myeloma cells. A very sensitive enzyme-linked immunosorbent assay (ELISA; 1 pg/mL) only rarely detected IL-10 in the sera of MM patients (3 of 89). On the contrary, serum IL-10 was detected in 60% of patients with plasma cell leukemia (12 of 20). These data show that IL-10 is an IL-6-unrelated growth factor for malignant plasmablastic cells. This cytokine could be involved in the late phase of MM in vivo.     

Cyclin D1 and p16INK4A are preferentially expressed in immature and mature myeloma cells, respectively

Myeloma cells consist of immature, intermediate and mature cells with respect to expression of VLA-5 (CD49e) and MPC-1 adhesion molecules. VLA-5⁻MPC-1⁻ immature myeloma cells respond to interleukin 6 (IL-6) to proliferate in vitro , but VLA-5⁺MPC-1⁺mature myeloma cells have almost no proliferative activity with higher secretory activity of M-protein in vitro . In order to further clarify the biological differences between these immature and mature myeloma cells, we examined survival of these cells with or without IL-6 in vitro , and investigated the underlying mechanism of the proliferative or non-proliferative character of these cells by examining expression of cell cycle regulators such as cyclin D1 and inhibitors for cyclin-dependent kinase (Cdk), p16^INK4A, p21^CIP1 and p27^KIP1 by RT-PCR and immunohistochemistry. In vitro survival of these myeloma cells was examined by flow cytometric quantification of fluorescein diacetate (FDA) and propidium iodide (PI) staining. Immature myeloma cells rapidly entered apoptosis without IL-6, but mature myeloma cells could survive without IL-6 as well as normal mature plasma cells. Immature myeloma cells as well as myeloma cell lines expressed cyclin D1 mRNA and protein, but not any Cdk inhibitors. On the other hand, mature myeloma cells did not express cyclin D1 but expressed p16, not p21 or p27, as well as normal mature plasma cells. Therefore these results show that immature myeloma cells constitutively express cyclin D1 and can proliferate, and mature myeloma cells as well as normal mature plasma cells preferentially express p16 and can survive for a long time without proliferation.     

In vitro growth pattern of myeloma cells in liquid suspension or semi-solid culture containing interleukin-6

We examined in vitro growth pattern of myeloma cells from 21 patients with multiple myeloma either in liquid suspension or methylcellulose semi-solid culture, both in the presence of interleukin-6 (IL-6). The survival or growth of myeloma cells in liquid culture was classified into four categories. Type 1: myeloma cells survived for only 2-3 weeks in eight patients; Type 2: survival for 1-2 months in seven patients; Type 3: survival for more than 3 months without an obvious increase in cell number in three patients; and Type 4: continuous proliferation (cell line) in three patients. As the clinical stage advanced, the survival of myeloma cells became longer. All three myeloma cell lines were obtained from patients in an advanced clinical stage. Human plasma added to the liquid culture induced the survival or growth of myeloma cells better than fetal calf serum in any survival or growth type. Myeloma colonies or clusters were generated in seven of 21 patients, though myeloma colony formation was restricted to Type 4 patients. IL-6 neither prolonged the survival of myeloma cells nor promoted their growth in vitro except for Type 4 cells. Moreover, IL-6 did not increase the success rate of generating myeloma colonies in vitro. However, IL-6 did elevate the number of myeloma clusters in Types 1, 2 and 3 patients examined. These results suggest that IL-6 has a minor proliferative effect on myeloma cells in semi-solid culture but not in liquid suspension culture except for cells from patients with aggressive myeloma.     

Hypercalcaemia and increased serum interleukin-6 levels induced by all-trans retinoic acid in patients with multiple myeloma

Summary. All-trans retinoic acid (ATRA) inhibits human myeloma cell growth in vitro, presumably through the down-regulation of interleukin 6 receptors (IL-6R). Based on these and other studies, we initiated a phase II clinical trial using ATRA in patients with advanced refractory multiple myeloma (MM). We report that three out of six treated patients developed severe hypercalcaemia following administration of ATRA, which was accompanied by a significant rise in serum IL-6 levels. Normal calcium levels were restored after the discontinuation of the drug and the administration of standard anti-hypercalcaemic care. We suspect that down-regulation of IL-6R resulted in increased serum IL-6 levels, leading to advanced bone resorption and hypercalcaemia. We conclude that the use of ATRA in patients with advanced MM is not warranted.     

Heterogeneity in therapeutic response of genetically altered myeloma cell lines to interleukin 6, dexamethasone, doxorubicin, and melphalan

Because there is no known genetic abnormality common to all patients with myeloma, it is important to understand how genetic heterogeneity may lead to differences in signal transduction, cell cycle, and response to therapy. Model cell lines have been used to study the effect that mutations in p53 and ras can have on growth properties and responses of myeloma cells. The U266 cell line has a single mutant p53 allele. Stable expression of wild-type (wt) p53 in U266 cells results in a significant suppression of interleukin (IL)-6 gene expression and in the concomitant suppression of cell growth that could be restored by the addition of exogenous IL-6. Expression of wt p53 also leads to cell cycle arrest and protection from doxorubicin (Dox)- and melphalan (Mel)-induced apoptosis. The addition of IL-6 resulted in cell cycle progression and blocked p53-mediated protection from apoptosis. ANBL6 is an IL-6-dependent cell line that is sensitive to dexamethasone (Dex), Dox, and Mel. IL-6 is able to protect ANBL6 cells from Dex- and Mel- but not Dox-induced apoptosis. To study the effect of an activating mutation in ras, the ANBL6 cell line transfected with either a constitutively activated N- or K-ras gene was used. Both N-ras12 and K-ras12 genes were able to protect ANBL6 cells from apoptosis induced by Dex, Dox, and Mel. These data show that changes in ras or p53 can alter the myeloma cell response to IL-6 and demonstrate that the genetic background can alter therapeutic responses.     

Transcriptional upregulation of p21/WAF/Cip1 in myeloid leukemic blasts expressing AML1-ETO

An inducible model for conditional expression of AML1-ETO in myeloid U-937 cells was generated previously to determine cellular effects of AML1-ETO and to identify target genes. Induction of AML1-ETO expression in U-937 resulted in reduced cell growth, G1 arrest and apoptosis. Microarray analysis showed more genes up-regulated than down-regulated (180 vs. 69). Clustering of AML1-ETO-positive and -negative cell lines was possible based on these differentially expressed genes. p21/WAF/Cip1 (CDKN1A) was up-regulated 4.6-fold upon induction of AML1-ETO which was confirmed in additional experiments. Knock-down of AML1-ETO by siRNA could reduce p21/WAF/Cip1 expression in Kasumi-1 cells. mRNA expression analysis of p21/WAF/Cip1 in a large cohort of acute myeloid leukemia patients demonstrated a significantly higher expression in AML1-ETO-positive leukemia. The increased expression of p21/WAF/Cip1 in primary leukemic blasts suggests that elevated p21/WAF/Cip1 levels may contribute to specific features observed in AML1-ETO positive leukemia.     

Interleukin-6 sensitizes multiple myeloma cell lines for apoptosis induced by interferon-alpha

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-alpha, 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-alpha 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-alpha. Addition of a PI-3-K inhibitor interfered with the enhancing effect of IL-6 on the apoptosis induced by IFN-alpha. 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.     

Role of interleukin-6 in the proliferation of human multiple myeloma cell lines OCI-My 1 to 7 established from patients with advanced stage of the disease

Interleukin-6 (IL-6) has been shown to stimulate the proliferation of multiple myeloma cells purified to a high degree from human bone marrow. IL-6 production in multiple myeloma has been attributed to cells belonging to the myeloma clone, thus supporting a mechanism of autostimulation. In addition, it has been shown that IL-6 may be produced by auxiliary cell populations of the bone marrow that are not part of the myeloma clone. A definitive separation of both putative sources for IL-6 may be difficult to achieve in fresh patient IL-6 growth requirement and production by pure myeloma cell populations using seven human myeloma cell lines (OCI-My 1 to 7) that were established from patients with advanced disease. The proliferative response of each line to recombinant IL-6 was measured in a clonogenic assay providing human plasma and methylcellulose as a viscous support and by 3H-thymidine uptake in liquid suspension culture. We observed marked heterogeneity, ranging from IL-6-dependent colony formation by OCI-My 4, to IL-6-independent growth. All lines expressed mRNA for the IL-6 receptor. Expression of IL-6 mRNA was analyzed after amplification by polymerase chain reaction and was present in five of seven lines. IL-6 protein was detected by enzyme-linked immunosorbent assay (ELISA) in the culture supernatants of two lines (OCI-My 3 and 2). Its functional activity was confirmed in a bioassay using the IL-6-dependent murine hybridoma line B 13.29. This activity was neutralized by anti-IL-6 antibody. Two lines did not express mRNA for IL-6. The remaining three lines expressed mRNA for IL-6, but did not secrete IL-6 protein. Immunoprecipitation experiments with lysates of one of these three lines did not detect the presence of IL-6 protein. These results suggest that autocrine stimulation by IL-6 may occur in some cell lines derived from patients with multiple myeloma. However, it does not represent a universal mechanism in myeloma cell growth.     

The gp 130 family cytokines IL-6, LIF and OSM but not IL-11 can reverse the anti-proliferative effect of dexamethasone on human myeloma cells

Summary. In order to understand the mechanisms supporting steroid escape in patients with multiple myeloma (MM), three IL-6 autocrine human myeloma cell lines, LP1, OPM2 and L363, have been treated with dexamethasone in the presence or absence of cytokines belonging to the gp 130 family: IL-6, LIF, OSM and IL-11. With pharmacological doses of dexamethasone, a dramatic growth arrest was observed in all the cell lines. IL-6 completely reversed this inhibition. Of note, this IL-6 induced reversion was still seen with very low amounts of IL-6 (12 pg/ml). Finally, whereas LIF and OSM had clear growth-promoting effects on OPM2 only, both cytokines (but not IL-11) reversed the dexa-methasone-induced growth arrest in all the cell lines. Therefore the high levels of IL-6 (ng/ml) observed in the MM intermediate milieu and the putative presence of LIF and OSM can easily counteract the effects of dexamethasone in vivo.     

IL-12-dependent innate immunity arrests endothelial cells in G0-G1 phase by a p21(Cip1/Waf1)-mediated mechanism

Innate immunity may activate paracrine circuits able to entail vascular system in the onset and progression of several chronic degenerative diseases. In particular, interleukin (IL)-12 triggers a genetic program in lymphomononuclear cells characterized by the production of interferon-γ and specific chemokines resulting in an angiostatic activity. The aim of this study is to identify molecules involved in the regulation of cell cycle in endothelial cells co-cultured with IL-12-stimulated lymphomonuclear cells. By using a transwell mediated co-culture system we demonstrated that IL-12-stimulated lymphomonuclear cells induce an arrest of endothelial cells cycle in G1, which is mainly mediated by the up-regulation of p21^Cip1/Waf1, an inhibitor of cyclin kinases. This effect requires the activation of STAT1, PKCδ and p38 MAPK, while p53 is ineffective. In accordance, siRNA-dependent silencing of these molecules in endothelial cells inhibited the increase of p21^Cip1/Waf1 and the modification in cell cycle promoted by IL-12-stimulated lymphomonuclear cells. These results indicate that the angiostatic action of IL-12-stimulated lymphomononuclear cells may lie in the capability to arrest endothelial cells in G1 phase through a mechanisms mainly based on the specific up-regulation of p21^Cip1/Waf1 induced by the combined activity of STAT1, PKCδ and p38 MAPK.     

Increased CD11/CD18 expression and altered metabolic activity on polymorphonuclear leukocytes from patients with polycythemia vera and essential thrombocythemia

Human myeloma cell lines are difficult to establish, and they usually originate from patients with extramedullary disease. We describe a new human myeloma cell line, TU-1, which was established from the bone marrow of a patient without extramedullary myeloma. The myeloma cells were initially maintained in a conditioned medium derived from another well-known myeloma cell line U-266. This conditioned medium contained interleukin-6 (IL-6) and oncostatin M (OSM), and possibly other unknown growth factors as well. In 3 months the TU-1 cell line proliferated autonomously and secreted IL-6 and OSM with a synergistic growth response. As we have previously shown the cell line acquired a p53 mutation in vitro, which may be an important factor causing autonomous proliferation. In patients with multiple myeloma OSM is frequently found in the serum and OSM has been associated with serum IL-6 and progressive disease. Our study demonstrates the close relationship of OSM and IL-6 also in vitro.     

Paracrine rather than autocrine regulation of myeloma-cell growth and differentiation by interleukin-6

To explore the mechanisms involved in the pathogenesis of human multiple myeloma (MM), we investigated the potential role of interleukin-6 (IL-6), a B-cell differentiation factor in humans, and a growth factor for rat/mouse heterohybridomas and murine plasmacytomas. Using a heterohybridoma assay, we found that two well-documented human myeloma cell lines, RPMI 8226 and U266, did not secrete IL-6 and did not express RNA messengers for IL-6. Neutralizing antibodies to IL-6 did not inhibit their proliferation, and recombinant IL-6 did not stimulate it. Taken together, these data show that IL-6 is not the autocrine growth factor of these human myeloma cell lines. A high production of IL-6 was found in the bone marrows of patients with fulminating MM, compared with patients with inactive or slightly active MM, or to healthy donors. This IL-6 production was assigned to adherent cells of the bone-marrow environment but not to myeloma cells. A spontaneous proliferation of myeloma cells freshly isolated from patients was observed in short-term cultures. Recombinant IL-6 was able to amplify it two- to threefold. The spontaneous proliferation of the myeloma cells was inhibited by anti-IL-6 antibodies and reinduced by recombinant IL-6. After 2 to 3 weeks of culture, the myeloma-cell proliferation progressively declined and no IL-6-dependent myeloma cell lines could be obtained despite repeated additions of fresh IL-6 and costimulation with other cytokines such as tumor necrosis factor (TNF)beta, or IL-1 beta. These data demonstrated a paracrine but not autocrine regulation of the growth and differentiation of myeloma cells by IL-6.