Michio M. Kawano

Interleukin-6, CD45 and the Src-Kinases in Myeloma Cell Proliferation

Abstract Multiple myeloma (MM) is a proliferative disorder of monoclonal plasma cells which accumulate in human bone marrow, and myeloma cells proliferate in response to a cytokine, interleukin-6 (IL-6). We recently found that MPC-1−CD49e− immature myeloma cells expressing CD45 form a proliferating population in MM. IL-6 activates at least two intracellular pathways including signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase 1/2 (ERK1/2) following the activation of Janus kinases (JAKs) via its receptor complexes composed of the IL-6 receptor α chain and gp130. Although the roles of CD45 have been extensively studied for antigen receptors in B and T cells, its physiological consequences in other hematopoietic cells remain largely unknown. Myeloma cells expressing CD45 antigens which contain the activation of src family protein-tyrosine kinases (PTKs) independent of IL-6 stimulation proliferate in response to IL-6, whereas the proliferation of CD45 cells which lack a considerable activity of the src family PTKs is not promoted by IL-6. The STAT3 and ERK1/2 pathways are similarly activated by IL-6 in both cells either expressing or not expressing CD45. In this review, we argue a novel mechanism of proliferation of myeloma cells, in that the activation of both STAT3 and ERK1/2 is not sufficient for IL-6-induced proliferation which further requires IL-6-independent activation of the src family kinases associated with CD45 phosphatase. We propose that the cellular context, such as CD45 expression and src family kinase activation, is crucial for myeloma cells to proliferate in response to IL-6.

Interleukin-6—Induced Proliferation of Human Myeloma Cells Associated with CD45 Molecules

Cytokines exert multiple biological functions through binding to their specific receptors that triggers activation of intracellular signaling cascades. The cytokine-mediated signals may produce variable and even opposing effects on different cell types, depending on cellular context, which also are dictated by the differentiation stage of the cell. Multiple myeloma is a monoclonal proliferative disorder of human plasma cells. Despite their clonal origin, myeloma cells appear to include mixed subpopulations in accordance with expression of their surface antigens, such as CD45, CD49e, and MPC-1. Although interleukin-6 (IL-6) is widely accepted as the most relevant growth factor for myeloma cells in vitro and in vivo, only a few subpopulations of tumor cells, such as CD45+MPC-1-CD49e- immature cells, proliferate in response to IL-6.We recently showed that IL-6 efficiently activated both signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase 1/2 (ERK1/2) in CD45- myeloma cell lines, although CD45- cells failed to proliferate in response to IL-6. In contrast, src family protein-tyrosine kinases (PTKs), the most important substrates for CD45 protein-tyrosine phosphatase (PTP) are found activated independently of STAT3 and ERK1/2 activation in CD45+ but not in CD45- myeloma cell lines.Therefore activation of both STAT3 and ERK1/2 is not sufficient for IL-6—induced proliferation of myeloma cells, which requires the src family kinase activation associated with CD45 expression. We propose a mechanism for IL-6—induced cell proliferation that is strictly dependent on the cellular context in myelomas.

Dehydroepiandrosterone can inhibit the proliferation of myeloma cells and the interleukin-6 production of bone marrow mononuclear cells from patients with myeloma.

The serum levels of an adrenal sex hormone, dehydroepiandrosterone sulfate (DHEA-S), are significantly more decreased in human myelomas compared with the reduction brought by physiologic decline with age. In order to clarify the effect of DHEA on myeloma cells, we investigated whether DHEA and DHEA-S could inhibit interleukin-6 (IL-6) production of bone marrow mononuclear cells and the proliferation of myeloma cells from patients with myeloma. DHEA-S and DHEA suppressed IL-6 production from a bone marrow stromal cell line, KM-102, as well as in bone marrow mononuclear cells from patients with myeloma. Furthermore, DHEA inhibited in vitro growth of the U-266 cell line and primary myeloma cells from the patients, as well as the in vivo growth of U-266 cells implanted i.p. in severe combined immunodeficiency-hIL6 transgenic mice. DHEA up-regulated the expression of peroxisome proliferator-activated receptor (PPAR), PPAR beta, but not PPARgamma or PPARalpha, and the expression of IkappaBalpha gene in myeloma cells and bone marrow stromal cells, which could explain the suppressive effect of DHEA on IL-6 production through the down-regulation of NF-kappaB activity. Therefore, these data revealed that DHEA-S, as well as DHEA, had a direct effect on myeloma and bone marrow stromal cells to inhibit their proliferation and IL-6 production, respectively.

Induction of Multilineage Markers in Human Myeloma Cells and Their Down-Regulation by Interleukin 6

Human primary myeloma cells are well known to be heterogeneous with regard to morphology and surface phenotype. We confirmed the heterogeneous expression of such multilineage markers as CD33, CD7, CD56, CD4, and CD86 in primary myeloma cells from 20 patients with multiple myeloma and in 8 human myeloma cell lines. CD33 expression in the Liu01 cell line, a subclone of U266 cells, and in vitamin D3-treated ILKM3 cells, correlated with a monocytoid morphology featuring convoluted nuclei and with increased C/EBPα expression. CD56+ myeloma cells from some myeloma patients and the CD56+ (but not the CD56-) myeloma cell lines expressed neuronal cell markers, such as neuron-specific enolase and β-tubulin III. CD7 expression in Liu01 cells and forskolin-stimulated U266 cells coincided with the presence of large cytoplasmic granules, and these cells featured increased expression of perforin messenger RNA and significant natural killer cell activity. Interleukin 6 (IL-6), a growth factor for myeloma cells, down-regulated CD33, CD7, or CD56 expression in primary myeloma cells, as well as in Liu01 cells. Therefore, these data suggest that human myeloma cells are capable of inducing the expression of multilineage markers and that IL-6 can down-regulate such expression.

PPARβ-mediated growth suppression of baicalein and dexamethasone in human myeloma cells

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CD19 Expression and Growth Inhibition of Tumours in Human Multiple Myeloma

Multiple myeloma (MM) is a proliferative disorder of monoclonal plasma cells which accumulate in human bone marrow (BM). CD19 is a hallmark differentiation antigen of the B cell lineage and positively regulates antigen receptor signal transduction in mature B cells. We have previously shown that malignant plasma cells (myeloma cells) isolated from the MM patients lack the CD19 expression, while non-malignant plasma cells isolated from the healthy donors do express the CD19 antigens. It is also intriguing that there exists both CD19 m and CD19 + plasma cells in some cases in pre-myeloma states including monoclonal gammopathy of undetermined significance (MGUS). It indicates that MGUS is usually composed of phenotypically non-malignant (CD19 + ) and malignant (CD19 m ) plasma cells. Furthermore, we recently demonstrate that, expression of the CD19 gene markedly inhibits the proliferation of human myeloma cell lines in vitro, and exhibits the reduced tumorigenicity in vivo and no anchorage-independent growth in vitro of a tumorigenic myeloma cell line. This inhibitory effect might result from the CD19-mediated intracellular signals because it is not observed in cells expressing the mutant CD19, which lacks the cytoplasmic domain. In this review, we suggest that loss of CD19 in MM could contribute to the proliferative advantage of the malignant plasma cell clones in this disease. Furthermore, we propose the usefulness of the phenotypic analysis of plasma cells in human plasma cell dyscrasia as a new diagnostic tool, and the CD19 gene as a potential target for the gene therapy in MM.

The regulatory mechanism of IL-6-dependent proliferation of human myeloma cells.

Abstract Multiple myeloma (MM) is a malignant tumor of plasma cells in the bone marrow. Interleukin 6 (IL-6) is an indispensable growth factor for myeloma cells. The heterogeneity of myeloma cells are the characteristics of MM, categorized into five sub-populations, two immature cells, MPC-1<PRE>-</PRE> CD49e<PRE>-</PRE> CD45<PRE>+/-</PRE>, intermediate cells, MPC-1<PRE>+</PRE> CD49e<PRE>-</PRE> CD45<PRE>+/-</PRE>, and mature cells, MPC-1<PRE>+</PRE> CD49e<PRE>+</PRE> CD45<PRE>+</PRE>. Only MPC-1<PRE>-</PRE> CD49e<PRE>-</PRE> CD45<PRE>+</PRE> immature cells (∼2% of total myeloma cells) respond to IL-6 to proliferate. CD45 protein tyrosine phosphatase is the determinant of IL-6 dependent cell growth of myeloma cells, although well studied IL-6 signal transducing factors, such as, IL-6Ra, gp130, Jak2, STAT3, and MAPK, are activated and involved in the process. Immature CD45<PRE>-</PRE> cells converted to CD45<PRE>+</PRE> cells after IL-6 stimulation both in U266 cells and sorted myeloma cells from the bone marrow aspirates of MM patients. CD45<PRE>-</PRE> cells are relatively resistant to serum starvation compared to CD45<PRE>+</PRE> cells. Because IL-6 level in the bone marrow is low even in MM patients, the CD45<PRE>-</PRE> phenotype of myeloma cells may protect the cells from apoptosis. These findings of a tuning effect of CD45 on myeloma cell proliferation may aid the study of IL-6 dependent proliferation of myeloma cells and lead to the development of new therapies for MM patients.