Multiple Myeloma Cells In Bone Marrow Research Articles

Mutually Exclusive Cyclin-Dependent Kinase 4/Cyclin D1 and Cyclin-Dependent Kinase 6/Cyclin D2 Pairing Inactivates Retinoblastoma Protein and Promotes Cell Cycle Dysregulation in Multiple Myeloma

Multiple myeloma, the second most common hematopoietic cancer, ultimately becomes refractory to treatment when self-renewing multiple myeloma cells begin unrestrained proliferation by unknown mechanisms. Here, we show that one, but not more than one, of the three early G(1) D cyclins is elevated in each case of multiple myeloma. Cyclin D1 or D3 expression does not vary in the clinical course, but that alone is insufficient to promote cell cycle progression unless cyclin-dependent kinase 4 (cdk4) is also elevated, in the absence of cdk6, to phosphorylate the retinoblastoma protein (Rb). By contrast, cyclin D2 and cdk6 are coordinately increased, thereby overriding the inhibition by cdk inhibitors p18(INK4c) and p27(Kip1) and phosphorylating Rb in conjunction with the existing cdk4. Thus, cyclin D1 pairs exclusively with cdk4 and cdk6 pairs only with cyclin D2, although cyclin D2 can also pair with cdk4 in multiple myeloma cells. The basis for this novel and specific cdk/D cyclin pairing lies in differential transcriptional activation. In addition, cyclin D1- or cyclin D3-expressing multiple myeloma cells are uniformly distributed in the bone marrow, whereas cdk6-specific phosphorylation of Rb occurs in discrete foci of bone marrow multiple myeloma cells before proliferation early in the clinical course and is then heightened with proliferation and disease progression. Mutually exclusive cdk4/cyclin D1 and cdk6/cyclin D2 pairing, therefore, is likely to be a critical determinant for cell cycle reentry and progression and may play a pivotal role in the expansion of self-renewing multiple myeloma cells.

Aberrant Autocrine and Paracrine CD40L Signaling Promotes Primary Myeloma Cell Survival and Attenuates Bortezomib-Induced Apoptosis.

In multiple myeloma (MM), impaired apoptosis is critical for clonal expansion of malignant MM cells in the bone marrow (BM), although the precise underlying mechanism has not been defined. During physiologic plasma cell differentiation, clonal expansion of antigen-activated B cells in the germinal center requires CD40 signalling, which mediates CD40L signals from activated T cells to promote B cell activation and survival. Terminal differentiation of plasma cells from activated B cells is then accompanied by a progressive reduction of CD40 expression, suggesting that the life span of normal plasma cells may be limited by the loss of CD40L signalling. Here we show that CD40L and CD40 are both aberrantly expressed in primary BM MM cells, but not in normal BM plasma cells, their likely normal counterparts. CD40L is also copiously expressed in multiple cell types in myeloma and normal bone marrows, indicating that autocrine and paracrine CD40L signalling may prolong the survival of MM cells in the bone marrow microenvironment. Supporting this hypothesis, disruption of CD40L signalling by an antagonizing CD40L antibody markedly accelerates apoptosis of BM MM cells ex vivo. In the majority of MM cases (N=50), CD40 is expressed at either greater than 90% of BM MM cells or lower than 30% of BM MM cells in vivo, but rarely in between. However, there is no apparent correlation between the frequency of CD40 expression in BM MM cells in vivo and immunoglobulin isotype or treatment history, including bortezomib therapy. Ex vivo, the level of CD40 expression in BM MM cells is enhanced in response to bortezomib, which rapidly activates CD40 transcription. Moreover, killing of primary BM MM cells by bortezomib ex vivo is attenuated by CD40L signalling, at least in part through sustaining NF-?B activation that is required for MM cell survival. Together, our findings suggest that autocrine and paracrine CD40L signalling is critical for the survival of MM cells in the bone marrow and may contribute to the development of drug resistance.

Mutually Exclusive Cdk4-Cyclin D1 and Cdk6-Cyclin D2 Pairing Inactivates Rb and Promotes Cell Cycle Dysregulation in Multiple Myeloma.

Multiple myeloma (MM), the second most common hematopoietic cancer, ultimately becomes refractory to treatment when self-renewing MM cells begin unrestrained proliferation by unknown mechanisms. Cell cycle reentry and progression is modulated by the balance between positive cell cycle regulators [cyclins and cyclin-dependent kinases (Cdk)] on the one hand and Cdk inhibitors (CKI) on the other. Here we show that one, but not more than one, of the three early G1 D cyclins is elevated in each case of MM. However, cyclin D1 or D3 overexpression rarely leads to phosphorylation of the retinoblastoma protein Rb and G1 cell cycle progression, unless cyclin-dependent kinase 4 (Cdk4) is correspondingly elevated in the absence of Cdk6. By contrast, cyclin D2 and Cdk6 are coordinately increased, thereby overriding inhibition by Cdk inhibitors, p18INK4c and p27Kip1, and phosphorylating Rb in conjunction with Cdk4. Thus, Cdk4 pairs exclusively with cyclin D1 whereas Cdk6 pairs exclusively with cyclin D2, although Cdk4 can also pair with cyclin D2 in MM cells. The basis for the exclusive Cdk4-cyclin D1 and Cdk6-cyclin D2 pairing is coordinated transcriptional activation. Cyclin D1 or D3 expression is scattered or uniform among bone marrow MM cells and at a constant frequency in the clinical course. By contrast, phosphorylation of Rb by Cdk6-cyclin D2 occurs in discrete foci of bone marrow MM cells early in the clinical course before cell proliferation, then is heightened with proliferation and disease progression. Mutually exclusive Cdk4-cyclin D1 and Cdk4/6-cyclin D2 pairing, therefore, critically controls cell cycle re-entry and G1 progression, which underlies the expansion of self-renewing MM cells in MM progression.

Valproic Acid Inihibts Histone Deacetylases and Proliferation and Induces Cell Cycle Arrest and Apoptosis in Multiple Myeloma.

Multiple myeloma remains an incurable disease in the majority of the patients and novel treatment strategies are urgently needed. A new class of drugs, the histone deacetylase (HDAC) inhibitors take influence in epigenetic modifications and have antiproliferative effects in some malignancies. Valproic acid (VPA) is an anticonvulsant drug and was recently shown to inhibit HDACs and suppress tumor growth. The drug is currently being evaluated in clinical studies in acute myeloid leukemia. Its effects on myeloma cells are unknown. The aim of this study was to evaluate the effects of VPA on proliferation, apoptosis and HDAC inhibition in multiple myeloma cell lines as well as in sorted human bone marrow multiple myeloma cells. Myeloma cell lines, OPM-2, NCI-H929, LP-1, and freshly isolated multiple myeloma cells from bone marrow aspirates were exposed to different concentrations of VPA for 4 to 72 hours. Cell proliferation, cell cycle distribution and apoptosis were assayed in reaction to the treatment. Proliferation decreased noticeably and apoptosis was induced in a dose-dependent manner in multiple myeloma cell lines as well as in freshly sorted primary myeloma cells. After 48 hours of incubation with VPA at 1 mM, approximately 46%, 52% and 25% of OPM-2, NCI-H929 and LP-1 cell lines had undergone specific apoptosis, respectively. Freshly sorted primary bone marrow myeloma cells from patients showed also specific apoptosis. In cell cycle analysis by flow cytometry, the population of cells in the G0/G1 phase increased, whereas cells in the S phase decreased in a time and dose dependent manner. Incubation of the cell line OPM-2, for example, with 1 mM VPA for 48 hours decreased the proportion of cells in the S phase from 39 % to 6 % of the total cell count and increased cells in the G0/G1 phase from 49 % to 85 %. Acetylation of histones and expression of cyclin D1 and the cell cycle regulators p21 and p27 were studied by western blot. Histone acetylation and p21 concentrations increased after VPA treatment whereas levels of p27 remained constant. A decrease in cyclin D1 concentrations was observed. Subapoptotic doses of VPA significantly decreased the production of VEGF in OPM-2 cell line. These data show that treatment with valproic acid effectively inhibits histone deacetylase activity, leading to the accumulation of acetylated histones in multiple myeloma cells. Parallel upregulation of cell cycle inhibitors like p21WAF1 was observed, together with a reduction of cyclin D1 levels. Myeloma cell proliferation was inhibited in a time and dose dependent manner and cell cycle arrest in the G0/G1 phase was induced by VPA treatment. VPA potently induced apoptosis in all human myeloma cell lines as well as in sorted primary multiple myeloma cells in a dose and time dependent manner. These results show for the first time that VPA acts as an HDAC inhibitor in multiple myeloma cells, induces G1 cell cycle arrest, potently inhibits tumor growth and markedly induces apoptosis. In addition to its direct antitumor effect, valproic acid may exert an antiangiogenic effect by reducing VEGF production in myeloma cells. These data provide the framework for clinical studies with valproic acid in multiple myeloma.

A clinically relevant SCID-hu in vivo model of human multiple myeloma

AbstractWe developed a novel in vivo multiple myeloma (MM) model by engrafting the interleukin 6 (IL-6)-dependent human MM cell line INA-6 into severe combined immunodeficiency (SCID) mice previously given implants of a human fetal bone chip (SCID-hu mice). INA-6 cells require either exogenous human IL-6 (huIL-6) or bone marrow stromal cells (BMSCs) to proliferate in vitro. In this model, we monitored the in vivo growth of INA-6 cells stably transduced with a green fluorescent protein (GFP) gene (INA-6GFP+ cells). INA-6 MM cells engrafted in SCID-hu mice but not in SCID mice that had not been given implants of human fetal bone. The level of soluble human IL-6 receptor (shuIL-6R) in murine serum and fluorescence imaging of host animals were sensitive indicators of tumor growth. Dexamethasone as well as experimental drugs, such as Atiprimod and B-B4-DM1, were used to confirm the utility of the model for evaluation of anti-MM agents. We report that this model is highly reproducible and allows for evaluation of investigational drugs targeting IL-6-dependent MM cells in the human bone marrow (huBM) milieu. (Blood. 2005;106:713-716)

Induction of Apoptosis in Multiple Myeloma by Ligands of Peroxisome Proliferator-Activated Receptor γ (PPAR-γ).

Peroxisome proliferator-activated receptor γ (PPAR-g) is a member of a nuclear receptor superfamily, which is expressed in different tumor tissues. Activation of PPAR-γ by its ligands has been shown to reduce tumor growth, interfere with tumor cell differentiation, and induce apoptosis in a variety of human malignancies including solid tumors like colon, breast, lung, liver, prostate cancer, as well as hematological malignancies like myeloid leukemia. Recently, it has been shown that both human B-lymphocytes and B-lymphomas express PPAR-γ and induce apoptosis. 15-deoxy-delta-12,14-prostaglandin J2 (15d-PGJ2) is a natural activator of PPAR-γ. Thiazolidinediones, including troglitazone, rosiglitazone (RGZ), and pioglitazone (PGZ), comprise a group of synthetic PPAR-γ agonists that are currently in use for the treatment of type 2 diabetes mellitus, and have revealed anti-tumor activity in vitro. We investigated in five human multiple myeloma cell lines (LP-1, U-266, RPMI-8226, OPM-2 and IM-9) and sorted human bone marrow myeloma cells whether treatment with PGZ, RGZ or 15d-PGJ2 inhibited tumor cell growth. Expression of PPAR-γ protein was demonstrated by western blot analysis in these cell lines. All 5 cell lines were sensitive to the PPAR-γ agonists. MTT assays revealed growth arrest induced by the natural activator of PPAR-γ 15d-PGJ2 and a lower antiproliferative effect with PGZ and RGZ in a dose dependent manner. At a dose of 50 μM 15d-PGJ2 cell proliferation was reduced to values between 0% and 26% in all multiple myeloma cell lines tested. In most cell lines the anti-proliferative effect was already detectable at 10 μM. At a dose level of 50 μM PGZ cell proliferation was reduced in MTT assay after 48 hours of incubation to 48% in LP-1, 52% in IM-9, 56% in OPM-2, 72% in U-266 and 77% in RPMI-8226. Comparable results were obtained with RGZ. Induction of apoptosis was indicated by annexin V staining. Cell lines were incubated with 50 μM of PPAR-γ agonists, a concentration which had been proven to be effective for growth inhibition in MTT assay before. Again, 15-dPGJ2 was more effective than PGZ and RGZ. All of the 15d-PGJ2 treated cell lines revealed specific apoptosis ranging between 60% and 92%. Apoptosis induced by PGZ in U-266, RPMI-8226-S, IM-9, and OPM-2 cell lines ranged between 17% and 43%, for RGZ it ranged between 20% and 50%. Furthermore, in sorted bone marrow plasma cells from myeloma patients induction of apoptosis was detected. Bone marrow multiple myeloma cells from five different patients were tested. The specific apoptosis rate induced by 15-dPGJ2 lay between 29% and 96%. Apoptosis induced by PGZ showed interindividual differences. In the myeloma cells from four patients the rate of specific apoptosis ranged between 9% and 28%, but in one patient induction of apoptosis was observed neither with PGZ nor with RGZ. For RGZ, the rate of apoptosis induced in the myeloma cells from the other four patients ranged between 7% and 26%. The rate of specific apoptosis induced by 15D-PGJ2 was not statistically different for sorted human bone marrow myeloma cells sensitive versus refractory to conventional chemotherapy with anthracyclines and alkylating agents (p = 0.8). This is one of the first studies evaluating PPAR-γ expression and its therapeutical implications in human multiple myeloma cells. Thiazolidinediones comprise anti-myeloma activity and should be explored further for the treatment of multiple myeloma.

Novel therapies for multiple myeloma.

Novel therapies for multiple myeloma.

Beta-lapachone, a novel plant product, overcomes drug resistance in human multiple myeloma cells.

To evaluate the anti-tumor potential of beta-lapachone in multiple myeloma (MM) cell lines (U266, RPMI8226, and MM.1S); MM cell lines resistant to dexamethasone (MM.1R), melphalan (RPMI8226/LR5), doxorubicin (RPMI8226/DOX40), and mitoxantrone (RPMI8226/ MR20); and MM cells from patients (MM1-MM4). Cytotoxicity of beta-lapachone was assessed by MTT and [3H]-thymidine uptake assays. Apoptosis was analyzed using propidium iodide staining, DNA fragmentation, TUNEL assay, caspase-9 colorimetric assay, and immunoblotting for caspase-3, poly (ADP-ribose) polymerase (PARP), and caspase-8 cleavage products. Paracrine growth of MM cells was assessed by [3H]-thymidine uptake in cultures of bone marrow stromal cells (BMSCs) and MM cells. Interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) secretion in the culture supernatants was measured by specific enzyme-linked immunosorbent assays (ELISAs). beta-lapachone showed significant cytotoxicity in MM cells (IC(50): 4-8 microM). In contrast, normal peripheral blood mononuclear cells (PBMCs) and BMSCs from MM patients were relatively resistant (IC(50): 8-16 microM). IL-6 did not protect against beta-lapachone-induced apoptosis in MM.1S cells, and dexamethasone showed additive cytotoxicity. beta-lapachone also decreased binding of MM.1S cells to BMSCs; abrogated IL-6 and VEGF secretion triggered by adhesion of BMSCs to MM.1S cells; reduced proliferation of MM.1S cells adherent to BMSCs; and decreased intracellular adhesion molecule-1 (ICAM-1) expression on MM.1S cells. Furthermore, beta-lapachone induced typical PARP cleavage, increased caspase-9 proteolytic activity, and activation of caspase-3, without activation of caspase-8 in U266 cells. These studies provide a framework for clinical evaluation of beta-lapachone to improve the outcome for patients with MM.

Increased uptake and accumulation of cobalamin by multiple myeloma bone marrow cells as a possible cause of low serum cobalamin

Increased uptake and accumulation of cobalamin by multiple myeloma bone marrow cells as a possible cause of low serum cobalamin

Detection of hypodiploidy using multi-parameter flow cytometric analysis: a prognostic indicator in multiple myeloma.

We analyzed 49 patients with multiple myeloma (MM) using propidium iodide (PI) staining and flow cytometry to assess DNA content of MM cells in bone marrow. In 12 patients the presence or absence of DNA content aneuploidy could not be determined by analysis of single-parameter DNA histograms alone. Using forward light scatter measurement, resolution of ploidy was readily obtained in all but three cases. Assuming the DNA content of the low light scatter population to be diploid, we were able to classify 7 cases as hypodiploid and 39 as hyperdiploid or diploid. Hyperdiploid or diploid patients survived a median of 24 months and hypodiploid patients only 2.5 months. We conclude that multiparameter analysis of DNA content and light scatter provides easier analysis for the presence or absence of aneuploidy. Patients with DNA content hypodiploidy have a short survival.

The 5T2 mouse multiple myeloma model: characterization of 5T2 cells within the bone marrow.

The transplantable C57BL/KaLwRij mouse 5T2 multiple myeloma (MM) is a new animal model for studies on MM in man. Histological examination of the 5T2 MM cells revealed their morphological heterogeneity. In this study we investigated whether this heterogeneity reflects subpopulations of 5T2 MM cells with different biological properties. 5T2 MM bone marrow cells were separated according to their sedimentation velocity (s.v.). When intravenously injected into syngeneic recipient mice, cells with s.v. of 8 mm h-1 led to the development of detectable 5T2 MM after 6 weeks; in contrast, 18 weeks elapsed before the same result was achieved with cells of s.v. lower than 5 mm h-1. Flow cytometric analysis revealed that 5T2 MM cells had an aneuploid DNA content and that most cycling 5T2 MM cells were large, their s.v. rate exceeding 9 mm h-1. It was further demonstrated that about half of all aneuploid cells carried on their membrane the 5T2 MM idiotype. The majority of the idiotype-positive cells had s.v. rate exceeding 6.5 mm h-1 (16%-39%) or lower than 3 mm h-1 (16%-19%). The 5T2 MM was shown to contain subpopulations of cells of different size, proliferation capacity and expression of their membrane 5T2 idiotype; this, most likely reflects cells in different stages of differentiation. The mouse 5T2 MM corresponds also in this respect with MM in man.

G-banding analysis of complex aneuploidy in multiple myeloma bone marrow cells

Chromosome studies with the banding technique have been performed in a considerable number of cases of myeloproliferative diseases, but technical difficulties have so far prevented detailed studies of chromosomal abnormalities in multiple myeloma. The karyotypes of bone marrow cells from two patients with multiple myeloma have been analyzed by a trypsin-Giemsa banding technique. Evidence is given for clonal evolution which in one patient has probably occurred by cell fusion and subsequent chromosome loss. Eight different marker chromosomes are characterized. Nonrandom chromosomal participation in the translocations and the existence of specific vulnerable points on chromosomes 1, 3, and 16 are suggested.

G-Banding Analysis of Complex Aneuploidy in Multiple Myeloma Bone Marrow Cells

Abstract Chromosome studies with the banding technique have been performed in a considerable number of cases of myeloproliferative diseases, but technical difficulties have so far prevented detailed studies of chromosomal abnormalities in multiple myeloma. The karyotypes of bone marrow cells from two patients with multiple myeloma have been analyzed by a trypsin-Giemsa banding technique. Evidence is given for clonal evolution which in one patient has probably occurred by cell fusion and subsequent chromosome loss. Eight different marker chromosomes are characterized. Nonrandom chromosomal participation in the translocations and the existence of specific vulnerable points on chromosomes 1, 3, and 16 are suggested.