Assessment of Apoptosis Regulating Factors BCL-2 and Fas Antigens on Malignant and Normal Plasma Cells

Multiple myeloma (MM) is characterised by slow proliferation of malignant plasma cells and their accumulation within the bone marrow. The dysregulation of programmed cell death (apoptosis) is a very important mechanism in the pathogenesis of this tumour. It prompted us to investigate the apoptosis regulating factors such as the pro-apoptotic Fas antigen and the anti-apoptotic protein BCL-2 on bone marrow malignant plasma cells in untreated patients with newly diagnosed MM and to compare them with their normal counterparts—plasma cells isolated from bone marrow of healthy individuals. Twenty-nine MM patients and 16 healthy persons were studied. Bone marrow mononuclear cells were isolated, indicated by monoclonal antibodies and analysed using the flow cytometry method. There was no statistically significant difference in BCL-2 expression in plasma cells between patients and control groups. However the percentage of BCL-2 positive cells was significantly related to the clinical stage of the disease. We detected statistically significant lower percentage of Fas positive cells in the patient group than in control.

We concluded that in MM at diagnosis the expression of BCL-2 in bone marrow malignant plasma cells was comparable to normal plasma cells but expression of Fas antigen on these cells was lower. It suggests that down regulation of Fas and normal regulation of BCL-2 may be implicated for myeloma cell survival and their escape from apoptosis in vivo.     

Evidence for the existence of circulating monoclonal B-lymphocytes in multiple myeloma patients

Multiple myeloma is characterized by the proliferation of a single clone of plasma cells producing a homogeneous immunoglobulin fraction. In this disease, plasma cells home essentially in the bone marrow. However, controversy exists whether peripheral blood B-lymphocytes in patients with multiple myeloma (MM) are part of the malignant clone. We investigated clonal immunoglobulin gene rearrangement (IgGR) in T-cell-depleted peripheral blood mononuclear cells as well as in bone marrow of these patients. Seven out of 17 MM patients demonstrated an identical IgGR in bone marrow and peripheral mononuclear cells, these patients were in an active stage of the disease. In nine patients in plateau phase, clonal IgGR could not be detected in peripheral blood. Peripheral mononuclear cells from ten patients with monoclonal gammopathies of undetermined significance (MGUS) were also examined and no IgGR was detected. The existence of monoclonal B-lymphocytes in the circulation of patients with MM suggests a mechanism whereby the malignant clone homes in the bone marrow through peripheral blood. These findings may also be used for the evaluation of patients with active myeloma and the determination of plateau phase.     

The relationship between proliferation and apoptosis in patients with monoclonal gammopathy of undetermined significance or multiple myeloma

Multiple myeloma (MM) is a clonal neoplastic lymphoproliferative disease affecting terminally differentiated B cells i.e. plasma cells characterized by slow proliferation activity and different resistance to apoptosis with latent accumulation of myeloma cells in the bone marrow. This process is induced by failure of normal tissue homeostatic mechanisms. We compared plasma cell proliferation and apoptic indices in various phases of MM and in monoclonal gammophaty of untetermined significance.     

Macrophages and mesenchymal stromal cells support survival and proliferation of multiple myeloma cells

Multiple myeloma (MM) is characterized by almost exclusive tropism of malignant cells for the bone marrow (BM) milieu. The survival and proliferation of malignant plasma cells have been shown to rely on interactions with nonmalignant stromal cells, in particular mesenchymal stromal cells (MSCs), in the BM microenvironment. However, the BM microenvironment is composed of a diverse array of cell types. This study examined the role of macrophages, an abundant component of BM stroma, as a potential niche component that supports malignant plasma cells. We investigated the proliferation of MM tumour cell lines when cultured alone or together with MSCs, macrophages, or a combination of MSCs and macrophages, using the carboxyfluorescein succinimidyl ester assay. Consistently, we observed increased proliferation of MM cell lines in the presence of either MSCs or macrophages compared to cell line-only control. Furthermore, the combined co-culture of MSCs plus macrophages induced the greatest degree of proliferation of myeloma cells. In addition to increased proliferation, MSCs and macrophages decreased the rate of apoptosis of myeloma cells. Our in vitro studies provide evidence that highlights the role of macrophages as a key component of the BM microenvironment facilitating the growth of malignant plasma cells in MM.     

Overexpression and involvement in migration by the metastasis-associated phosphatase PRL-3 in human myeloma cells

Multiple myeloma (MM) is characterized by accumulation and dissemination of malignant plasma cells (PCs) in the bone marrow (BM). Gene expression profiling of 2 MM cell lines (OH-2 and IH-1) indicated that expression of PRL-3, a metastasis-associated tyrosine phosphatase, was induced by several mitogenic cytokines. Cytokine-driven PRL-3 expression could be shown in several myeloma cell lines at both the mRNA and protein levels. There was significantly higher expression of the PRL-3 gene in PCs from patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering myeloma (SMM), and myeloma than in PCs from healthy persons. Among 7 MM subgroups identified by unsupervised hierarchical cluster analysis, PRL-3 gene expression was significantly higher in the 3 groups denoted as "proliferation," "low bone disease," and "MMSET/FGFR3." PRL-3 protein was detected in 18 of 20 BM biopsies from patients with MM. Silencing of the PRL-3 gene by siRNA reduced cell migration in the MM cell line INA-6, but had no detectable effect on proliferation and cell-cycle phase distribution of the cells. In conclusion, PRL-3 is a gene product specifically expressed in malignant plasma cells and may have a role in migration of these cells.     

Zoledronic acid exerts its antitumor effect in multiple myeloma interfering with the bone marrow microenvironment

Multiple myeloma (MM) is a B-cell malignancy characterized by an excess of monotypic plasma cells which localize almost exclusively in the bone marrow provoking bone destruction via the activation of the osteoclasts. The bone marrow microenvironment, mainly through stromal cells, is strictly involved in the evolution of the disease supporting MM cell growth and survival [1]. MM plasma cells reside in the bone marrow by binding to adhesion molecule of extracellular matrix (ECM) and stromal cells. The activation of some signaling pathways within the stromal cells increases the production of several cytokines which in turn favors the myeloma cell proliferation and survival [2-6], and enhance the drug resistance by anti-apoptotic mechanisms [1,7-9]. Novel therapeutic agents target not only the myeloma cells but also the interaction between MM cells and the bone marrow microenvironment [8]. Bisphosphonates (Bps) interfere as well with bone microenvironment inhibiting the survival of stromal cells and hampering the contact between plasma and stromal cells.In this review we will revise preclinical evidences, and the potential mechanisms of the antitumor activity of zoledronic acid.     

Zoledronic acid exerts its antitumor effect in multiple myeloma interfering with the bone marrow microenvironment

Multiple myeloma (MM) is a B-cell malignancy characterized by an excess of monotypic plasma cells which localize almost exclusively in the bone marrow provoking bone destruction via the activation of the osteoclasts. The bone marrow microenvironment, mainly through stromal cells, is strictly involved in the evolution of the disease supporting MM cell growth and survival [1]. MM plasma cells reside in the bone marrow by binding to adhesion molecule of extracellular matrix (ECM) and stromal cells. The activation of some signaling pathways within the stromal cells increases the production of several cytokines which in turn favors the myeloma cell proliferation and survival [2–6], and enhance the drug resistance by anti-apoptotic mechanisms [1,7–9]. Novel therapeutic agents target not only the myeloma cells but also the interaction between MM cells and the bone marrow microenvironment [8]. Bisphosphonates (Bps) interfere as well with bone microenvironment inhibiting the survival of stromal cells and hampering the contact between plasma and stromal cells. In this review we will revise preclinical evidences, and the potential mechanisms of the antitumor activity of zoledronic acid.     

CD28-mediated regulation of multiple myeloma cell proliferation and survival

Although interactions with bone marrow stromal cells are essential for multiple myeloma (MM) cell survival, the specific molecular and cellular elements involved are largely unknown, due in large part to the complexity of the bone marrow microenvironment itself. The T-cell costimulatory receptor CD28 is also expressed on normal and malignant plasma cells, and CD28 expression in MM correlates significantly with poor prognosis and disease progression. In contrast to T cells, activation and function of CD28 in myeloma cells is largely undefined. We have found that direct activation of myeloma cell CD28 by anti-CD28 mAb alone induces activation of PI3K and NFkappaB, suppresses MM cell proliferation, and protects against serum starvation and dexamethasone (dex)-induced cell death. Coculture with dendritic cells (DCs) expressing the CD28 ligands CD80 and CD86 also elicits CD28-mediated effects on MM survival and proliferation, and DCs appear to preferentially localize within myeloma infiltrates in primary patient samples. Our findings suggest a previously undescribed myeloma/DC cell-cell interaction involving CD28 that may play an important role in myeloma cell survival within the bone marrow stroma. These data also point to CD28 as a potential therapeutic target in the treatment of MM.     

Detection and quantitation of malignant cells in the peripheral blood of multiple myeloma patients.

One of the distinguishing features of multiple myeloma (MM) is the proliferation of plasma cells that home to the bone marrow (BM). However, there still remains some uncertainty concerning the presence of related malignant cells in the peripheral blood of myeloma patients. Using consensus oligonucleotide primers, we amplified the third complementary determining region (CDR3) of rearranged immunoglobulin heavy chain alleles from MM marrow samples by polymerase chain reaction (PCR). From the sequences of the products, we derived allele-specific oligonucleotides (ASO), and these were used in subsequent amplification reactions to detect malignant clones in the peripheral blood of myeloma patients. This method is highly specific and sensitive to 1 malignant cell in the background of 10(5) normal cells. Using this method we detected circulating malignant cells in 13 of 14 previously untreated MM patients. Furthermore, by applying ASO-PCR to artificial titrations of initial BM DNA sample into normal peripheral blood lymphocyte (PBL) DNA we were able to generate standard curves and quantitate the amount of tumor in the patient PBL. We observed a wide variation in the amount of circulating tumor between patients. In addition, we found that the incidence of circulating tumor cells was independent of BM tumor burden and stage of disease. The detection and quantitation of circulating tumor cells in the PBL of MM patients may offer an alternative assessment of the disease and may be an important consideration in the use of peripheral stem cells in bone marrow transplantation.     

Towards a new age in the treatment of multiple myeloma

Multiple myeloma (MM) is an incurable disease characterized by the proliferation of end-stage B lymphocytes (plasma cells, PCs). As a consequence of myeloma growth in the bone marrow, a number of signaling pathways are activated that trigger malignant PC proliferation, escape from apoptosis, migration, and invasion. Thanks to new insights into the molecular pathogenesis of MM, novel approaches aimed at targeting these abnormally activated cascades have recently been developed and others are under study. These strategies include the inhibition of membrane receptor tyrosine kinases, inhibition of the proteasome/aggresome machinery, inhibition of histone deacetylases, inhibition of farnesyltransferases, targeting of molecular chaperones, and others. We will herein review and discuss these novel biological approaches with particular emphasis on those based on biochemical pathways which drive cell signaling. By providing the rationale for innovative therapeutic strategies, the above mechanisms represent targets for new compounds being tested in the management of this disease.     

Presentation of multiple myeloma occurring in the humerus after strain: a case report

Clinical Rheumatology - Multiple myeloma (MM) is a malignant tumor originating from plasma cells that synthesize immunoglobulin in an abnormal amount and invade the bone marrow. The presenting...     

B‐Lymphocyte stimulator: a new biomarker for multiple myeloma

Multiple myeloma (MM) is a common malignant tumor, characterized by unlimited proliferation of abnormal plasmocytes in bone marrow. Considering the biological function of B‐Lymphocyte stimulator (BLyS) and its receptors in B cell, we examined BLyS and its receptors expression in MM cells. Our studies confirmed that BLyS and its receptors are expressed in MM cells, including KM3, CZ‐1, and primary MM cells, playing an important role in the survival and proliferation of MM cells. Additionally, we provide evidence that BLyS protein is located in the MM cell plasma membrane. We also found that IFN‐γ and IL‐6 can induce BLyS expression on MM cells, while after the treatment of BAY11‐7082, an IkB‐α phosphorylation inhibitor, IFN‐γ induced up regulation of BLyS was completely inhibited, suggesting that nuclear factor κB (NF‐κB) might be involved in the mechanism of the regulation of BLyS levels in response to cytokines. Finally, linear correlation analysis of the Lactate Dehydrogenase concentration and beta 2‐microglobulin level with BLyS, and expressions of BLyS mRNA in MM patients revealed a significant correlation between them (P < 0.01 in all case), showing that BLyS could be a biomarker for the diagnosis and treatment of MM.     

Induction of serum stimulation and plasma cell proliferation during chemotherapy of multiple myeloma

Sequential sera from 45 patients with multiple myeloma (MM) and from 6 patients with solid tumors but normal bone marrows who received cyclophosphamide, 15 mg/kg/day for 4 days, were assayed for their effects on tritiated thymidine (3H-TdR) incorporation by normal bone marrow cells and malignant plasma cells. Pretreatment sera from 23 of the 45 patients with MM inhibited normal marrow cell proliferation relative to the effects of normal sera. Of these 45 sera, 30 inhibited plasma cell proliferation. This humoral inhibition was overcome by the induction of humoral stimulation at a predictable time during chemotherapy. The sera obtained sequentially from patients with MM and patients with normal bone marrows increased 3H-TdR uptake by both cell types by days 12-15 of therapy. Sequential changes in malignant marrow plasma cell 3H-TdR labeling indices paralleled the changes in serum activity, with an increased tumor cell growth fraction occurring at the time of peak serum stimulatory activity. The relationship between serum stimulation and malignant plasma cell proliferation was confirmed in vitro.     

Biological aspects of angiogenesis in multiple myeloma

Multiple myeloma (MM) is a hematological malignancy characterized by the aberrant expansion of malignant plasma cells within the bone marrow (BM). One of the hallmarks of this disease is the close interaction between myeloma cells and neighboring cells within the BM. Angiogenesis, through the activation of endothelial cells, plays an essential role in MM biology. In the current review, we describe the angiogenesis process in MM by identifying the interacting cells, the pro- and anti-angiogenic cytokines modulated, and the extracellular matrix degrading proteases liable to participate in the pathophysiology. Finally, we highlight the impact of hypoxia (through hypoxia-inducible factor-1) and constitutive activation of nuclear factor-κB in this tumor-induced neo-vascularization.     

Bisphosphonates in Multiple Myeloma: Preclinical and Clinical Data

Multiple myeloma (MM) is a plasma cell malignancy characterized by the high capacity to induce bone destruction. Osteolysis is the hallmark of bone lesions in MM patients due to a severe uncoupled and unbalanced bone remodeling with an increase in osteoclast formation and activity, and the suppression of osteoblast formation and function. Bisphosphonates (BPs) are potent osteoclast inhibitors that are approved and widely used for the treatment for MM bone disease and the prevention of skeletal-related events in MM patients. In the last years, several preclinical data suggest that nitrogen-containing BPs such as pamidronate and zoledronic acid show a significant anti-MM effects either directly on MM cell proliferation and survival or indirectly targeting the bone marrow microenvironment cells such as osteoclasts, endothelial cells, and δγ T lymphocytes. More recently, a potential antitumoral effect has been also demonstrated in clinical trials in MM patients showing that the long-term treatment with zoledronic acid may have an impact on the overall survival. This review summarizes both preclinical and clinical evidence supporting the use of BPs in MM patients.     

Insulin-like growth factor I induces migration and invasion of human multiple myeloma cells

Multiple myeloma (MM) is an incurable form of cancer characterized by accumulation of malignant plasma cells in the bone marrow. During the course of this disease, tumor cells cross endothelial barriers and home to the bone marrow. In latter stages, myeloma cells extravasate through blood vessels and may seed a variety of organs. Insulin-like growth factor I (IGF-I) is one of several growth factors shown to promote the growth of MM cells. In the current study, we have assessed the ability of IGF-I to serve additionally as a chemotactic factor affecting the mobility and invasive properties of these cells. Results indicate that IGF-I promotes transmigration through vascular endothelial cells and bone marrow stromal cell lines. Analysis of endogenous signaling pathways revealed that protein kinase D/protein kinase Cmicro (PKD/PKCmicro) and RhoA were both activated in a phosphatidylinositol 3-kinase (PI-3K)-dependent manner. Inhibition of PI-3K, PKCs, or Rho-associated kinase by pharmacologic inhibitors abrogated migration, whereas mitogen-activated protein kinase (MAPK), Akt, and p70S6 kinase inhibitors had no effect. These results suggest that IGF-I promotes myeloma cell migration by activation of PI-3K/PKCmicro and PI-3K/RhoA pathways independent of Akt. The identification of IGF-I as both a proliferative and migratory factor provides a rational basis for the development of targeted therapeutic strategies directed at IGF-I in the treatment of MM.     

Multiple myeloma cells catalyze hepatocyte growth factor (HGF) activation by secreting the serine protease HGF-activator

Multiple myeloma (MM) is a common hematologic neoplasm consisting of malignant plasma cells, which expand in the bone marrow. A potential key signal in the evolution of MM is hepatocyte growth factor (HGF), which acts as a potent paracrine and/or autocrine growth factor and survival factor for MM cells. Proteolytic conversion of HGF into its active form is a critical limiting step in HGF/MET signaling. Here, we show that malignant MM plasma cells convert HGF into its active form and secrete HGF-activator (HGFA), a serine protease specific for HGF activation. By using serine protease inhibitors and neutralizing antibodies, we demonstrate that HGFA produced by the MM cells is responsible for their ability to catalyze HGF activation. We, therefore, suggest that autocatalyzation of HGF conversion by MM cells is an important step in HGF/MET-induced myeloma growth and survival, which may have implications for the management of this incurable form of cancer.     

Endothelial progenitor cells in multiple myeloma neovascularization: a brick to the wall

Multiple myeloma (MM) is characterized by the clonal expansion of plasma cells in the bone marrow that leads to events such as bone destruction, anaemia and renal failure. Despite the several therapeutic options available, there is still no effective cure, and the standard survival is up to 4 years. The evolution from the asymptomatic stage of monoclonal gammopathy of undetermined significance to MM and the progression of the disease itself are related to cellular and molecular alterations in the bone marrow microenvironment, including the development of the vasculature. Post-natal vasculogenesis is characterized by the recruitment to the tumour vasculature of bone marrow progenitors, known as endothelial progenitor cells (EPCs), which incorporate newly forming blood vessels and differentiate into endothelial cells. Several processes related to EPCs, such as recruitment, mobilization, adhesion and differentiation, are tightly controlled by cells and molecules in the bone marrow microenvironment. In this review, the bone marrow microenvironment and the mechanisms associated to the development of the neovasculature promoted by EPCs are discussed in detail in both a non-pathological scenario and in MM. The latest developments in therapy targeting the vasculature and EPCs in MM are also highlighted. The identification and characterization of the pathways relevant to the complex setting of MM are of utter importance to identify not only biomarkers for an early diagnosis and disease progression monitoring, but also to reveal intervention targets for more effective therapy directed to cancer cells and the endothelial mediators relevant to neovasculature development.