Effect of vincristine on bone marrow cells of patients with multiple myeloma. A cytokinetic study

The cytokinetic changes induced by Vincristine (VCR) on bone marrow erythroblasts, myeloid cells and neoplastic plasma cells have been studied in four patients with plasma cell malignancies using combined DNA cytofluorometry and in vitro tritiated thymidine cytoautoradiography. The changes observed 9 h after the administration of the drug were in accordance with its S-phase specificity. The magnitude of the stathmokinetic effect was in fact roughly proportional to the proliferative activity of the different cell lines, i.e., marked on the erythroblasts, less evident on the myeloid cells and still lower on the plasma cells. In this last cell population VCR has also blocked or partially impaired the DNA synthesis. Nine days after VCR, the plasma cells were recruited into the proliferative cycle while the regeneration of the hemopoietic cells was already exhausted. Repeated administrations of VCR spaced at about 9 day intervals are more and more effective on the plasma cell population, since the S place specificity of the drug against the recruited plasma cells is potentiated. On the contrary, the regeneration of the hemopoietic cells is protected by this time interval.     

Molecular cytogenetic aberrations in Tunisian patients with multiple myeloma identified by cIg-FISH in fixed bone marrow cells

Cytogenetic studies in multiple myeloma (MM) are hampered by the hypo-proliferative nature of plasma cells. In order to circumvent this problem, we have used a combination of immunolabeling of cytoplasmic Ig light chains (λ or κ) and FISH (cIg-FISH), which allowed a comprehensive detection of the most common and/or recurrent molecular cytogenetic aberrations on fixed bone marrow cells of 70 Tunisian patients. Translocations involving the chromosome 14q32 region were observed in 32 cases (45.7%), including 18 cases with a t(11;14), 8 cases with a t(4;14), and 2 cases with a t(14;16). Deletions of the 13q14 region (D13S319/RB1) were detected in 18.6%, and deletions of the 17p13 region (TP53) in 5.7% of the cases, respectively. Of all patients with a D13S319/RB1 deletion, 61.5% also carried a 14q32 translocation, whereas TP53 deletions were associated with a t(11;14) in 2 cases (50%) and a D13S319 deletion in 1 case (25%). Our results suggest that there is a correlation between the presence of 14q32 translocations and chromosome 13q14 deletions in MM patients and that cIg-FISH is more sensitive as compared to conventional karyotyping in detecting molecular cytogenetic abnormalities in this disease.     

Communication between bone marrow mesenchymal stem cells and multiple myeloma cells: Impact on disease progression

Multiple myeloma (MM) is a hematological malignancy characterized by the accumulation of immunoglobulin-secreting clonal plasma cells at the bone marrow (BM). The interaction between MM cells and the BM microenvironment, and specifically BM mesenchymal stem cells (BM-MSCs), has a key role in the pathophysiology of this disease. Multiple data support the idea that BM-MSCs not only enhance the proliferation and survival of MM cells but are also involved in the resistance of MM cells to certain drugs, aiding the progression of this hematological tumor. The relation of MM cells with the resident BM-MSCs is a two-way interaction. MM modulate the behavior of BM-MSCs altering their expression profile, proliferation rate, osteogenic potential, and expression of senescence markers. In turn, modified BM-MSCs can produce a set of cytokines that would modulate the BM microenvironment to favor disease progression. The interaction between MM cells and BM-MSCs can be mediated by the secretion of a variety of soluble factors and extracellular vesicles carrying microRNAs, long non-coding RNAs or other molecules. However, the communication between these two types of cells could also involve a direct physical interaction through adhesion molecules or tunneling nanotubes. Thus, understanding the way this communication works and developing strategies to interfere in the process, would preclude the expansion of the MM cells and might offer alternative treatments for this incurable disease.     

The role of the bone marrow microenvironment in multiple myeloma

Multiple myeloma (MM) is a malignant disease that results from an excess of monotypic plasma cells in the bone marrow (BM). This malignancy is characterised by complex karyotypic aberrancies. In 60% of all MM there are recurrent primary translocations involving the heavy chain gene locus. The MM cells strongly interact with the BM microenvironment, which is composed of endothelial cells, stromal cells, osteoclasts, osteoblasts, immune cells, fat cells and the extracellular matrix. This interaction is responsible for the specific homing in the BM, the proliferation and survival of the MM cells, the resistance of MM cells to chemotherapy, the development of osteolysis, immunodeficiency and anaemia. New therapeutic agents target both the MM, as well as the interaction MM cell - BM microenviroment.     

Results of the study of the activity of bone marrow nucleolar organizers in patients with multiple myeloma

Data obtained from karyotyping and estimation of nucleolar organizer (NO) activity in bone marrow cells from 9 patients with multiple myeloma (MM) and from 8 donors are presented. Chromosomes of the 14th and 1st pairs in patients with MM are confirmed to be more frequently involved in rearrangements. It is proved that activity of NO in myeloma cells is rather high as compared to that of erythroid and granulocyte cells, that is associated with their participation in paraprotein synthesis.     

In vitro facilitating role of polygonatum sibiricum polysaccharide in osteogenic differentiation of bone marrow mesenchymal stem cells from patients with multiple myeloma

Background

Bone marrow mesenchymal stem cells (BMMSCs) were proved to play a vital role in multiple myeloma (MM). Polygonatum sibiricum polysaccharide (PSP) was found to have anti-tumor pharmacological effects, yet its interaction with BMMSCs remained poorly understood. Therefore, we explore the effect of PSP on osteogenic differentiation of BMMSCs.

Methods

BMMSCs were isolated by density gradient centrifugation. CD90 and CD34 were detected by flow cytometry (FCM). Osteogenic marks were detected by quantitative real-time PCR (qRT-PCR) and Western blotting (WB). The vitality of cells treated with different concentrations of PSP was observed by Cell Counting Kit-8 (CCK-8). ALP staining kit was used to detect the activity of alkaline phosphatase (ALP). Alizarin red staining detected the formation of mineralized nodules. Osteoblast-associated genes were evaluated by qRT-PCR and WB. The phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) signaling pathways were tested by WB.

Results

The BMMSCs showed good growth under an inverted microscope. FCM showed that CD34 and CD45 was low-expressed, whereas CD44, CD90 and CD105 was highly expressed. Compared with the Control group, the expressions of Runx2 and ALP in cells were significantly increased. CCK-8 showed that different concentrations of PSP had no significant effect on the viability of BMMSCs. BMMSCs treated with 25 mg/l PSP were stained the most deeply by ALP. Mineralized nodules in PSP groups dramatically increased, and hit a peak under the action of 25 mg/l PSP. PSP up-regulated p-PI3K, p-AKT, and p-mTOR, but had no significant effect on PI3K, AKT, and mTOR.

Conclusion

PSP induced osteogenic differentiation of BMMSCs from MM patients.

     

Adhesion of multiple myeloma cells to the bone marrow microenvironment: implications for future therapeutic strategies

Multiple myeloma is characterized by excess plasma cells within the bone marrow in association with monoclonal antibody protein in the serum and/or urine. Tumor cells localize within the marrow via an interaction of cell-surface adhesion molecules with their respective ligands on marrow stromal cells and extracellular matrix proteins. Stimulation of myeloma cells via these cell-surface molecules, either directly or via tumor cell adhesion to stromal cells, can induce autocrine or paracrine tumor cell growth mediated by interleukin 6. It might therefore be possible to develop innovative treatment strategies either to inhibit interleukin 6 production or to interrupt interleukin 6 signal transduction in multiple myeloma.     

Frequent detection of Human Herpes Virus-8 in bone marrow of Jordanian patients of multiple myeloma

The association between Human Herpes Virus-8 (HHV-8), also called Kaposi's sarcoma associated herpesvirus (KSHV), and the pathogenesis of multiple myeloma remains controversial. Many past studies conducting on different populations have come to contradicting conclusions. In this study, we attempted to investigate the presence of HHV-8 in Jordanian multiple myeloma patients. We carried out nucleic acid amplification reactions targeting specific viral DNA sequences on 35 fresh bone marrow aspirate samples from 17 patients with multiple myeloma, 9 patients with various hematological malignancies and 9 normal subjects. HHV-8 specific sequences were detected in 7 out of 17 multiple myeloma patients (41%) using primers specific for the open reading frame region 26 (ORF26). All patients with other hematological malignancies as well as the normal subjects did not harbour the virus. These findings support the previous reports of frequent detection of HHV-8 in bone marrow of multiple myeloma patients.     

Unique biomechanical interactions between myeloma cells and bone marrow stroma cells

We observed that BMSCs (bone marrow stromal cells) from myeloma patients (myeloma BMSCs) were significantly stiffer than control BMSCs using a cytocompression device. The stiffness of myeloma BMSCs and control BMSCs was further increased upon priming by myeloma cells. Additionally, myeloma cells became stiffer when primed by myeloma BMSCs. The focal adhesion kinase activity of myeloma cells was increased when cells were on stiffer collagen gels and on myeloma BMSCs. This change in myeloma stiffness is associated with increased colony formation of myeloma cells and FAK activation when co-cultured with stiffer myeloma BMSCs or stiffer collagen. Additionally, stem cells of RPMI8226 cells became stiffer after priming by myeloma BMSCs, with concomitant increases of stem cell colony formation. These results suggest the presence of a mechanotransduction loop between myeloma cells and myeloma BMSCs to increase the stiffness of both types of cells via FAK activation. The increase of stiffness may in turn support the growth of myeloma cells and myeloma stem cells.     

Human bone marrow stroma-dependent myeloma sister cell lines MOLP-6 and MOLP-7 derived from a patient with multiple myeloma

Human bone marrow stroma (BST)-dependent myeloma sister cell lines MOLP-6 and MOLP-7 were established from the peripheral blood of a multiple myeloma (MM) patient with IgA kappa type MM (stage IIIB). The growth of the cell lines is constitutively dependent on BST cells; none of the cytokines tested nor the culture supernatant of the BST cells could support the growth. Both cell lines showed typical plasma cell morphology with abundant cytoplasm and one to four nuclei under Wright staining. The immunoprofiles of MOLP-6 and MOLP-7 correspond to that seen typically in primary MM cells: positive for cytoplasmic immunoglobulin (Ig) chains, a heavy and kappa light chains, CD9, CD28, CD40, CD44, CD45, CD56, and PCA-1; the cells were negative for surface Igs and various other B-cell, T-cell and myelomonocyte associated markers. Both cell lines also expressed adhesion molecules including HCAM (CD44), VLA-4 (CD49d/CD29), VLA-6 (CD49f/CD29), ICAM-1 (CD54), NCAM (CD56), LFA-3 (CD58) and L-selectin (CD62L). The doubling time of MOLP-6 and MOLP-7 was 48 and 168 hours, respectively. In addition to this growth characteristic, the maximum cell density of each cell line was obtained at 1.7 x 10(6) cells/ml and 9.7 x 10(5) cells/ml, respectively. The characteristics of each cell line may reflect intraclonal variation of the proliferative capacity. The MOLP-6 together with the MOLP-7 sister will be useful model systems for the investigation of the biology of myeloma.     

Soluble rank ligand produced by myeloma cells causes generalised bone loss in multiple myeloma

Patients with multiple myeloma commonly develop focal osteolytic bone disease, as well as generalised osteoporosis. The mechanisms underlying the development of osteoporosis in patients with myeloma are poorly understood. Although disruption of the RANKL/OPG pathway has been shown to underlie formation of focal osteolytic lesions, its role in the development of osteoporosis in myeloma remains unclear. Increased soluble RANKL in serum from patients with myeloma raises the possibility that this molecule plays a key role. The aim of the present study was to establish whether sRANKL produced by myeloma cells contributes directly to osteoporosis. C57BL/KaLwRij mice were injected with either 5T2MM or 5T33MM murine myeloma cells. 5T2MM-bearing mice developed osteolytic bone lesions (p<0.05) with increased osteoclast surface (p<0.01) and reduced trabecular bone volume (p<0.05). Bone volume was also reduced at sites where 5T2MM cells were not present (p<0.05). In 5T2MM-bearing mice soluble mRANKL was increased (p<0.05), whereas OPG was not altered. In contrast, 5T33MM-bearing mice had no changes in osteoclast surface or trabecular bone volume and did not develop osteolytic lesions. Soluble mRANKL was undetectable in serum from 5T33MM-bearing mice. In separate experiments, RPMI-8226 human myeloma cells were transduced with an human RANKL/eGFP construct, or eGFP alone. RPMI-8226/hRANKL/eGFP cells, but not RPMI-8226/eGFP cells, stimulated osteoclastic bone resorption (p<0.05) in vitro. Sub-cutaneous injection of NOD/SCID mice with RPMI-8226/hRANKL/eGFP or RPMI-8226/eGFP cells resulted in tumour development in all mice. RPMI-8226/hRANKL/eGFP-bearing mice exhibited increased serum soluble hRANKL (p<0.05) and a three-fold increase in osteoclast number (p<0.05) compared to RPMI-8226/eGFP-bearing mice. This was associated with reduced trabecular bone volume (27%, p<0.05), decreased trabecular number (29%, p<0.05) and increased trabecular thickness (8%, p<0.05). Our findings demonstrate that soluble RANKL produced by myeloma cells causes generalised bone loss, suggesting that targeting RANKL may prevent osteoporosis in patients with myeloma.     

Modification of the bone marrow MSC population in a xenograft model of early multiple myeloma

Multiple myeloma (MM) is a hematological malignancy characterized by clonal proliferation of abnormal plasma cells. MM dysregulates the homeostasis of the bone niche cells like osteoclasts and osteoblasts, responsible for the bone maintenance leading to bone loss and hypercalcemia, as well as the normal immune cells leading to immunodeficiency and anemia. Osteoblasts are part of the cell population differentiating from mesenchymal stem cells (MSC). MSC also gives rise to other cell types such as adipocytes and chondrocytes. It has been observed that adipocytes support MM growth by increasing its survival and chemo-resistance. As adipocytes originate from MSC, the understanding of early modifications in the MSC population during the disease progression is of paramount importance and may help for early diagnosis of MM. Herein, we have evaluated the modification of the MSC population in the bone niche in an in vivo model of MM. Our results showed that before an observable engraftment of MM in the bone niche, the proportion of MSC population is significantly decreased, while a significant increase in adipocyte related genes such as PPARγ and CEBPα expression appears, with no difference in osteogenic differentiation. These results suggest that the bone niche is switching to a “fatty” marrow which would create an adequate microenvironment for MM. This led us to screen for and identify modulated adipokines in the sera of this in vivo MM-mice model. Such changes could reflect early signs of MM and potentially be exploited as detection biomarkers of the disease.     

高表达microRNA-155的骨髓间充质干细胞对免疫调节的影响

目的探讨骨髓间充质干细胞（BMSCs）中miR-155表达水平改变后,通过诱导树突状细胞（DC）实现对免疫调节能力的影响。 方法实验分为control组、miR-155 agomir NC组、miR-155 agomir组、miR-155 antagomir NC组和miR-155 antagomir组,通过脂质体转染特异性调控BMSC中miR-155表达量后诱导DC 48 h,检测该诱导过程对DC的成熟度和迁移能力的影响;经诱导的DC与T细胞共培养72 h后检测T细胞增殖能力。多组间分析采用One-?way ANOVA进行统计学分析,两组间采用t检验进行统计学分析。 结果流式柱形直观图可见miR-155 angomir组T细胞增殖能力低于其他组。提高miR-155表达水平后,MSCs诱导的DC细胞成熟的表面标志CD40表达量由100%下降至85%（t = 33.71,P < 0.05）;CD86表达水平由100%下降至75%（t = 57.00,P < 0.05）。miR-155 agomir组的BMSCs诱导的DC的迁移能力较其对照组减弱（t = 7.35,P < 0.05）。提高BMSCs中miR-155表达水平后,其诱导的DC的NF-κβ信号通路蛋白表达下降（t = 23.32,P < 0.05）;AKT信号通路蛋白表达量下降（t?= 22.21,P < 0.05）。 结论BMSCs高表达miR-155后,可以通过抑制NF-κβ和AKT途径诱导耐受性DC的产生,通过诱导DC减少T细胞的增殖从而对免疫调节进行影响。     

Reduced bone marrow stem cell pool and progenitor mobilisation in multiple myeloma after melphalan treatment

The content of stem cells was analysed in bone marrow samples from 75 multiple myeloma patients. In unstimulated bone marrow the percentage of CD34+cells was significantly reduced in 11 patients previously treated with melphalan-prednisolone (MP)(median= 0.15%) compared to median 0.87% in 31 untreated patients (P=0.0001). The bone marrow cellularity in the two groups did not differ. There was no correlation between the number of courses or total dose of melphalan and content of CD34+cells in the bone marrow. The clonogenicity as, well as the ability to expand the marrow stem cell pool during growth factor treatment were also reduced in MP treated patients compared to untreated patients. Analysis of different subsets of CD34+ cells revealed no influence on the pre B cell compartment in the bone marrow by MP treatment, but the committed stem cells (CD34+CD38+) were reduced more than the uncommitted stem cells (CD34+CD38—) in the MP treated group compared to the untreated patients. Mobilisation to and harvest of total number of CD34+ cells from peripheral blood was also reduced in the MP treated group. There was, however, no difference in the distribution between CD34+CD38+and CD34+CD38—populations in the leukapheresis products in the untreated and the melphalan-treated group, suggesting selective mobilisation of CD34+CD38+ cells and/or differentiation of CD34+ CD38-cells during growth factor stimulation. We conclude that melphalan decreased the number of stem cells in the bone marrow, the ability to expand the stem cell pool and mobilise stem cells to the pheripheral blood.     

Steroid-carrier proteins in patients with multiple myeloma

Patients with multiple myeloma have transcortin levels lower than normal. This is due in essence to a subgroup of patients producing IGG heavy chains with lambda light chains. Patients producing IGG with predominantly kappa light chains have almost normal transcortin levels. On the other hand, the binding activity of the steroid binding beta globulin (SB beta G) of the kappa type of multiple myeloma is significantly higher than the steroid binding of the lambda type of multiple myeloma. The serum levels of vitamin D binding protein (DBP) fall in the normal range.     

Monitoring of bone resorption and bone formation in multiple myeloma

The article deals with the clinical value of monitoring of serum markers of osteoresorption (ICTP) and bone formation (PICP) in multiple myeloma. In a group of patients treated by conventional chemotherapy and group of patients treated by high dose chemotherapy with autologous peripheral blood stemm cell transplantation (APBSTC).