多发性骨髓瘤患者骨髓基质细胞IL-1β.IL-6.SCF.TPO表达的研究

 【摘要】　目的　研究多发性骨髓瘤 （MM） 患者骨髓基质细胞多种细胞因子在mRNA水平的表达情况，探讨不同因子在MM发生、发展中可能存在的作用。方法　采用半定量RT-PCR方法在mRNA水平上检测MM患者骨髓基质细胞白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）、干细胞因子（SCF）、血小板生成素（TPO）的表达。结果　IL-1β、IL-6的表达：MM组与正常对照组和非MM组相比差异均具有统计学意义（P＜0.01）。SCF的表达：自体移植后患者组与正常对照组相比差异无统计学意义（P＞0.05），初发MM组和难治复发MM组与正常对照组和非MM组相比差异均有统计学意义（P＜0.05）。TPO的表达：MM组与正常对照组相比差异具有统计学意义（其中初发组P＜0.01，其余两组P＜0.05）。初发组与非MM组相比差异有统计学意义（P＜0.01），难治复发MM组和移植后患者组与非MM组相比差异无统计学意义。结论　体外培养的MM患者骨髓基质细胞 IL-1β、IL-6、SCF和TPO存在表达异常，这些因子在疾病进展中可能起重要作用。     

The IL-6 receptor antagonist SANT-7 overcomes bone marrow stromal cell-mediated drug resistance of multiple myeloma cells

The bone marrow micro-environment produces a number of different survival factors that are important for the malignant growth and drug resistance of multiple myeloma (MM) cells. One of the main factors reported to be essential for survival and growth of MM cells in some experimental systems is IL-6. Therefore, the development and testing of substances that interfere with IL-6 or IL-6 receptor (IL-6R) function might have therapeutic value for the treatment of MM. We analyzed the effect of the IL-6R antagonist SANT-7 on growth and survival of the IL-6--dependent MM cell lines INA-6 and XG-1 as well as primary MM cells from 7 patients co-cultured with bone marrow stromal cells (BMSCs). In particular, we were interested in whether SANT-7 enhances the growth-inhibitory effects of dexamethasone (Dex) and all-trans-retinoic acid (ATRA). None of the drugs when tested as a single substance, including SANT-7, induced major growth inhibition if MM cells were co-cultured with primary human BMSCs. However, when Dex and ATRA were given in combination with SANT-7, strong growth inhibition was achieved in cell lines and primary MM cells. This effect was due to cell-cycle arrest and induction of apoptosis.     

APE1表达增强与多发性骨髓瘤细胞对马法兰耐药的关系研究

目的：探讨脱嘌呤/脱嘧啶核酸内切酶（apurinic/apyrimidinic endonucle ase,APE1）在多发性骨髓瘤（multiple myeloma,MM）细胞中的表达及其与马法兰耐药的关系.方法：采用双荧光抗体免疫标记法结合激光共聚焦显微镜和免疫细胞化学观察KM3细胞、32例MM患者和10例正常自愿者骨髓标本APE1蛋白定位及表达,并通过免疫细胞化学和Western blot检测0～15 μmol/L马法兰作用KM3细胞1～2 d后APE1表达的变化.结果：APE1和CD38蛋白在MM细胞存在共表达,APE1蛋白表达尤以细胞核周围表达明显;APE1阳性表达在正常对照组、MM初治组和MM复发或难治组间依次增高,P＜0.05;马法兰可诱导KM3细胞APE1表达增强,并与其作用时间及剂量成正比.结论：APE1蛋白表达强度与MM疗效有关,且马发兰作用可诱导其表达增强,提示APE1基因表达增强可能在MM对马发兰耐药中起一定作用.     

APE1表达增强与多发性骨髓瘤细胞对马法兰耐药的关系研究

目的:探讨脱嘌呤/脱嘧啶核酸内切酶(apurinic/apyri midinic endonucle-ase,APE1)在多发性骨髓瘤(multiple my-eloma,MM)细胞中的表达及其与马法兰耐药的关系。方法:采用双荧光抗体免疫标记法结合激光共聚焦显微镜和免疫细胞化学观察KM3细胞、32例MM患者和10例正常自愿者骨髓标本APE1蛋白定位及表达,并通过免疫细胞化学和West-ern blot检测0~15μmol/L马法兰作用KM3细胞1~2d后APE1表达的变化。结果:APE1和CD38蛋白在MM细胞存在共表达,APE1蛋白表达尤以细胞核周围表达明显;APE1阳性表达在正常对照组、MM初治组和MM复发或难治组间依次增高,P<0·05;马法兰可诱导KM3细胞APE1表达增强,并与其作用时间及剂量成正比。结论:APE1蛋白表达强度与MM疗效有关,且马发兰作用可诱导其表达增强,提示APE1基因表达增强可能在MM对马发兰耐药中起一定作用。肿瘤防治杂志,2005,12(19):1445-1448     

APE1/Ref-1与肿瘤的研究进展

脱嘌呤脱嘧啶核酸内切酶1又称氧化还原因子-1,是一种双功能酶,不仅具有核酸内切酶活性,发挥DNA修复功能,而且具有氧化还原功能,调控多种重要转录因子的活性。目前研究发现APE1/Ref-1在人体多种肿瘤中表达且与肿瘤的发生、发展及预后相关。APE1/Ref-1可能成为极具潜力的肿瘤基因治疗的新靶点。本文就APE1/Ref-1的结构、功能及在肿瘤研究方面的进展作了一系列的回顾与总结。     

APE表达水平与胃癌临床病理因素的相关性分析

目的：研究胃癌患者不同胃黏膜组织无嘌呤无嘧啶核酸内切酶（apurinic/apyrimidinic endonuclase APE）的表达水平与临床病理因素的相关性.方法：利用已构建好的包含208例胃癌患者胃癌、正常胃黏膜和转移淋巴结的组织芯片,用免疫组化方法检测不同组织APE表达水平,结合患者的临床资料进行统计分析.结果：正常组织和转移淋巴结中细胞核、细胞质APE表达水平与临床各病理因素之间没有明确相关性;胃癌组织中胞核表达与肿瘤浸润深度（P=0.000）、有无淋巴结转移（P=0.010）、TNM分期（P=0.000）有明显相关,浸润深度越深、出现淋巴结转移和TNM分期越晚,则胞核表达越弱,胞核表达还显示出与性别有一定相关性,男性表达水平较女性高（P=0.048）,胞质表达水平则与性别无明确相关性,只显示与淋巴结转移（P=0.017）和TNM分期（P=0.019）有关,有淋巴结转移和TNM分期较晚的患者胞质表达水平较弱.结论：随着胃癌的进展,肿瘤分期越晚、浸润深度越深和出现淋巴结转移,则胃癌组织中胞质和胞核的APE表达水平越低.     

Hierarchical regulation of interleukin production: Induction of interleukin 6 (IL-6) production from bone marrow cells and marrow stromal cells by interleukin 3 (IL-3)

IL-3 stimulated the production of IL-6 from a bone marrow-adherent cell population, macrophages and from hemopoietic supportive stromal cell lines. It also induced IL-6 production from a stem cell-enriched population of bone marrow cells which did not produce IL-6 without stimulation. In contrast, stimulation with IL-6 of all the cell populations studied in the present experiments did not induce IL-3 production. These results indicate a hierarchical network in the regulation of interleukin production, and existence of a positive feedback mechanism; IL-3 induces IL-6 production which in turn stimulates stem cells into cycle and induces stem cells to respond to IL-3.     

In vitro growth of hematopoietic progenitors and stromal bone marrow cells from patients with multiple myeloma

In the present study we have determined the content of hematopoietic and stromal progenitors in multiple myeloma (MM) bone marrow, and assessed their in vitro growth. Marrow cells were obtained from 17 MM patients at the time of diagnosis, and from 6 hematologically normal subjects. When mononuclear cells (MNC) from MM marrow were cultured, reduced numbers of hematopoietic progenitors were detected and their growth in long-term cultures was deficient, as compared to cultures of normal cells. When cell fractions enriched for CD34⁺ Lin⁻ cells were obtained, the levels of hematopoietic progenitors from MM marrow were within the normal range, and so was their growth kinetics in liquid suspension cultures. The levels of fibroblast progenitors in MM were not statistically different from those in normal marrow; however, their proliferation potential was significantly reduced. Conditioned media from MM-derived MNC and stroma cells contained factors that inhibited normal progenitor cell growth. Our observations suggest that hematopoietic progenitors in MM marrow are intrinsically normal; however, their growth in LTMC may be hampered by the presence of abnormal accessory and stroma cells. These results suggest that besides its role in the generation of osteolytic lesions and the expansion of the myeloma clone, the marrow microenvironment in MM may have a negative effect on hematopoiesis.     

Zoledronic acid down-regulates adhesion molecules of bone marrow stromal cells in multiple myeloma: a possible mechanism for its antitumor effect

BACKGROUND: Myeloma plasma cells interact with the bone marrow microenvironment which, in turn, supports their growth and protects them from apoptosis. In vitro studies have demonstrated the antitumor potential of zoledronic acid (ZOL) on myeloma cell lines, but few data are available on its effects on bone marrow stromal cells (BMSCs). The aim of the current study was to evaluate the antiproliferative and apoptotic effect of ZOL on BMSCs, as well as its effect on the expression of adhesion molecules. METHODS: BMSCs, obtained from bone marrow mononucleated cells of 8 patients with multiple myeloma, were treated with increasing concentrations of ZOL for 3 days. Cytotoxic effect was analyzed by 3-(4-5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide; thiazolyl blue (MTT) assay whereas the induction of apoptosis was evaluated by flow cytometric detection of fluorescein isothiocyanate-labeled annexin V, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and nuclear changes. Moreover, expression of CD106, CD56, CD50, CD49d, CD44, and CD40 was analyzed by flow cytometry. Data were evaluated by the Friedman test. RESULTS: After 3 days of exposure at concentrations of 10(-4) to 10(-5) M, ZOL induced a decrease in proliferation (P < 0.0001) and an increase in apoptosis (P < 0.002). Analysis of culture supernatants showed that myeloma BMSCs expressed interleukin (IL)-6, negligible levels of tumor necrosis factor-alpha, and no IL-1beta. In vitro exposure to the lowest concentrations of ZOL decreased IL-6 production by BMSCs. Among the adhesion molecules, CD106, CD54, CD49d, and CD40, which were strongly expressed at baseline, showed a statistically significant reduction compared with controls after exposure to ZOL. CONCLUSIONS: ZOL interfered with myeloma BMSCs by reducing proliferation, increasing apoptosis, and modifying the pattern of expression of adhesion molecules, especially those involved in plasma cell binding. These effects on BMSCs might explain the antitumor activity of ZOL.     

非小细胞肺癌组织无嘌呤无嘧啶核酸内切酶蛋白表达及其临床意义

CLC组织APE/Ref-1过表达提示患者预后不良.     

趋化因子受体MCP-1/CCR2在多发性骨髓瘤中基质细胞与瘤细胞的异常表达

 [摘要] 目的 观察趋化因子（MCP-1）及其趋化因子受体（CCR2）在多发性骨髓瘤（MM）患者骨髓单个细胞,骨髓基质细胞中的表达。方法 选择2011年5月—2011年9月山西医科大学第一附属医院血液科住院患者,15例经国内统一标准确诊为多发性骨髓瘤的初发病人,10例为一般血液病患者。①实验方法,取样本进行骨髓瘤细胞与骨髓基质细胞的分离培养,在含13%小牛血清的RPMI1640培养液中培养,每2天更换1次培养液,本实验用的细胞均处于对数生长期。②实验评估,采用流式细胞术检测骨髓瘤患者骨髓瘤细胞,骨髓基质细胞中MCP-1、CCR2。结果 实验评估方法显示约2/3的多发性骨髓瘤患者骨髓均表达MCP-1、CCR2,而对照组的骨髓中趋化因子MCP-1、CCR2基本无表达。结论 大部分多发性骨髓瘤患者骨髓均表达MCP-1、CCR2。 MCP-1及其特异性受体CCR2是MM细胞表面表达的主要趋化因子,对MM的疾病的进展起着一个比较重要的作用。     

IL-1beta expression in IgM monoclonal gammopathy and its relationship to multiple myeloma.

We have shown that IL-1beta is not detectable in normal plasma cells but is produced by plasma cells from virtually all patients with multiple myeloma (MM). To extend our earlier work, IL-1beta expression was determined in 13 newly diagnosed patients with IgM monoclonal gammopathy. Eleven patients with Waldenstrom's macroglobulinemia (WM) and two patients with IgM MM were investigated for IL-1beta expression by in situ hybridization (ISH). All patients with WM had bone marrow biopsies consistent with the diagnosis, an IgM M-protein in the serum, and subsequently required chemotherapy. Seven of 11 patients with WM had an M-protein >3 g/dl and five patients had bone surveys performed that were negative for osteolytic disease. Two patients were diagnosed with IgM MM because of the presence of significant osteolytic disease on a metastatic bone survey. ISH for kappa, lambda, and IL-1beta expression was performed on bone marrow aspirates from each of the 13 patients. None of the neoplastic cells from the 11 patients with WM showed detectable IL-1beta expression by ISH. However, the neoplastic cells from both patients with IgM MM expressed IL-1beta mRNA at high levels. This aberrant IL-1beta production may explain the presence of bone lesions in the patients with IgM MM.     

Adherence of multiple myeloma cells to bone marrow stromal cells upregulates vascular endothelial growth factor secretion: therapeutic applications.

Increased angiogenesis has recently been recognized in active multiple myeloma (MM). Since vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are two key mediators of angiogenesis, we characterized the production of VEGF, b-FGF and interleukin-6 (IL-6) (a MM growth and survival factor) in MM cell lines and Epstein-Barr virus (EBV) transformed B cell lines from MM patients, patient MM cells, as well as bone marrow stromal cells (BMSCs) from normal healthy donors and MM patients. We detected secretion of VEGF, but no bFGF and IL-6, in MM cell lines (MM.1S, RPMI 8226 and U266); EBV transformed B cell lines from MM patients (IM-9, HS-Sultan and ARH77); MM cell lines resistant to doxorubicin (RPMI-DOX40), mitoxantrone (RPMI-MR20), melphalan (RPMI-LR5) and dexamethasone (MM.1R); and patient MM cells (MM1 and MM2). BMSCs from MM patients and normal donors secreted VEGF, b-FGF and IL-6. Importantly, when MM cells were adhered to BMSCs, there was a significant increase in VEGF (1.5- to 3.1-fold) and IL-6 (1.9- to 56-fold) secretion. In contrast, the bFGF decreased in co-cultures of BMSCs and MM cells. Paraformaldehyde fixation of BMSCs or MM cells prior to adhesion revealed that VEGF was produced both from BMSCs and MM cells, though it may come primarily from BMSCs in some cultures. IL-6 was produced exclusively in BMSCs, rather than MM cells. Moreover, when MM cells were placed in Transwell insert chambers to allow their juxtaposition to BMSCs without cell to cell contact, induction of VEGF and IL-6 secretion persisted, suggesting the importance of humoral factors. Addition of exogenous IL-6 (10 ng/ml) increased VEGF secretion by BMSCs. Conversely, VEGF (100 ng/ml) significantly increased IL-6 secretion by BMSCs. Moreover, anti-human VEGF (1 microg/ml) and anti-human IL-6 (10 microg/ml) neutralizing antibodies reduced IL-6 and VEGF secretion, respectively, in cultures of BMSCs alone and co-cultures of BMSCs and MM cells. Finally, thalidomide (100 microM) and its immunomodulatory analog IMiD1-CC4047 (1 microM) decreased the upregulation of IL-6 and VEGF secretion in cultures of BMSCs, MM cells and co-cultures of BMSCs with MM cells. These data demonstrate the importance of stromal-MM cell interactions in regulating VEGF and IL-6 secretion, and suggest additional mechanisms whereby thalidomide and IMiD1-CC4047 act against MM cells in the BM millieu.     

Etodolac induces apoptosis and inhibits cell adhesion to bone marrow stromal cells in human myeloma cells

OBJECTIVES: Cyclooxygenase-2 (COX-2) is reported to regulate apoptosis and to be an important cellular target for therapy. METHODS: We examined whether etodolac, meloxicam, and thalidomide inhibited proliferation and induced apoptosis in myeloma cell lines (RPMI 8226 and MC/CAR cells). RESULTS: Etodolac induced apoptosis more strongly compared with thalidomide or meloxicam. Etodolac induced down-regulation of Bcl-2 protein and mRNA, activation of Caspase-9, -7 and -3, cIAP-1 and Survivin, and loss of mitochondrial membrane potential in a dose-dependent manner. In addition, when myeloma cells were coincubated with 50 microM etodolac on bone marrow stromal cells (BMSCs), myeloma cell adhesion to BMSCs was significantly inhibited compared with thalidomide or meloxicam coincubation, and the adhesion molecules VLA-4, LFA-1 (CD11a), CXCX4, and CD44 were suppressed on myeloma cells treated with etodolac. Moreover, 50-100 microM racemate of etodolac significantly inhibited the proliferation of myeloma cells compared to 100 microM R-etodolac or S-etodolac. CONCLUSIONS: Etodolac induced loss of mitochondrial membrane potential and apoptosis via a COX-2-independent pathway, suppressed the expression of adhesion molecules, and inhibited myeloma cell adhesion to BMSCs compared with thalidomide or meloxicam. The activities of etodolac potentially extend to the treatment of patients with myeloma resistant to standard chemotherapy, including thalidomide.     

CD45 expression by bone marrow plasma cells in multiple myeloma: clinical and biological correlations.

Multiple myeloma (MM) is characterized by accumulation of clonal plasma cells (PCs). CD45, a key regulator of antigen-mediated signaling and activation in lymphocytes, is present in early stages of PCs development. We studied CD45 expression on MM PCs by flow cytometry, correlating it to important biological disease characteristics. Additionally, we examined the expression of various adhesion molecules on PCs. A total of 75 patients with untreated MM (29), relapsed MM (17), smoldering MM (12), and monoclonal gammopathy of undetermined significance (MGUS) (17) were studied. The proportion of PCs expressing CD45 was higher among those with early disease (MGUS or smoldering MM) compared to those with advanced disease (new or relapsed MM) (43 vs 22%; P=0.005). Among those with advanced disease, patients with bone lesions had a lower percentage of CD45-positive (CD45+) PCs; 14 vs 34% (P=0.02). Patients with high-grade angiogenesis had a lower percentage of CD45+ PCs; 13 vs 31% (P=0.03). The median overall survival for the CD45+ group (>20% PCs positive) was 39 vs 18 months for the CD45-negative (CD45-) group (P=0.07). The expression of CD138, CD56 and CD54 were higher among the CD45- PCs. This study demonstrates important biological correlates of CD45 expression on myeloma cells.     

Suppressor of cytokine signaling-1 expression by infectivity-enhanced adenoviral vector inhibits IL-6-dependent proliferation of multiple myeloma cells

Multiple myeloma (MM) accounts for 10% of hematological malignant disorders. Its refractory nature indicates the necessity of developing novel therapeutic modalities. Since interleukin 6 (IL-6) is one of the major growth factors for MM cells, we expressed suppressor of cytokine signaling-1 (SOCS-1), one of the blockades of IL-6 receptor downstream signaling, to suppress the proliferation of MM cells. Because MM cells are resistant to conventional adenoviral vector infection, we utilized infectivity-enhanced adenoviral vectors with an RGD4C motif in the adenoviral fiber-knob region (RGD-modified vector). In infectivity analysis, RGD-modified vectors were superior to unmodified controls in the majority of the MM cell lines tested. The overexpression of SOCS-1 using infectivity-enhanced adenoviral vectors achieved growth suppression in IL-6-dependent MM cells, but not in the IL-6-independent cells. IL-6-induced STAT3 phosphorylation was suppressed in IL-6-dependent cells, indicating that the signal transduction cascade of the IL-6 receptor signaling was blocked. In aggregate, SOCS-1 overexpression with RGD-modified adenoviral vectors achieved the antiproliferative effect in IL-6-dependent MM cells. These results provide an initial proof-of-principle of the anticancer effect of SOCS-1 expression vector as well as a promise for the future development of therapeutic modality for MM based on this vector.