重组人血管内皮抑素对血管新生的影响研究

目的 探讨重组人血管内皮抑素（Endostar,恩度）对血管内皮细胞趋化、迁移、粘附、增殖及小管形成等与血管新生相关生物学行为的影响。方法 以原代培养的人脐静脉内皮细胞（HUVEC）为细胞模型,通过Boyden小室荧光定量分析、划痕试验、HUVEC荧光定量粘附分析、CFSE染色流式细胞术、CCK-8定量检测、小管形成试验和Matrigel栓试验研究恩度对HUVEC与血管新生相关的生物学行为的影响。结果 恩度在5～50 000ng／ml范围内可抑制血管内皮生长因子诱导HUVEC的迁移运动,且浓度为50ng／ml和500ng／ml时效果最明显;恩度在5～50 000ng／ml间可呈剂量依赖的方式抑制HUVEC向损伤部位的迁移。与0ng／ml相比,50、500和5000ng／ml 恩度处理的HUVEC的粘附率、增殖率及HUVEC形成网状小管结构的数量、面积和长度均降低,差异均有统计学意义（P＜0.05）; Matrigel栓实验结果显示,恩度在50～5000ng／ml间对SCID小鼠体内血管新生有明显的抑制作用。结论 恩度在细胞水平能抑制HUVEC与血管新生相关的生物学行为,包括HUVEC的趋化、迁移、粘附、增殖和小管形成;在动物水平能抑制SCID小鼠体内的血管新生,据此推断恩度能抑制血管新生。     

Bone morphogenetic protein 9 is a potent synergistic factor for murine hemopoietic progenitor cell generation and colony formation in serum-free cultures.

The effect of the recently cloned cytokine bone morphogenetic protein 9 (BMP-9) on colony formation and generation in vitro clonable hematopoietic progenitors (CFU-C) in serum-free liquid cultures (LC) of both normal and post-5-fluorouracil murine bone marrow cells was studied in the presence of various other cytokines. In LC, BMP-9 concentrations of 100 ng or more per ml led to complete inhibition of Steel Factor (SF) + interleukin-11 (IL-11) or IL-12 supported CFU-C generation, which was partly abrogated when IL-3 was additionally included. We found this inhibitory effect of BMP-9 to be mediated by an increased TGF-beta1 elaboration and TGF-beta1 mRNA expression in bone marrow cells with increasing BMP-9 concentrations. In the presence of neutralizing antibodies (Ab) against TGF-beta1, BMP-9 concentrations of 3 ng or higher synergized with IL-3, SF+IL-3, SF+IL-11/12, or IL-3+SF+IL-11/12 to increase CFU-C generation. Similarly, high BMP-9 concentrations dramatically inhibited primary colony formation induced by SF+IL-11/12, whereas in the presence of TGF-beta1 neutralizing Ab only 3 ng or more BMP-9 per ml stimulated both the time of colony appearance, the colony size and colony numbers in the presence of IL-3, M-CSF, GM-CSF, SF, SF+Flt3-L, SF+IL-3, SF+IL-11/12 or IL-3+SF+IL-11/12. BMP-9 neither stimulated CFU-C generation nor colony formation as a single factor, nor did it synergize with thrombopoietin (Tpo), erythropoietin (Epo), Flt3-L, IL-11, IL-12 or G-CSF. The effect of BMP-9 on its target cells was direct as demonstrated using single-sorted stem cells. These observations demonstrate that BMP-9 plays a dual role in regulating proliferation of primitive hemopoietic progenitor cells. Thus, in addition to its ability to enhance TGF-beta1 elaboration in bone marrow cells, it acts as a potent synergistic activity that is different from SF, Flt3-L, IL-11 or IL-12. BMP-9 mRNA was exclusively detected in the liver of adult mice, whilst no expression was found in stromal cell lines propagated from day-16 fetal liver or neonatal or adult bone marrow. 125I-BMP-9 bound specifically to a high percentage of blast cells in lineage-depleted post-fluorouracil bone marrow cells and to megakaryocytes in normal and post-fluorouracil bone marrow, indicating that BMP-9R are expressed on these cells. The dissociation between the site of BMP-9 production and its target cells in the bone marrow makes BMP-9 a hemopoietic hormone.     

RNA干扰技术下调CEACAMl基因表达对胶质瘤血管生成的影响

目的：利用RNA干扰技术下调胶质瘤细胞中癌胚抗原相关细胞黏附分子1（ CEACAM1）基因的表达情况,探讨胶质瘤细胞CEACAM1表达变化后其培养上清液诱导人脐静脉内皮细胞（ HUVEC）增殖、血管生成的情况及其可能机制。方法设计并化学合成针对CEACAM1的3对siRNA,脂质体法瞬时转染SHG44细胞,收集细胞培养上清液作为条件培养基。分别采用RT－PCR检测RNA干扰后细胞中CEACAM1及血管内皮生长因子（ VEGF） mRNA表达的变化情况,ELISA法检测上清液中VEGF蛋白的含量。 MTT比色法及Transwell侵袭实验检测共培养刺激后HUVEC增殖及迁移能力的变化,体外血管生成实验检测体外血管生成能力。结果与正常对照组和阴性对照组相比,转染CEACAM1－siRNA后胶质瘤细胞中CEACAM1 mRNA的表达水平下调（P＜0．01）,以CEACAM1－siRNA3组最为显著。 RNA干扰后SHG44细胞VEGF mRNA及上清液中的VEGF蛋白含量均减少,HUVEC的增殖受到抑制,迁移及体外血管生成能力下降。结论 CEACAM1－siRNA能够下调SHG44细胞中CEACAM1 mRNA的表达,并通过减少VEGF分泌,从而影响HUVEC的增殖、迁移和体外血管生成能力。     

沉默FOXM1拮抗肝细胞生长因子对胃癌细胞SGC7901增殖、迁移和侵袭的诱导作用

目的:探索转录因子FOXM1在肝细胞生长因子(HGF)诱导胃癌细胞增殖、迁移和侵袭过程中的作用.方法:体外培养胃癌细胞SGC7901,根据不同处理将细胞分组:空白对照组、20 ng/ml HGF组、40 ng/ml HGF组、60 ng/ml HGF组、siRNA-scramble+HGF组、siRNA-FOXM1+HGF组.采用MTT法检测各组SGC7901细胞的增殖,Transwell法检测各组细胞的迁移和侵袭情况,qRT-PCR和Western blot法分别检测各组细胞中FOXM1 mRNA和蛋白水平.结果:与空白对照组相比,不同浓度HGF均能够促进胃癌细胞SGC7901增殖、迁移和侵袭,同时上调FOXM1的表达水平,并且呈浓度依赖性关系.沉默HOXM1表达能够明显抑制HGF对SGC7901细胞的增殖、迁移和侵袭的诱导作用.结论:HGF对胃癌细胞增殖、迁移和侵袭的诱导作用可能与上调FOXM1表达有关,沉默FOXM1表达能够抑制HGF的作用.     

In vitro production of rabbit macrophage tumor cell cytotoxin

Rabbit pulmonary lavage cells, consisting mostly of macrophages (90-95%), cultured in the presence of LPS, secreted tumor cell cytotoxin that was similar to tumor necrosis serum cytotoxin. A similar cytotoxin was produced by phorbol-ester-pretreated rabbit bone marrow cells and by blood mononuclear cells when cultured in the presence of LPS. All cytotoxins had molecular weights of approximately 48,000 daltons by gel filtration and eluted from DEAE-Sephadex between 0.28 and 0.32 M NaCl. All were stable to 56 degrees C for 60 min, but labile to 70 degrees C for 20 min. B16C3 melanoma cells and mouse embryo fibroblasts were resistant to the cytotoxins. By 3 h in culture, all effector cells secreted detectable cytotoxin levels. Kinetics of cytotoxin production differed for effector cells derived from the different tissues. No additional cytotoxin production could be demonstrated after 30 h in pulmonary lavage or bone marrow cell cultures, despite a change to fresh medium with LPS. Actinomycin D (I microgram/ml) added with LPS inhibited cytotoxin production (greater than 95%) by pulmonary lavage cells. Delaying addition of actinomycin D after LPS treatment demonstrated that messenger RNA production for cytotoxin was completed by 2 to 6 h.     

Effect of cytokines on the toxicity of cytostatic drugs to normal bone marrow cells in vitro

We evaluated in vitro how growth factors influenced the effect of cytostatic drugs on normal hematopoietic progenitor cells. Bone marrow was obtained from 15 donors for bone marrow transplantation. After separation the mononuclear fraction was incubated with granulocyte colony-stimulating factor (G-CSF) at 5 and 50 ng/ml, with granulocyte/macrophage colony-stimulating factor (GM-CSF) at 1 and 10 ng/ml, and with interleukin 3 (IL-3) at 0.5 and 5 ng/ml for 24 h prior to incubation with cytostatic drugs. These incubations were performed with 0.05 μM mitoxantrone and 0.2 μM daunorubicin for 1 h, and cells were thereafter cultured for colony-forming units – granulocyte/macrophage (CFU-GM) in soft agar for 10–12 days. Incubation with 0.05 μM cytosine arabinoside was performed continuously throughout the culture period. The proliferation of normal hematopoietic progenitor cells stimulated with GM-CSF at 10 ng/ml and with IL-3 at 5 ng/ml was significantly increased to 218% and 215% colonies, respectively, as compared with the control stimulated with conditioned medium only. Stimulation with G-CSF, on the other hand, did not induce any significantly enhanced proliferation relative to the control. Daunorubicin applied in combination with G-CSF at 5 ng/ml or with IL-3 at 0.5 ng/ml exerted a significantly higher degree of cytotoxicity on normal hematopoietic progenitor cells, resulting in 21% and 30% surviving colonies as compared with the 38% recorded for daunorubicin alone (P<0.05). Neither G-CSF nor IL-3 at a higher concentration nor GM-CSF exerted a significantly altered degree of toxicity relative to cells incubated with daunorubicin alone. The cytotoxic effect exerted on normal hematopoietic cells by mitoxantrone or ara-C was unchanged or significantly decreased after stimulation with growth factors as compared with the effect on cells incubated with cytostatic drugs alone. We conclude that G-CSF and IL-3 augment the effect of daunorubicin on normal hematopoietic progenitor cells. Received: 6 May 1997 / Accepted: 4 February 1998     

In vitro growth characteristics of virally transformed murine myeloid cells.

Bone marrow from normal BALB/c mice, mice with myeloid leukemia induced by Soule myeloid leukemia virus, and mice with virally induced mammary carcinoma was cultured in semisolid agar. Bone marrow from either leukemic or mammary tumor-bearing mice produces more clones in vitro in the presence of a specific colony-stimulating factor. However, in all cases, the myeloid progenitor cells have similar requirements for the colony-stimulating factor. The optimum condition for growth in all instances is 7% fetal calf serum + 7% horse serum + 7% tryptose phosphate broth. Decrease in the concentration of these three constituents has a less drastic effect on in vitro proliferation of bone marrow cells from leukemic mice. Some cells from Soule virus-induced leukemias even grew in the absence of serum. The combination of suboptimal amounts of serum and colony-stimulating factor is used as a tool for detecting cells with altered growth characteristics in bone marrow of leukemic mice. During the progression of the leukemia, there is an increase in the amount of transformed colony-forming cells per 5 X 10(4) bone marrow cells. The increase is already noticeable 4 weeks after inoculation, when no clinical signs of the leukemia are present, and reaches a maximum of about 20%.     

59. Protectivc effect of melatonin on genetic damage by chemical mutagen and the influence on cell prolife-ration kenetics

Objective: In this study, we observed the effect of melatonin on the frequency of sister chromatid exchange, micronucleus formation of binuclear cell in lymphocyte from human peripheral blood in vitro, micronucleus formation of mouse bone marrow polycychromatic erythrocyte in vivo, which were induced by chemical mutagen, and lymphocyte proliferation kenetics in vitro. Methods: ① Lymphocytes were cultured in vitro in the presence of 0.01,0.10,1.00 mmol/L melatonin, mitomycin C(MMC) (positive control), 0.5% ethanol (negative control)and 0.01,0.10,1.00 mmol/L melatonin plus MMC for 72 h at 37℃±1℃. Lymphocytes were examined for the frequence of SCE, mitotic index, cell proliferation cycle, cell cycle ratio and proliferation index. ② Lymphocytes were cultured in vitro in the presence of 0.01,0.10,1.00 mmol/L melatonin, mitomycin C(MMC) (positive control), 0.5% ethanol (negative control) and 0.01,0.10,1.00 mmol/L melatonin plus MMC for 44 h at 37℃±1℃. Then each culture was given cytochalasin B, which was cultured to 72 h. Binuclear lymphocytes were examined for the micronucleus rate. ③ The mice were administered with 0.1, 1.0,10.0 mg/kg*bw melatonin and distillated water (negative control) respectively for 7 d, then were given melatonin plus cyclophosphamide (CP) (positive control) for 2 d since the eighth day. The rate of micronulclei of mouse bone marrow polycychromatic erythrocyte was examined. Results: ① The frequences of sister chromatid exchange of lymphocytes which were cultured in the presence of 0.01,0.10,1.00 mmol/L melatonin compared with negative control exhibited no statistical significance. ② The SCE of cells treated with melatonin plus MMC compared with positive control were markedly decreased. ③ The mitotic indices of lymphocytes cultured in the presence of 0.10,1.00 mmol/L melatonin were lower than negative control. The proliferation index was significant lower than negative control only in the culture exposed to 1.00 mmol/L melatonin. ④ The proliferation cycle of lymphocyte cultured in the presence of 1.00 mmol/L melatonin exhibited an increase in the percent of cells in their first division, and concomitant significant decrease in their third or later division. which were increased in their second division in cells treated with melatonin of three concentration respectively. The ratio between the first and third or later division, between the second and third or later division of lymphocytes exposed to 1.00 mmol/L melatonin were higher than negative control. ⑤ The micronucleus rates of binuclear lymphocytes treated with 0.01,0.10,1.00 mmol/L melatonin plus MMC were lower than positive control, of which the inhibitory rates of micronuclei were 14.37%, 22.17% and 31.34%, respectively. ⑥ The micronucleus rates of mouse bone marrow polycychromatic erythrocytes exposed to 0.1,1.0,10.0 mg/kg*bw melatonin plus CP were lower markedly than positive control, in which it exhibited a significant and concentration－depended decrease, of which the inhibitory rates of micronuclei were 28.89%, 47.73% and 69.96%, respectively. Conclusion: In vitro assay. It seems that melatonin has not only no genotoxic, but also anti-mutagenic effect on lymphocyte. It can inhibit the increase of frequency of SCE in lymphocyte and micronucleus rate of binuclear lymphocyte induced by MMC, and micronucleus rate of mouse bone marrow induced by CP at some concentiations. It can inhibit the lymphocyte proliferation of mitogen stimulated and has some influence on cell proliferation kenetics at higher concentrations (1.00 mmol/L).     

An inhibitor of murine stem cell proliferation produced by normal human bone marrow

Media conditioned by normal human bone marrow cells contain a specific inhibitor of haemopoietic stem cell proliferation. Molecular ultrafiltration and dose response studies indicate that it is similar to a previously described factor obtained from freshly isolated or long-term cultured murine bone marrow cells. It is suggested that the mechanisms involved in the control of murine and human stem cell proliferation may be essentially identical.     

Role of humoral factors in granulopoiesis: Colony promoting factor (CPF) and its target “pre-CFU-C’ in long-term bone marrow culture

Supernatant of long-term bone marrow cultures contains colony promoting factor (CPF). The factor has no colony stimulating activity itself but augments granulocyte-macrophage colony formation in semi-solid culture of bone marrow cells in the presence of colony stimulating factor (CSF). CPF was specifically absorbed by bone marrow cells but not by thymocytes. Incubation of bone marrow cells with CPF alone resulted in the increase of the number of granulocyte-macrophage progenitor cells (CFUc). CPF-containing supernatant did not stimulate the proliferation of pluripotent stem cells (CFUs) but inhibited CFUs proliferation. These results suggest that CPF is a separate factor from CSF or from CFUs regulators, and that CSF-non-responsive cells (pre-CFUc) in the bone marrow mature into CSF-responsive CFUc by the stimulus of CPF.     

The Comparison of Biologic Characteristics between Mice Embryonic Stem Cells and Bone Marrow Derived Dendritic Cells

OBJECTIVE This research was to induce dendritic cells (DCs)from mice embryonic stem cells and bone marrow mononuclear cells in vitro, and then compare the biologic characteristics of them.METHODS Embryonic stem cells (ESCs) suspending cultured in petri dishes were induced to generate embryonic bodies (EBs).Fourteen-day well-developed EBs were transferred to histological culture with the same medium and supplemented 25 ng/ml GM-CSF and 25 ng/ml IL-3. In the next 2 weeks, there were numerous immature DCs outgrown. Meantime, mononuclear cells isolated from mice bone marrow were induced to derive dendritic cells by supplementing 25 ng/ml GM-CSF and 25 ng/ml IL-4, and then the morphology, phenotype and function of both dendritic cells from different origins were examined.RESULTS Growing mature through exposure to lipopolysaccharide (LPS), both ESC-DCs and BM-DCs exhibited dramatic veils of cytoplasm and extensive dendrites on their surfaces, highly expressed CD11c, MHC-Ⅱ and CD86 with strong capacity to stimulate primary T cell responses in mixed leukocyte reaction (MLR).CONCLUSION ESC-DC has the same biologic characteristics as BM-DC, and it provides a new, reliable source for the functional research of DC and next produce corresponding anti-tumor vaccine.     

Interactions of human prostatic epithelial cells with bone marrow endothelium: binding and invasion

Prostate cancer shows a propensity to form secondary tumours within the bone marrow. Such tumours are the major cause of mortality in this disease. We have developed an in vitro system to study the binding of prostate epithelial cells to bone marrow endothelium (BME) and stroma (BMS). The metastatic prostate cancer cell line, PC3 (derived from a bone metastasis), was seeded onto confluent layers of BME and its binding characteristics compared to human umbilical vein endothelial cells (HUVEC), lung endothelium (Hs888Lu) and BMS. The PC3 cell line showed significantly increased binding to BME (P< 0.05) compared to endothelium derived from HUVEC and lung or BMS with maximal binding occurring at 1 h. Following pre-incubation with a beta1 integrin antibody PC3 binding to BME was inhibited by 64% (P< 0.001). Antibodies directed against the integrins beta4, alpha2, alpha4, alpha5 and the cellular adhesion molecules P-selectin, CD31, VCAM-1 and sialy Lewis X showed no effect on blocking PC3 binding. Primary prostatic epithelial cells from both malignant (n = 11) and non-malignant tissue (n = 11) also demonstrated equivalent levels of increased adhesion to BME and BMS compared to HUVEC, peaking at 24 h. Further studies examined the invasive ability of prostate epithelial cells in response to bone marrow endothelium using Matrigel invasion chamber assays. In contrast to the previous results, malignant cells showed an increase (1000 fold) in invasive ability, whilst non-malignant prostate epithelia did not respond. We have shown that both malignant and non-malignant prostate epithelial cells can bind at equivalent levels and preferentially to primary human bone marrow endothelium in comparison to controls. However, only malignant prostate epithelia show increased invasive ability in response to BME.     

Role of Hepatocyte Growth Factor in Hemopoiesis

Hepatocyte growth factor (HGF) is a polypeptide that stimulates proliferation, motility, and morphogenesis of various cells, particularly epithelial cells. There is considerable evidence that HGF is a regulator in hemopoiesis not only in mice but also in humans. In mice, HGF and c-met (its receptor) mRNA are coexpressed in the fetal liver in the middle and late stages, when hemopoiesis is most active. HGF and c-met mRNA are also expressed in the stromal cells of both fetal liver and bone marrow. Human HGF (2 to 20 ng/ml) enhances colony-forming units in culture (CFU-C) counts and cobblestone colony counts in the long-term cultures of the fetal liver and bone marrow, although HGF has no effect on freshly isolated bone marrow or fetal liver cells in the CFU-C assay. However, when the bone marrow or fetal liver cells are cocultured with HGF in the presence of IL-3, CFU-C counts increase. In humans, it has also been shown that HGF in the presence of erythropoietin induces the formation of erythroid burst-forming unit (BFU-E) colonies from CD34⁺ cells purified from the bone marrow, peripheral blood, or cord blood. This review discusses the role of HGF as a regulator in hemopoiesis.     

奥沙利铂联合热疗对血管生成的抑制作用

目的探讨奥沙利铂(L-OHP)联合热疗在体内外对血管生成的抑制作用。方法采用四甲基偶氮唑盐(MTY)法观察L-OHP联合热疗对人脐静脉内皮细胞(HUVEC)和人结肠癌LOVO细胞增殖的影响;采用transwell实验观察L-OHP联合热疗对HUVEC迁移的影响;采用鸡胚绒毛尿囊膜(CAM)模型观察L-OHP对CAM新生血管的抑制作用。结果0.5～16μs/ml L-OHP均具有抑制HUVEC增殖的作用,细胞存活率为80.1%～42.5%,其增殖速度与L-OHP浓度呈负相关(r=-0.943, P=0.005);在此浓度范围内,L-OHP对LOVO细胞增殖的抑制明显低于对HUVEC的抑制。0.5、1和16μg/ml L-OHP与热疗对HUVEC增殖的抑制具有协同作用,2、4和8μg/ml L-OHP与热疗具有相加作用。0.25～2μg/ml L-OHP具有抑制HUVEC迁移的作用,细胞迁移抑制率为18.7%～53.0%。1、4μg/ml L-OHP对CAM新生血管具有明显的抑制作用,抑制率分别为70.0%和100.0%。结论小剂量L-OHP在体内外具有抑制血管生成的作用,联合热疗对抑制HUVEC增殖具有协同或相加效应。     

长春瑞滨联合西妥昔单抗抑制血管生成作用的实验研究

目的 探讨长春瑞滨联合西妥昔单抗对体内外血管生成的抑制作用.方法 以人肺腺癌细胞株A549为对照,采用四甲基偶氮唑蓝(MTT)法,观察长春瑞滨联合西妥昔单抗对人脐静脉内皮细胞(HUVEC)增殖能力的影响;应用Transwell小室模型、体外小管形成实验及流式细胞术,分别观察长春瑞滨联合西妥昔单抗对HUVEC迁移、小管形成及细胞凋亡的影响;应用鸡胚绒毛尿囊膜(CAM)模型,观察药物对体内CAM血管生成的抑制作用.结果 0.1、0.4和0.8 ng/ml长春瑞滨对HUVEC增殖的抑制率分别为25.8%、39.2%和54.0%,0.25 μg/ml西妥昔单抗对HUVEC增殖的抑制率为19.7%.0.1 ng/ml长春瑞滨+0.25 μg/ml西妥昔单抗和0.4 ng/ml长春瑞滨+0.25 μg/ml西妥昔单抗对HUVEC增殖的抑制率分别为29.5%和46.4%,联合用药有次加作用;0.8 ng/ml长春瑞滨+0.25 μg/ml西妥昔单抗对HUVEC增殖的抑制率为64.6%,联合用药有协同作用.0.1、0.4和0.8 ng/ml长春瑞滨分别联合0.25 μg/ml西妥昔单抗具有抑制HUVEC迁移的作用,抗HUVEC迁移率分别为51.9%、68.2%和95.0%,联合应用有协同作用.0.1 ng/ml长春瑞滨+0.25 μg/ml西妥昔单抗或0.4 ng/ml长春瑞滨+0.25 μg/ml西妥昔单抗对抑制HUVEC小管形成有次加或协同作用,抗HUVEC小管形成率分别为38.8%和57.7%;0.8 ng/ml长春瑞滨+0.25 μg/ml西妥昔单抗对抑制HUVEC小管形成有协同作用,抗HUVEC小管形成率为78.9%.0.8 ng/ml长春瑞滨+0.25 μg/ml西妥昔单抗诱导HUVEC的凋亡率为59.9%,有协同作用.长春瑞滨联合西妥昔单抗具有协同抑制CAM血管生成的作用.结论 小剂量长春瑞滨和西妥昔单抗在体内外具有抗血管生成作用,联合用药具有次加或协同作用.     

RANKL directly induces bone morphogenetic protein-2 expression in RANK-expressing POS-1 osteosarcoma cells

The POS-1 murine model of osteolytic osteosarcoma was used to elucidate the molecular and cellular mechanisms involved in the development of primary bone tumors and associated lung metastasis. The POS-1 cell line is derived from an osteosarcoma tumor which develops spontaneously in C3H mice. The POS-1 cell line was characterized in vitro by mineralization capacity and expression of bone markers by semi-quantitative RT-PCR, compared to primary osteoblasts and bone marrow cells. POS-1 cells showed no mineralization capacity and exhibited an undifferentiated phenotype, expressing both osteoblastic and unexpected osteoclastic markers (TRAP, cathepsin K and RANK). Thereby, experiments were performed to determine whether RANK was functional, by studying the biological activity of murine RANKL through the receptor RANK expressed on POS-1 cells. Results revealed a RANKL-induced increase in ERK phosphorylation, as well as BMP-2 induction at the mRNA and protein levels, and a decrease of POS-1 cell proliferation in the presence of 10 ng/ml RANKL. BMP-2 induction is dependent on the ERK 1/2 signal transduction pathway, as its expression is abolished in the presence of UO126, a specific synthetic inhibitor of the ERK 1/2 pathway. Moreover, a 2-fold molar excess of soluble RANK blocks the RANKL-induced BMP-2 expression, demonstrating that the biological effects of RANKL observed in POS-1 cells are mediated by RANK. This is the first report describing a functional RANK expressed on osteosarcoma cells, as shown by its ability to induce signal transduction pathways and biological activity when stimulated by RANKL.     

Antiproliferative Activity of Mammalian Lignan Derivatives Against the Human Breast Carcinoma Cell Line, ZR-75-1

The effect of each of twelve mammalian lignun derivatives on the growth of human mammary tumor ZR-75-1 cells was examined. At a concentration less than 10 μ g/ml, tumor cell growth was inhibited from 18-68%. The effect of 2,3-dibenzylbutane-1,4-diol (hattalin) was found to be strongest, inhibiting growth by 50% at a concentration (EC₅₀ of 2.1 μ g/ml. Hattalin inhibited membrane Na^%, K^%-ATPase of canine kidney cortex. It also inhibited the ATPase of the plasma membrane fraction from both cultured cells and a section of human breast cancer tissue at a concentration ranging from 0.5 to 2.0 mM. However, only a few percent of membrane ATPase from either ZR-75-1 cells or breast carcinoma tissue was inhibited by 2.0 mM of ouabain, suggesting that the target ATPase of hattalin was other than ouabainsensitive ATPase. The relative incorporation of [³H]thymidine per 1 10⁵ cells into the acid-precipitable fraction of ZR-75-1 cells was not affected by 1-50 μg/ml of hattalin, while a marked decrease resulted from 1-10 μg/ml of 5-fluorouracil (5-FU). These results suggest that the suppressive effect of hattalin on tumor cell growth my not occur through inhibition of DNA synthesis but rather partly by inhibition of the plasma membrane ATPase other than Na^% and K^% -dependent ones.     

Enhancement of vinblastine-induced cytotoxicity by lysolecithin and phosphatidylinositol

Vinblastine inhibited the growth of cultured KB cells 3 days after drug treatment by 55% and 67% at 2.7 ng/ml and 3.5 ng/ml of the medium, respectively. Lysolecithin and phosphatidylinositol showed only a marginal inhibitory effect on the growth of KB cells at respective concentrations of 35–125 μg/ml and 50–150 μg/ml of the medium. Lysolecithin, however, enhanced the cytotoxicity of vinblastine. Depending upon the concentrations of lysolecithin (35–125 μg/ml), the growth of KB cells was inhibited by 60–91% and 86–98% at respective vinblastine concentrations of 2.7 ng/ml and 3.5 ng/ml. Enhancement of vinblastine-induced cytotoxicity also occurred similarly for phosphatidylinositol. The mechanism could be explained partly by an elevated amount of intracellular vinblastine. Other possible mechanisms can only be speculated.     

Apoptotic cytotoxic effects of a histone deacetylase inhibitor, FK228, on malignant lymphoid cells.

Histone deacetylases are promising targets for cancer treatment. Here we studied the in vitro effects of a potent histone deacetylase inhibitor, FK228 (formerly FR901228), on human leukemia / lymphoma cells and cell lines compared with normal hematopoietic cells. In a lymphoma cell line, Raji, a nanomolar concentration of FK228 induced G1 arrest and / or apoptotic cell death, depending on the concentration and exposure time. Growth of lymphoid cell lines including Raji (N = 13) was inhibited by 50% (IC(50)) after 2-day treatment at concentrations of 0.83 to 1.87 ng / ml. Viability of clinical samples from patients with acute lymphoblastic leukemia was decreased by 50% at 0.78 +/- 0.46 ng / ml, whereas the IC(50) values for normal mononuclear cells from peripheral blood and bone marrow were 2.3 +/- 0.96 and 7.8 +/- 1.0 ng / ml, respectively. The IC(50) values for normal progenitor cells were 3.1, 4.4 and 7.8 ng / ml for BFU-E, CFU-GM and CFU-Mix, respectively. Expression levels of HDAC-1 and HDAC-3 proteins, which varied among cell lines, but were stable during the treatment with FK228, did not correlate with the sensitivity to FK288. This novel agent might be useful in the treatment of lymphoid malignancies, because the above concentrations are clinically achievable in vivo according to a recent clinical study.