Recombinant interferon-gamma inhibits the growth of IL-6-dependent human multiple myeloma cell lines in vitro.

Recombinant human IFN-gamma (100-1000 U/ml) inhibited the IL-6-induced growth of 2 human IL-6-dependent multiple myeloma (MM) cell lines U-1958 and U-266-1970 in vitro. In contrast, the U-1996 line, independent of IL-6 for maintenance at a slow growth rate but responding to IL-6 by increased proliferation, and the IL-6-independent U-266-1984 were refractory to the anti-proliferative effect of IFN-gamma. The effect of IFN-gamma in the sensitive MM cell lines was cytostatic in U-266-1970, and cytostatic and cytotoxic in U-1958. Northern blot analysis revealed that the growth inhibition of the IL-6-dependent MM cell line U-1958 was not due to down-regulation of IL-6 receptor mRNA expression and that the differential sensitivity to IFN-gamma was not due to differences in IFN-gamma receptor expression. The growth inhibition was not a consequence of an IFN-gamma-induced terminal differentiation as flow cytometric analyses demonstrated an arrest in all phases of the cell cycle. IFN-alpha inhibited the growth in 3 of the 4 cell lines tested. The results thus suggest that the particular MM phenotype, which includes IL-6 dependency for survival and growth, may also be characterized by IFN-gamma sensitivity. Furthermore, the study demonstrates that MM cell lines are not simultaneously sensitive to IFN-gamma and alpha, indicating that the mechanisms of action of the two types of IFN are distinct.     

Lentivirus-delivered ZEB-1 small interfering RNA inhibits lung adenocarcinoma cell growth in vitro and in vivo

Purpose

To explore the relationship between the expression of ZEB1 gene and the proliferation ability of lung adenocarcinoma cells.

Methods

Immunohistochemistry, Western blot and Real-time PCR were used to detect the expression of ZEB1 gene in lung adenocarcinoma tissue and cell lines compared with adjacent noncancerous region and the human lung fibroblast cell HLF cells. The lentivirus RNA interference technique was used to knock down the expression of ZEB1 in lung adenocarcinoma A549 and H1299 cell lines. Cell cycle and cell apoptosis were measured by FCM assay. In vivo, four groups of 4-week-old nude mice were subcutaneously injected with the stably transfected (ZEB-si, scr-si) cells at a single site to investigate the effect of ZEB1-siRNA in the nude mice tumor growth. In situ apoptosis was detection by TUNEL assay.

Results

ZEB-1 was highly expressed in lung adenocarcinoma tissue and cell lines compared with adjacent noncancerous region and the human lung fibroblast cell HLF cells. ZEB1-siRNA could decrease lung adenocarcinoma cell proliferation by delaying S-phase entry and induce cell apoptosis, which led to the inhibition of the tumorigenicity of A549 and H1299 cell lines. Further investigation showed that injecting the ZEB1-siRNA cells into the nude mice could significantly decrease the tumor growth.

Conclusion

Knockdown of ZEB-1 expression by lentivirus-delivered siRNA may provide a novel therapeutic target for the treatment of lung cancer.     

Paeonol inhibits tumor growth in gastric cancer in vitro and in vivo

AIM:To investigate the anti-tumor effects of paeonol in gastric cancer cell proliferation and apoptosis in vitro and in vivo.METHODS:Murine gastric cancer cell line mouse forestomach carcinoma(MFC) or human gastric cancer cell line SGC-7901 was cultured in the presence or absence of paeonol.Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay,and cell cycle and apoptosis by flow cytometry and TUNEL staining.Tumor growth after subcutaneous implantation of MF...     

Troglitazone inhibits tumor growth in hepatocellular carcinoma in vitro and in vivo

Peroxisome proliferator-activated receptor gamma (PPARgamma) has been implicated in the differentiation and growth inhibition of cancer cells. We examined the effects of PPARgamma activation by troglitazone on hepatocellular carcinoma (HCC) cell growth, proliferation, and apoptosis in vitro and in vivo. We also studied relationships between PPARgamma activation and cyclooxygenase-2 (COX-2) expression. Human HCC cell lines Huh7 and Hep3B were cultured in the presence or absence of troglitazone. Cell growth was determined via WST-1 assay, proliferation by cell cycle analysis and proliferating cell nuclear antigen (PCNA) Western blotting, and apoptosis by flow cytometry and TUNEL. Tumor growth after subcutaneous implantation of Huh7 cells in nude mice was monitored, and the effects of treatment with troglitazone were determined. In resected HCCs, PPARgamma expression was less compared with the histologically normal surrounding liver. In cultures of Hep3B and Huh7 cells, basal expression of PPARgamma was relatively low, but troglitazone caused dose-dependent induction of PPARgamma expression. Cell cycle analysis revealed a decreased proportion of cells in S phase, with arrest at G0/G1. Concomitant downregulation of PCNA and an increase in TUNEL staining, cells were consistent with decreased proliferation and induction of apoptosis by troglitazaone. Troglitazone-mediated PPARgamma activation also suppressed COX-2 expression and induced p27 in HCC cells. Administration of troglitazone to Huh7 tumor-bearing mice significantly reduced tumor growth and caused tumor regression. In conclusion, collectively, these results indicate that PPARgamma could be a regulator of cell survival and growth in HCC. PPARgamma therefore represents a putative molecular target for chemopreventive therapy or inhibition of liver cancer growth.     

RNA干扰对肝素酶在肝癌细胞中表达及其肿瘤生长抑制的作用

目的:采用RNA干扰(RNAi)技术抑制肝癌细胞系HepG2肝素酶(Hpa)的表达,体内观察其对肿瘤生长的抑制作用.方法:以脂质体介导的方法将构建的2个抗Hpa短发卡式RNA(Hpa-shRNA)真核表达载体稳定转染人肝癌细胞株HepG2(高表达Hpa),并设空载体对照组;流式细胞技术鉴定转染稳定性,Western blot及RT-PCR检测肝素酶蛋白和mRNA表达;将干扰组和对照组的肿瘤细胞皮下接种裸鼠,比较各组肿瘤的体质量和体积大小,免疫组化法检测肝素酶在肿瘤组织的表达.结果:与空载体组相比,干扰后的HepG2细胞中Hpa mRNA和蛋白表达明显降低,干扰组裸鼠的肿瘤生长速度明显变慢,统计结果表明干扰组在抑制肿瘤生长方面与空载体组、空白对照组存在显著差异(P＜0.05),两干扰组无显著差异(P＞0.05).结论:应用RNAi技术沉默Hpa基因可以有效下调肝癌肿瘤细胞中Hpa mRNA及蛋白的表达,抑制体内肿瘤生长.     

重组腺相关病毒介导的RNA干扰抑制血管生长素表达对肺腺癌细胞成瘤能力的影响

目的 通过腺相关病毒(AAV)介导的RNA干扰(RNAi)抑制肺腺癌细胞A549血管生长素(ANG)表达,观察其对癌细胞生长和成瘤能力的影响.方法 构建H1启动子驱动的表达针对ANG的小干扰RNA(siRNA)重组腺相关病毒(AAV-shANG),转染A549细胞,同时以正常A549细胞以及转染AAV-Null的A549细胞作为对照,观察重组腺相关病毒介导的RNAi抑制ANG表达对A549细胞生长、致瘤、肿瘤细胞增殖、凋亡及肿瘤微血管密度的影响.用t检验分析各组间差别,所有数据均用x±s表示.结果 体外实验结果表明重组腺相关病毒AAV-shANG成功构建,重组腺相关病毒转染A549细胞72 h后ANG蛋白表达水平明显低于A549细胞组及AAV-Null组;转基因A549细胞细胞周期分析结果提示,正常A549细胞、AAV-Null转染细胞和AAV-shANG转染细胞的增殖指数(PI)分别为0.32±0.29、0.35±0.38和0.31±0.43,差异不明显.体内实验结果表明,AAV-shANG转染细胞组胸腺缺陷小鼠的成瘤体积、瘤质最均明显低于两对照组.各组微血管密度分别为9.4±1.5、9.8±2.1和5.7±1.9,提示AAV-shANG转染细胞组与正常A549细胞和AAV-Null转染细胞组有明显差异.各组凋亡细胞的百分率分别为(7.7±3.1)%、(8.5±5.4)%和(17.1±8.6)%.AAV-shANG转染细胞组凋亡细胞的百分率明显高于正常A549细胞组和AAV-Null转染细胞组,各组细胞增殖核抗原(PCNA)阳性表达率分别为(84.8±9.7)%、(85.8±9.8)%、(70.4±10.1)%,AAV-shANG转染细胞组PCNA表达率低于两对照组.结论 AVV-siRNA表达能显著抑制肿瘤细胞ANG表达及肿瘤细胞增殖,促进肿瘤细胞凋亡,抑制肿瘤生长.     

重组人生长激素对大鼠骨髓间质细胞作用的实验研究

目的 探讨重组人生长激素(rhGH)在体外对骨髓间质细胞(MSCs)的作用及机制.方法 四甲基偶氮唑盐微量酶反应比色法测定不同浓度、不同时间点rhGH对MSCs作用的光密度值;逆转录-聚合酶链反应测定rhGH作用及去除rhGH作用后胰岛素样生长因子1(IGF-1)mRNA的表达.结果 rhGH在10 μg/L浓度以上对MSCs有促生长作用,最佳浓度为200 μg/L,生长率为144.74%;200 μg/L的rhGH在24 h始促MSCs生长,随时间延长逐渐增强,72 h生长率为144.10%.IGF-1mRNA含量在rhGH作用24、48和72 h逐渐增加,去除rhGH后IGF-1 mRNA表达虽有所减少,但仍随时间延长逐渐增加,2周达高峰.结论 rhGH在体外可促进MSCs生长,其最适浓度为200 μg/L;IGF-1介导是rhGH促MSCs生长的途径之一.     

腺病毒介导的肿瘤生长抑制因子4和白细胞介素-24双基因共表达对肺腺癌放疗增敏的体内外实验研究

目的 研究肿瘤生长抑制因子4 (ING4)和白细胞介素-24 (IL-24) 双基因共表达腺病毒载体对肺腺癌细胞SPC-A-1的体内外放疗增敏作用及其潜在的作用机制.方法 用Western blot法检测ING4和IL-24在SPC-A-1细胞中的表达;四甲基偶氮唑盐比色(MTT)法和流式细胞仪分别检测Ad-ING4-IL-24联合放疗对SPC-A-1肺腺癌细胞的生长抑制作用和促凋亡作用.应用抽签法将25只裸鼠随机均分为5组:PBS组、腺病毒(Ad)组、双基因组、放疗组及联合组,除放疗组外其余各组均采用瘤体内注射干预用药,隔日1次,共注射6次.第1次治疗前及开始治疗后隔日测量各组瘤体的长径(L)和短径(W),计算瘤体体积(V=L×W2/2),绘制瘤体体积-时间变化曲线.采用 SPC-A-1细胞株建立肺腺癌裸鼠模型,观察Ad-ING4-IL-24联合放疗对移植瘤的抑制作用.免疫组织化学法检测天冬氨酸特异性半胱氨酸蛋白酶-3(Caspase-3)、B细胞淋巴瘤/白血病-2 (Bcl-2)、Bcl-2相关X蛋白(Bax)及血管内皮细胞生长因子(VEGF)基因的表达.采用金正均法计算q值并判断其放疗增敏作用.结果 Western blot法检测结果显示,目的 基因在SPC-A-1细胞中成功表达;MTT和流式细胞仪检测结果显示,Ad-ING4-IL-24联合放疗组对SPC-A-1细胞的生长抑制和促细胞凋亡作用[(86.2±0.8)%、(60.9±1.0)%]明显高于Ad-ING4-IL-24双基因组[(49.8±0.3)%、(26.3±1.3)%]和放疗组[(44.4±2.2)%、(33.3±0.8)%](生长抑制率比较,F=550.88,P＜0.01;凋亡率比较,F=614.08,P＜0.01),Ad-ING4-IL-24联合放疗具有放疗增敏协同作用(q=1.20);裸鼠体内瘤重抑瘤率Ad-ING4-IL-24组、放疗组及Ad-ING4-IL-24联合放疗组分别为49.5%(样本5只)、35.4%(样本5只)和79.8%(样本5只),Ad-ING4-IL-24联合放疗能明显抑制移植瘤生长,具有放疗增敏协同作用(q=1.18);免疫组织化学法检测结果显示,Ad-ING4-IL-24联合放疗可明显上调Bax和Caspase-3基因表达,下调Bcl-2及VEGF基因表达.结论 Ad-ING4-IL-24具有放疗增敏作用,是理想的放疗增敏剂,其作用机制可能与诱导肿瘤细胞凋亡和抑制血管形成有关.

Abstract：

Objective To study the radiosensitivity of the recombinant adenoviral vector (called Ad-ING4-IL-24) carrying and co-expressing inhibitor of growth 4 (ING4) and interleukin-24 (IL-24) to human lung adenocarcinoma and the underlying mechanisms. Methods The expression levels of ING4 and IL-24 were detected by Western blot. The growth-suppressing and apoptosis-inducing effect of Ad-ING4-IL-24 combined with radiotherapy on SPC-A-1 lung carcinoma cells were assessed by MTT assay and FCM respectively. The 25 nude mice were randomly divided into 5 groups of 5 mice ecah: PBS group,Ad group,Ad-ING4-IL-24 group,radiotherapy group and joint group (Ad-ING4-IL-24 combined radiotherapy). Mice in all groups except radiotherapy group were intratumorally injected every other day for 6 cycles. The short and long axes of the tumor were measured dynamically, tumor volume was calculated as: V=L×W2/2, changes in tumor volume were graphed. The human lung carcinoma model was established with SPC-A-1 cells in nude mice. The ratios of tumor-suppression and q were calculated. The expression of Caspase-3, Bcl-2, Bax, VEGF in tumor samples were detected by immunohistochemistry. Results The expressions of ING4 and IL-24 were successfully expressed in SPC-A-1 cells. MTT assay and FCM showed that the levels of cell-growth inhibition and apoptosis induction in Ad-ING4-IL-24 combined with radiotherapy group [(86.2±0.8)%,(60.9±1.0)%] were higher than in Ad-ING4-IL-24 group [(49.8±0.3)%,(26.3±1.3)%] and in radiotherapy group [(44.4±2.2)%,(33.3±0.8)%] (ratio of cell-growth inhibition, F=550.88,P＜0.01; ratio of induced apoptosis F=614.08,P＜0.01). Ad-ING4-IL-24 combined with radiotherapy showed an enhanced radiosensitivity effect on human lung adenocarcinoma(q=1.20). In Ad-ING4-IL-24 group, radiotherapy group and Ad-ING4-IL-24 combined with radiotherapy group, the weight inhibition ratio was 49.5% (5 nude mice), 35.4%(5 nude mice), 79.8%(5 nude mice) respectively. Ad-ING4-IL-24 combined with radiotherapy had a synergetic and enhanced radiosensitivity effect on inhibiting the growth of transplanted tumor(q=1.18). According to immunohistochemistry, Ad-ING4-IL-24 was shown to up-regulate the expression of Bax and Caspase-3 but down-regulate the expression of Bcl-2 and VEGF. Conclusion Ad-ING4-IL-24 had an enhanced radiosensitivity effect on human lung adenocarcinoma, and therefore acted as a radiotherapy sensitizer, which may be related to its effect on apoptosis-induction and antiangiogenesis.     

In vitro chemosensitivity of human lung cancer cell lines

Human lung cancer cell lines, established from patients with both small cell cancer (SCLC) and non-small cell cancer (non-SCLC; squamous cell, large cell, anaplastic, and adenocarcinoma), were tested for their in vitro chemosensitivity to a panel of drugs. Drug sensitivity was assayed by either soft agar clonogenicity or a novel dye-exclusion assay. Eleven non-SCLC lines (eight continuous, three recently cultured) and five SCLC lines (all continuous) were tested. Four of eight continuous non-SCLC lines cloned sufficiently to permit limited in vitro drug testing, as did two of the five SCLC lines. All 16 cell lines could be tested for multiple drugs using the dye-exclusion assay. Drug concentrations for the nonclonogenic assay more closely approximated the area under the concentration-time curve for a given concentration of each agent. There was considerable variation in the relative sensitivity of the cell lines and the patterns of individual drug sensitivity. The majority of non-SCLC cell lines were refractory to most drugs. Cell lines derived from two previously treated SCLC patients and from three untreated SCLC patients showed greater sensitivity. Concurrent clonogenic and dye-exclusion assays showed similar drug rankings but different absolute values for percent survival. The nonclonogenic dye-exclusion assay is more rapid than the soft agar clonogenic assay (4 days vs. 2-3 weeks), could be performed on all cell lines tested, and appears to reflect the clinical diversity of human lung cancer.     

Hematoporphyrin derivative‐mediated photodynamic therapy inhibits tumor growth in human cholangiocarcinoma in vitro and in vivo

Aim: To investigate the effects of hematoporphyrin derivative‐mediated photodynamic therapy (HPD‐PDT) on cell growth in human cholangiocarcinoma in vitro and in vivo, as well as the underlying mechanisms of these effects. Methods: 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay was used to evaluate growth status of human cholangiocarcinoma cell line (QBC939). Hoechst 33258 staining and flow cytometry assays were applied to determine cell apoptosis. Western blotting analysis was performed to detect the release of cytochrome c in QBC939 cells, and caspases enzymatic assay was used to investigate the activation of caspase‐3, ‐8, and ‐9. Further, tumor growth after subcutaneous implantation of QBC939 cells in nude mice was monitored. Results: HPD‐PDT inhibits QBC939 cell growth via cell apoptosis in vitro, and initiates cell mitochondria apoptosis pathway by the release of cytochrome c and the activation of caspase‐9 and ‐3. Moreover, HPD‐PDT also inhibits subcutaneous tumor growth of QBC939 cells and reduces tumor cell mitosis in nude mice. Conclusion: HPD‐PDT inhibits tumor growth of human cholangiocarcinoma, suggesting that HPD‐PDT is useful in cholangiocarcinoma therapy.     

Anti-CD19 inhibits the growth of human B-cell tumor lines in vitro and of Daudi cells in SCID mice by inducing cell cycle arrest

In this report, we extend our previous findings that IgG or F(ab')2 fragments of HD37 anti-CD19 antibody (Ab) in combination with the immunotoxin (IT), RFB4-anti-CD22-deglycosylated ricin A chain (dgA) (but neither reagent alone), prolonged the survival of SCID mice with disseminated human Daudi lymphoma (SCID/Daudi mice) to 1 year at which time they still remained tumor-free. We explored the mechanisms by which the HD37 Ab exerts antitumor activity in vivo by studying its activity in vitro. We found that it has antiproliferative activity (IC50 = 5.2 - 9.8 x 10(-7) mol/L) on three CD19+ Burkitt's lymphoma cell lines (Daudi, Raji, and Namalwa) but not on a weakly CD19-positive (CD19lo) pre-B cell tumor (Nalm-6). The inhibitory effect was manifested by cell cycle arrest, but not apoptosis. Results using three additional anti-CD19 Abs, suggest that the affinity of the antibody and possibly the epitope which it recognizes may effect its capacity to transmit a signal that induces cell cycle arrest. Hence, therapeutically useful Abs may exert anti-tumor activity by a variety of mechanisms, each of which should be evaluated before undertaking clinical trials in humans.     

Effects of interleukin-4 on the in vitro growth of human lymphoid and plasma cell neoplasms

Recombinant human interleukin-4 (rhIL-4) has pleiotropic biologic effects including stimulation of normal B-cell growth. We investigated the effects of rhIL-4 on the in vitro growth of 35 fresh human lymphoid and plasma cell malignancies. Inhibition of growth was seen in 52% and 60% of multiple myeloma and lymphoma specimens, respectively. Growth was stimulated relative to control in 8.6% (3 of 35) of the tumors tested. Immunophenotyping showed that tumors displaying growth stimulation expressed surface markers associated with a poor clinical prognosis. Our findings support the clinical evaluation of rhIL-4 as a therapeutic agent in selected patients with lymphoid and plasma cell neoplasms.     

Monoclonal antibody to transferrin receptor blocks transferrin binding and inhibits human tumor cell growth in vitro.

A murine hybridoma has been obtained that produces a monoclonal antibody against the human transferrin receptor. In contrast to previously characterized monoclonal antibodies that recognize the transferrin receptor, this antibody, designated 42/6, blocks the binding of transferrin to its receptor and inhibits the growth of the human T leukemic cell line, CCRF-CEM, in vitro. Inhibition of cell growth was dose dependent, and as little as 2.5 micrograms of purified antibody per ml had a detectable effect, even though transferrin was present in the tissue culture medium in large molar excess. Cells grown in the presence of antibody for 7 days accumulated in S phase of the cell cycle. The addition of iron to antibody-treated cultures in the form of ferric complexes or ferrous sulfate did not overcome the growth inhibitory effects of the anti-transferrin-receptor antibodies. This result suggests that either transferrin is the only means by which CCRF-CEM leukemic cells can be provided with sufficient iron in vitro or that other factors in addition to iron starvation are involved in the antibody-mediated growth inhibition. The inhibition of cell growth by 42/6 monoclonal antibody suggests that monoclonal antibodies against proliferation-associated cell surface antigens, such as the transferrin receptor, may be useful pharmacological reagents to modify cell growth in vitro.     

Recombinant human nerve growth factor with a marked activity in vitro and in vivo

Recombinant human nerve growth factor (rhNGF) is regarded as the most promising therapy for neurodegeneration of the central and peripheral nervous systems as well as for several other pathological conditions involving the immune system. However, rhNGF is not commercially available as a drug. In this work, we provide data about the production on a laboratory scale of large amounts of a rhNGF that was shown to possess in vivo biochemical, morphological, and pharmacological effects that are comparable with the murine NGF (mNGF), with no apparent side effects, such as allodynia. Our rhNGF was produced by using conventional recombinant DNA technologies combined with a biotechnological approach for high-density culture of mammalian cells, which yielded a production of approximately 21.5 +/- 2.9 mg/liter recombinant protein. The rhNGF-producing cells were thoroughly characterized, and the purified rhNGF was shown to possess a specific activity comparable with that of the 2.5S mNGF by means of biochemical, immunological, and morphological in vitro studies. This work describes the production on a laboratory scale of high levels of a rhNGF with in vitro and, more important, in vivo biological activity equivalent to the native murine protein.     

Cimetidine inhibits in vivo growth of human colon cancer and reverses histamine stimulated in vitro and in vivo growth.

The effect of histamine and cimetidine on the growth of four human colon cancer cell lines was studied. Histamine significantly stimulated the uptake of tritiated thymidine in vitro in a dose dependent manner, to a maximum of 120% and 116% of controls for C170 and LIM2412, respectively. This effect was antagonised by cimetidine, but not diphenhydramine. Histamine also stimulated a dose dependent increase in cyclic adenosine monophosphate accumulation in C170 cells, antagonised by cimetidine. When grown as subcutaneous xenografts in Balb/c nu/nu mice, cimetidine had a significant inhibitory effect on the same two cell lines. The final volume of C170 tumours in animals given cimetidine was 44% of controls. This response was dose dependent, plateauing at a cimetidine dose of 50 mg/kg/day. The final volume of LIM2412 tumours in animals given cimetidine was 60% of controls. Histamine administered locally by a mini-osmotic pump stimulated C170 tumour growth to 164% of controls, was antagonised by cimetidine at a dose of 200 mg/kg/day, but not by lower concentrations. Histamine has a trophic effect on at least two colorectal cancer cell lines in vivo and in vitro. As this effect is antagonised by cimetidine, it may be mediated via tumour histamine type 2 receptors.     

Vasoactive intestinal peptide inhibits human small-cell lung cancer proliferation in vitro and in vivo

Small-cell lung carcinoma (SCLC) is an aggressive, rapidly growing and metastasizing, and highly fatal neoplasm. We report that vasoactive intestinal peptide inhibits the proliferation of SCLC cells in culture and dramatically suppresses the growth of SCLC tumor-cell implants in athymic nude mice. In both cases, the inhibition was mediated apparently by a cAMP-dependent mechanism, because the inhibition was enhanced by the adenylate cyclase activator forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine in proportion to increases in intracellular cAMP levels, and the inhibition was abolished by selective inhibition of cAMP-dependent protein kinase. If confirmed in clinical trials, this antiproliferative action of vasoactive intestinal peptide may offer a new and promising means of suppressing SCLC in human subjects, without the toxic side effects of chemotherapeutic agents.     

An endothelin receptor B antagonist inhibits growth and induces cell death in human melanoma cells in vitro and in vivo

Activation of the endothelin receptor B (ETRB) in cultured melanocyte precursors promotes cell proliferation while inhibiting differentiation, two hallmarks of malignant transformation. We therefore tested whether ETRB has a similar role in malignant transformation of melanoma. When tested in culture, we find that the selective ETRB antagonist BQ788 can inhibit the growth of seven human melanoma cell lines, but not a human kidney cell line. This inhibition often is associated with increases in pigmentation and in the dendritic shape that is characteristic of mature melanocytes. In three cell lines we also observe a major increase in cell death. In contrast, the endothelin receptor A (ETRA) antagonist BQ123 does not have these effects, although all the cell lines express both ETRA and ETRB mRNA. Extending these studies in vivo, we find that administration of BQ788 significantly slows human melanoma tumor growth in nude mice, including a complete growth arrest in half of the mice treated systemically. Histological examination of tumor sections suggests that BQ788 also enhances melanoma cell death in vivo. Thus, ETRB inhibitors may be beneficial for the treatment of melanoma.     

In vitro growth inhibition of a broad spectrum of tumor cell lines by activated human dendritic cells

Dendritic cells (DCs) are critical subsets of leukocytes providing antigen presentation for initiation of humoral and cellular immune responses. Their role as effector cells in tumor resistance, however, is less known. We report here that human DCs generated by culturing plastic-adherent peripheral blood monocytes in the presence of granulocyte-monocyte colony-stimulating factor (GM-CSF) and interleukin-4 have potent growth-inhibition activity in vitro on a wide spectrum of human tumor lines of different tissue origin. Proinflammatory stimuli lipopolysaccharide (LPS) and interferon-gamma, but not tumor necrosis factor-alpha and CD40 signaling, can further enhance DC-mediated inhibition of tumor growth. The growth inhibition requires contact between DCs and tumor cells while LPS treatment enhances the antitumor activity in DC culture supernatants. Our results suggest that in addition to their predominant role as regulatory cells, activated DCs are also potential effector cells in tumor immunity.     

Recombinant human interleukin-4 inhibits the production of granulocyte-macrophage colony stimulating factor by blood mononuclear cells

Summary. The effect of recombinant human (rh) interleukin-4 (rIL-4) on human blood BFU-E was investigated using two populations of cells: platelet-depleted low-density mononuclear cells (FH, Pl⁻ cells), as unpurified cells, and highly purified BFU-E. When FH, Pl⁻ cells were cultured with rherythropoietin (rEp), rIL-4 inhibited BFU-E growth in a dose-dependent manner. However, the addition of rIL-4 did not affect rh-interleukin-3 (rIL-3) supported BFU-E growth. Limiting dilution analysis (LDA) of FH, Pl⁻ cells showed that rIL-4 suppressed endogenous production of burst-promoting activity (BPA) by accessory cells. Highly purified BFU-E were used as target cells to measure BPA in the conditioned medium (CM) that was prepared by FH, Pl⁻ cells. When 100 purified BFU-E were cultured in 0·5 ml clots with 20% (vol/vol) of the CM, the number of BFU-E colonies was increased by the CM. The increase was significantly reduced by the addition of the CM prepared in the presence of rIL-4, but anti-IL-4 blocked the effect of rIL-4. The concentration of IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) in CM was determined by an enzyme-linked immunoadsorbent assay (ELISA). The spontaneous production of GM-CSF but not IL-3 was detected, and this was significantly decreased in the presence of rIL-4. Anti-GM-CSF but not anti-IL-3 inhibited CM supported BFU-E growth, indicating that the main BPA in the CM is GM-CSF and that rIL-4 suppresses the spontaneous production of GM-CSF by accessory cells. From these studies, we conclude that rIL-4 has a unique mechanism as a negative regulator on erythropoiesis through the inhibition of BPA production by blood mononuclear accessory cells.