Neuronal differentiation in human neuroblastoma cells by nerve growth factor following TrkA up-regulation by interferon-gamma

BACKGROUND: TrkA mRNA expression has been reported to be related to favorable outcome of neuroblastoma (NB). Previously, we found that interferon-gamma (IFN-gamma) can enhance TrkA mRNA expression in NB cell lines. In the present study, we examined the effect of nerve growth factor (NGF) on IFN-gamma-induced TrkA protein to clarify the relationship between TrkA and cell differentiation of NB. PROCEDURE: The effect of IFN-gamma on the TrkA mRNA expression was screened in six human NB cell lines and a freshly prepared sample, SK-rib, from a stage IV patient. Using two of them, we examined their morphological change during simultaneous loading of NGF and IFN-gamma. Tyrosine phosphorylation pattern after 5 min of NGF stimulation was also examined in immunoblot analysis with anti-gp140(trkA) antibody and antiphospho tyrosine antibody. RESULTS: After a 4-day treatment with 500 IU/ml IFN-gamma, TrkA mRNA increased in five cell lines and SK-rib cells in association with growth inhibition. Although the degree of morphological differentiation did not increase in proportion to the TrkA expression induced by IFN-gamma, continuous loading of both IFN-gamma and NGF caused marked morphological differentiation in a cultured KP-N-RT cell line and SK-rib cells during 10 days. Moreover, 5 min of NGF stimulation after IFN-gamma treatment caused the phosphorylation of TrkA protein and downstream proteins. CONCLUSIONS: IFN-gamma could induce the functional NGF receptor even in the aggressive phenotype of NB.     

Inhibition of normal human granulopoiesis by neuroblastoma cells

In vitro inhibition of marrow granulopoiesis was produced by a well-characterized human neuroblastoma cell line (CHP 134). A standard double layer, semi-solid agar system was employed in the experiments. The inhibition was present whether the neuroblastoma cells were mixed with the marrow cells or whether they were separated in a contiguous agar layer. Irradiation of the neuroblastoma cells lessened the inhibitory effect but did not eradicate it. Medium conditioned by the neuroblastoma cells had a mild, but not statistically significant, suppressive effect upon granulopoiesis. Additional studies to define the precise mechanism of suppression are underway.     

Continuous infusion of recombinant interleukin-2 and lymphokine-activated killer cells in refractory malignancies

Adoptive immunotherapy with recombinant interleukin-2 (rIL-2) and lymphokine-activated killer (LAK) cells has been reported to effect the regression of tumor in patients with a variety of malignancies. Responses have occurred in patients treated with high-dose bolus rIL2 as well as lower-dose continuous intravenous infusion. Ten patients who had been extensively pretreated with systemic chemotherapy with or without additional radiation therapy were treated with continuous infusion rIL2 and LAK cells. Five patients received rIL2 alone for 96-120 hours prior to leukapheresis in addition to rIL2 at the time of LAK cell infusion. Three patients received LAK cells that had been cultured for 14 days in an automated tissue culture system. No responses were seen in this ten-patient cohort. While the lack of response in these patients may be related to any one or more of several variables, patients who have been heavily pretreated may not respond as well as patients who have received little to no systemic chemotherapy.     

Distribution of natural antibody against human neuroblastoma among children with or without neuroblastoma

BACKGROUND: This study was conducted to examine the distribution of a natural antibody against neuroblastoma (NB) among Japanese children and to clarify the clinical significance of the presence of this antibody in the sera during treatment in patients with International Neuroblastoma Staging System Stage 4 NB. PROCEDURE: Human sera were obtained from 8 healthy volunteers, 82 patients with non-malignant surgical diseases, and 35 patients with NB including 3 with Stage 1 disease, 6 with Stage 2, 7 with Stage 3, 17 with Stage 4, and 2 with Stage 4S. This natural antibody was quantified by flow cytometry and its anti-tumor activity was measured by complement-dependent cytotoxicity (CDC) using TGW cells, a human NB cell line, as the target. RESULTS: IgM antibody and CDC activity against NB could be detectetd in all sera from healthy volunteers and from patients with non-malignant surgical dis eases who were age >1 year. The amount of IgM antibody and CDC activity in sera from patients with Stage 4 NB at diagnosis consistently was low, most likely because of massive absorption by tumor cells. In this group of patients, the increased CDC activity detected during treatment was indicative of a favorable factor for survival. CONCLUSIONS: A natural antibody against NB appears to exist in the sera of Japanese children. The sequential assessment of the levels of this antibody in the sera from Stage 4 NB patients during treatment may serve as a prognostic indicator.     

Natural killer cell immunodeficiency in siblings: defective killing in the absence of natural killer cytotoxic factor activity in natural killer and lymphokine-activated killer cytotoxicities

A immunodeficiency of natural killer cells as effectors for natural killer and lymphokine-activated killer cytotoxicities was first demonstrated in siblings. Two of three male siblings persistently lacked natural killer activity against K562 target cells as assayed by a 51Cr-release assay: percent lysis values were less than 1.0% as compared to the normal lymphocyte values of 43.5% +/- 6.2% (mean +/- SD). Their lymphocytes did not develop natural killer cell activity by changing effector to target ratios, prolonging the incubation time, or stimulating them with interferon-alpha or interleukin 2. Numbers of lymphocytes bearing Leu-7, CD16, or NKH-1 were normal but those of Leu-7-, CD16+ cells were decreased as estimated by flow cytometry. Single cell-in-agarose assays showed normal numbers of natural killer cells capable of binding to a target cell but incapable of killing it. They had depressed levels of lymphokine-activated killer activity, which was totally eliminated by the treatment with OKT3 and complement. This result indicates that the patients' natural killer cells are also defective in the capacity to work as effectors for lymphokine-activated killer activity. The patients' natural killer cells did not produce natural killer cytotoxic factor activity. Antibody-dependent cellular cytotoxicity and cytotoxic T lymphocyte cytotoxicity were normal. These results demonstrate a selective natural killer cell deficiency as effectors for natural killer and lymphokine-activated killer cytotoxicities with a familial tendency, in which there is defective killing with the absence of natural killer cytotoxic factor activity.     

Cytokine-stimulated human natural killer cytotoxicity: response to rotavirus-infected cells

The ability of rotavirus-infected cells to stimulate leukocytes to release a cytokine which enhanced the subsequent leukocyte cytotoxicity to a second set of [51Cr] labeled rotavirus-infected cells was analyzed. Human interferon increased leukocyte cytotoxicity to Simian rotavirus (SA-11)-infected target cells. Similarly, 11 of 12 supernates of SA-11-stimulated peripheral blood leukocyte cultures increased the killing of SA-11-infected cells (P less than 0.005). This resulted in a calculated cytokine-dependent cellular cytotoxicity value of 9.6 +/- 1.9%. Three of five of the supernates tested contained measurable levels of interferon (12-48 unit/ml). In contrast, SA-11-stimulated colostral leukocyte culture supernates neither increased leukocyte cytotoxicity nor contained measurable levels of interferon.     

Recognition of an in vivo immune response to human neuroblastoma modulation of antigen expression by retinoic acid

Neuroblastoma is one of the most common solid tumors of childhood and is notable for its ability to spontaneously regress and, in some instances, to differentiate to less malignant ganglioneuromas. Since immune mechanisms may account for these phenomena, identification of in vivo immune responses to tumor cell surface antigens may be important to the progression of the disease. As determined by analysis on the fluorescence-activated cell sorter, sera from 10 of 18 neuroblastomas patients were found to contain antibodies to a cell surface antigen present on subpopulations of cells from human neuroblastoma cell lines maintained in vitro. Eight human neuroblastoma cell lines were examined and found to vary in reactivity with sera. Induction of differentiation of cell lines with retinoic acid (RA) in vitro resulted in most cell lines bearing higher percentages of positive cells but with a decreased mean cell fluorescence. Preliminary Western blot analysis of lysates of the human cell lines NMB/N7, SMS-KAN, and SK-N-MC showed two principal antigen bands on reducing gels. Comparison of sera from different individuals on lysates of cell lines showed reactivity principally with bands of 105-110 kD and 65-70 kD and an additional minor band of slightly lower molecular weight with the higher titer sera. The ability of different sera to recognize a common antigen pattern suggests that this represents an immunodominant cell surface antigen. Examination of reactivity of other cell lines in this system showed that positive sera reacted with all neuroblastoma lines examined, one neuroepithelioma (SK-N-MC), two melanoma lines (MeWo, G361), and one adrenal-derived adenocarcinoma (SW-13).     

ATRA诱导神经母细胞瘤SMS-KCNR分化与N-myc癌基因表达

目的 观察全反式维甲酸（ＡＴＲＡ）诱导神经母细胞瘤（ＮＢ）细胞分化时Ｎ－ｍｙｃ癌基因的变化以及与时间的关系。方法 用不同浓度的ＡＴＲＡ处理ＮＢ细胞系ＳＭＳ－ＫＣＮＲ,用相差显微镜观察细胞形态学改变。采用Ｎｏｒｔｈｅｒｎ印迹杂交方法检测ＮＢ细胞分化前后Ｎ－ｍｙｃ癌基因的ｍＲＮＡ表达水平。结果 用１０＾－６ｍｏｌ／Ｌ的ＡＴＲＡ处理２４～１２０ｈ,ＮＢ细胞的胞体减小,神经突起延长。在加ＡＴＲＡ２４ｈ后,     

Etoposide toxicity on human neuroblastoma cells in vitro is enhanced by preceeding hyperthermia

BACKGROUND: Thermal enhancement has been proven in vitro for the cytotoxic effect of alkylants and platinum compounds, not, however, for etoposide, which acts synergistically to these drugs. PROCEDURE: Our in vitro study on a neuroblastoma cell line confirmed previous results in other tumor models that the cytotoxicity of etoposide (12.8% as compared to untreated controls) is not enhanced by simultaneous heating to 40 or 42 degrees C for 1 hr (11.9%), as jugded by colony forming assay. RESULTS: The same temperature applied 24 hr before the drug resulted in a significant decrease of colonies (6.1%). Double treatment with etoposide within a 24-hr-interval yielded a similar result (5.6%). The colony number could be further decreased by adding hyperthermia 24 hr before the second treatment (1.3%). CONCLUSIONS: We demonstrate in vitro that the enhancing effect of increased temperature on the cytotoxicity of etoposide on neuroblastoma cells is not absent, but depends on scheduling. The temperature range used is achievable in total body hyperthermia. Thus, our results are relevant for possible treatment of disseminated neuroblastoma.     

VP16CREB-induced differentiation of neuroblastoma

Objective  Drug-induced differentiation is commonly used as a therapeutic modality for the treatment of neuroblastoma tumors. Increased level of cyclic adenosine 3′, 5′-monophosphate (cAMP) mediates terminal differentiation in some neuroblastoma cell lines through activation of several signaling networks, including cAMP response element binding protein (CREB). Objective was to test whether cAMP-induced differentiation in a murine neuroblastoma cell line (NBP2) is partly mediated by CREB. Methods  Fluorescent microscopy was used to document neuron-like morphological changes imparted by a constitutively active CREB (VP16CREB). Real time PCR (RT-PCR) was performed to verify changes in the expression of cAMP/CREB responsive genes. Results  It was found that transient expression of VP16CREB into NBP2 cells resulted in morphological changes that were characteristics of terminally differentiated neurons. Furthermore, increased expression of cAMP responsive genes was compromised in cells resisting VP16CREB-mediated differentiation. Conclusion  A constitutively active CREB induces terminal differentiation in a subset of NBP2 cell population. Altered expression of cAMP responsive genes may account for differentiation resistant phenotype in NBP2 cells.     

双酚A对神经母细胞瘤SK-N-SH细胞株移植瘤生长的影响

目的环境内分泌干扰物对人类健康的影响已引起全球的关注。本研究旨在观察双酚A（BPA）对神经母细胞瘤生长的影响及其可能机制。方法常规培养SK-N—SH细胞，接种于双侧卵巢切除（OVX）的裸小鼠皮下。可触摸到肿瘤结节时，将裸鼠随机分为3组：①对照组（OVX＋溶剂，OVX，n=9）；②E2组（OVX＋E2，500ug·kg^-1·d^-1，n=11）；③BPA组（OVX＋BPA，200mg·kg^-1·d^-1，BPAgroup，n=10）。每周2次检测皮下移植瘤体积（TV）和裸鼠体重。第18d，解剖处死所有裸鼠，称移植瘤重量。免疫组化方法检测肿瘤组织微血管内皮细胞的表达情况，在光学显微镜高倍视野下分别计数肿瘤组织的微血管密度（MVD））。用Western Blot方法检测肿瘤组织血管内皮细胞生长因子（VEGF）蛋白的表达情况。结果实验终点时，OVX组、E2组和BPA组的TV分别为（2．62±0．54）cm^3、（5．07±0．93）cm^3和（4．19±0．68）cm^3，BPA组较OVX组增高59％（P〈0．05）。3组裸鼠体重没有明显差异（P〉0．05）。3组瘤重分别为（1．64±0．18）g、（2．58±0．19）g、（2．96±0．27）g，BPA组较OVX组增高82％（P〈0．05）；3组中Ⅷ因子相关抗原均染色，E2组（22．73±1．46）及BPA组（27．76±2．24）MVD较对照组（15．14±1．12）明显增高（P〈0．01），VEGF的蛋白表达亦较对照组增高（P〈0．05）。结论BPA可促进人神经母细胞瘤裸鼠皮下移植瘤生长，微血管形成可能参与了促生长作用。     

Expression patterns of microRNAs are altered in hypoxic human neuroblastoma cells

Background/Purpose  

There was a report that microRNA (miRNA) controls multiple genes. In addition, there are some reports that the presence of neoplastic cells that are hypoxic because of rapid tumor development is related to prognosis. As a step toward identifying the role of miRNA in hypoxic tumor cells, the present study was designed to determine which miRNAs have increased expression and which have decreased expression in hypoxic neuroblastoma cells.     

三氧化二砷联合化疗药物对神经母细胞瘤SK-N-SH细胞系杀伤作用的研究

目的观察三氧化二砷(As_2O_3)联合化疗药物对神经母细胞瘤SK-N-SH细胞系的毒性作用和细胞周期的影响,为临床寻找新的治疗手段和合理用药提供依据。方法采用流式细胞术检测单药,即As_2O_3、长春瑞滨、多西他赛、依托泊苷(VP16)或顺铂(DDP),或As_2O_3分别与另一种药物双药联合(同步给予As_2O_3和另一种药物;先予As_2O_3使SK-N-SH细胞周期阻滞在G2/M期后再予另一种药物)作用于SK-N-SH细胞24 h、48 h后不同处理组的凋亡率和细胞周期分布的差异。结果同步给药双药组的凋亡率均高于相应的单药组的凋亡率;As_2O_3与M期特异性化疗药物(长春瑞滨或多西他赛)联合用药时,非同步给药组即先予As_2O_3使SK-N-SH细胞周期阻滞在G2/M期后再加用长春瑞滨或多西他赛组的凋亡率高于相应单药组的凋亡率,As_2O_3与非M期特异性化疗药物(VP16或DDP)联合用药时,同步给药组即As_2O_3与VP16或DDP同步给药组的凋亡率高于相应单药组的凋亡率。联合用药组随着细胞凋亡率的变化,细胞周期的分布也发生了显著的变化,表现为随着SK-N-SH细胞凋亡率的增高,处于G2/M期的细胞比例减少。结论 As_2O_3与化疗药物联合用药或许能提高它们对SK-N-SH细胞的杀伤效果;当As_2O_3与M期特异性化疗药物(长春瑞滨或多西他赛)联合用药时,宜先采用As_2O_3的周期阻滞作用将NB细胞周期阻滞在G2/M期后再加M期特异性化疗药物的非同步给药方式;而当As_2O_3与非M期特异性化疗药物(VP16或DDP)联合用药时,宜采用同步给药的方式以提高杀伤效果。