Increased N-myc expression following progressive growth of human neuroblastoma

Many human neuroblastoma tumors and cells in culture contain amplified N-myc DNA and this is associated with tumor stage. We have analyzed pairs of neuroblastoma cell cultures derived from two patients at the time of diagnosis and after tumor progression following the initiation of therapy. Cell cultures derived after progression have increased expression of N-myc RNA. In one pair this increase is associated with increased N-myc DNA amplification; in the other, amplification decreases and activation of N-myc is most likely the result of a regulatory change. Analysis of the pattern of DNA amplification in these cell cultures demonstrates additional changes that might be associated with tumor progression.     

Targeted inhibition of NMYC by peptide nucleic acid in N-myc amplified human neuroblastoma cells: cell-cycle inhibition with induction of neuronal cell differentiation and apoptosis

We developed an antisense peptide nucleic acid (PNA) targeted against a unique sequence in the terminus of the 5'-UTR of N-myc, designed for selective inhibition of NMYC in neuroblastoma cells. Fluorescent microscopy showed carrier-free delivery of the PNA to two human neuro-blastoma cell lines: GI-LI-N (N-myc-amplified) and GI-CA-N (N-myc-unamplified). Only in the former, PNA treatment determined 70% cell-viability reduction (at 48 h). In N-myc-amplified GI-LI-N cells, the PNA determined NMYC-translation inhibition (Western blotting), accumulation of cells in G1, induction of differentiation and apoptosis. Selectivity of the PNA was demonstrated by altering three point mutations. These findings should encourage development of a PNA-based tumor-specific agent for neuroblastoma (or other neoplasms) with N-myc overexpression.     

Glucocorticoids induce neuroendocrine cell differentiation and increase expression of N-myc in N-type human neuroblastoma cells

Human neuroblastoma cell lines comprise cellular counterparts of normal differentiation phenotypes arising from the developing neural crest Three distinct cell types have been isolated from cell lines: N-type cells with properties of embryonic sympathoadrenoblasts, S-type cells resembling nonneuronal Schwannian/glial/melanoblastic precursors, and I-type stem cells that can differentiate into either N- or S-type cells. Sympathoadrenoblasts from the normal neural crest further differentiate into neuronal or neuroendocrine cells. In this study, we show that malignant N-type neuroblasts likewise can differentiate futher along these same pathways. Retinoic acid and forskolin induce a neuronalphenotype, denoted morphologically by cell aggregation and increased neurite formation and biochemically by increases in neurofilament proteins, tyrosine hydroxylase, and secretogranin II and decrease inchromogranin A. By contrast, dexamethasone, a synthetic glucocorticoid, induces a chromaffin cell phenotype characterized by increased cell flattening, loss of neuritic processes, increased chromogranin A and tyrosine hydroxylase proteins, and decreased amounts of secretogranin II and neurofilaments. N-myc gene expression is upregulated by glucocorticoids; dexamethasone-treated N-type cells show significant (2.3- to 7.8-fold) increases in N-myc mRNA and protein steady-state levels. This effect is specific for glucocorticosteroids, is blocked by addition of the steroid receptor antagonist RU486, and involves direct activation of the N-myc promoter. These findings are the first to show that glucocorticoids upregulate N-myc expression in human neuroblastoma cells.     

Effect of human interferons on morphological differentiation and suppression of N-myc gene expression in human neuroblastoma cells

The activity of human interferons (HuIFNs) to induce morphological changes and the suppression of N-myc gene expression on human neuroblastoma cells (GOTO and KP-N-RT) was evaluated. Morphological differentiation, characterized as the extension and bifurcation of neurites, the formation of multinucleated giant cells and the formation of neurite networks, was induced by treatment with recombinant HuIFN-gamma (rHuIFN-gamma) and also with natural HuIFN-gamma on human neuroblastoma cells (GOTO and KP-N-RT). But recombinant HuIFN-alpha A and recombinant HuIFN-beta did not induce any changes. The rHuIFN-beta and rHuIFN-gamma inhibited the growth of GOTO and KP-N-RT cells more strongly than the rHuIFN-alpha A did. The expression of N-myc gene was suppressed in GOTO cells treated with rHuIFN-gamma. The suppressive effect of rHuIFN-gamma was dependent on the duration of the treatment. However, rHuIFN-alpha A and rHuIFN-beta did not suppress N-myc gene expression. Moreover, both morphological differentiation and the suppressive effect on N-myc gene expression by rHuIFN-gamma were inhibited in the presence of cycloheximide. These results suggest that the morphological changes and N-myc gene expression in neuroblastoma cells are closely related. Furthermore, this decreased N-myc gene expression during the morphological differentiation may be related to the proteins induced by HuIFN-gamma.     

N-myc oncogene RNA expression in neuroblastoma

Tumor specimens from 33 patients with neuroblastoma were assayed for amplification of the N-myc oncogene and RNA expression to determine whether N-myc RNA expression levels correlated with N-myc gene amplification and clinical outcome. N-myc gene amplification was detected in one stage II tumor, one stage IV-S tumor, and seven stage III or IV tumors. In each case, N-myc RNA expression roughly paralleled N-myc gene amplification. However, enhanced N-myc RNA expression was not confined to tumors with N-myc gene amplification: all of the early (stage I and II) tumors, five stage IV-S tumors, and 12 advanced (stage III and IV) tumors had levels of N-myc RNA that were elevated up to 50-fold. While N-myc gene amplification correlated with prognosis, there was no such correlation with levels of N-myc RNA expression. The precise role of the N-myc gene in the pathogenesis of neuroblastoma remains unclear.     

Cell type-specific expression and negative regulation by retinoic acid of the human N-myc promoter in neuroblastoma cells.

Expression of the N-myc oncogene is an important determinant of tumor behavior in human neuroblastoma. To study the regulation of N-myc, we have subcloned fragments of the 5' flanking region of the human N-myc gene upstream of the chloramphenicol acetyl transferase (CAT) reporter gene, and assayed for promoter activity in transient transfections into neuroblastoma and other cell lines. Upstream sequences were found to possess promoter activity to within 121 bp of the major cap site (-121). Negative regulatory elements were identified in regions approximately 2 kb and 500 bp upstream from the major cap site, as well as 150-1000 bp downstream. Promoter constructs containing downstream elements from bp +150 to +1000 were active in N-myc-expressing neuroblastoma cell lines, but not in non-expressing Epstein-Barr virus (EBV)-transformed 729-6 B-cell or HeLa cell lines, while those lacking this element were active in all cell types tested. All tested constructs retaining promoter activity showed decreased activity in parallel with the down-regulation of endogenous N-myc in response to treatment of transfected cells with retinoic acid. These studies suggest that N-myc regulation may be controlled at different levels, and provide a basis for further characterization of N-myc regulation in neuroblastoma.     

Genistein modulates neuroblastoma cell proliferation and differentiation through induction of apoptosis and regulation of tyrosine kinase activity and N-myc expression

Genistein is a specific inhibitor of protein tyrosine kinase (PTK) and is considered as a therapeutic candidate for various cancers. In this paper we investigate the effects of genistein on cell proliferation and differentiation in neuroblastoma (NB) cell lines and its possible mechanism of action. Genistein substantially inhibited the growth of five (N2A, JC, SKNSH, MSN and Lan5) of the six tumor cell lines examined in a dose-dependent manner with an IC50 value of approximately 5 microg/ml. The exception was GC cells. N2A cells were treated with genistein for 6 days and exhibited morphological features of differentiation, as evidenced by the development of dendritic extensions. Terminal deoxynucleotidyl transferase (TDT) histochemical staining showed a significant elevation in darkly stained nuclei in genistein-treated N2A cells compared with controls, indicating the occurrence of apoptosis. Fluorescent quantitation of DNA fragments confirmed apoptosis in genistein-treated N2A cells. To further elucidate the possible mechanisms by which genistein modulates NB cell growth and differentiation we investigated the effect of genistein on the activities of PTK and mitogen-activated protein (MAP) kinase and N- myc proto-oncogene expression in N2A cells. The results showed that genistein down-regulated intrinsic PTK activity by approximately 33% and inhibited insulin-like growth factor (IGF)-stimulated PTK activity by 75%. The effect of genistein on the intrinsic activity of MAP kinase was insignificant. In addition, genistein significantly reduced N-myc expression in a dose-dependent fashion. Our study suggests that genistein arrests cell growth and induces NB cell differentiation by mediating apoptosis and modulating PTK activity and N-myc proto- oncogene expression.      

Induction of apoptosis by flavopiridol in human neuroblastoma cells is enhanced under hypoxia and associated with N-myc proto-oncogene down-regulation.

PURPOSE: Neuroblastoma is the most common extracranial solid tumor of children that arises from the sympathetic nervous system. Survival rates for neuroblastoma patients is low despite intensive therapeutic intervention, and the identification of new effective drugs remains a primary goal. The cyclin-dependent kinase inhibitor, flavopiridol, has demonstrated growth-inhibitory and cytotoxic activity against various tumor types. Our aim was to investigate flavopiridol effects on advanced-stage, N-myc proto-oncogene (MYCN)-amplified human neuroblastomas and the modulation of its activity by hypoxia, a critical determinant of tumor progression and a major challenge of therapy. EXPERIMENTAL DESIGN: Cell viability was monitored by 3-(4,5 dimethyl-2 thiazolyl)-2,5 diphenyl-2H tetrazolium bromide (MTT) and trypan blue dye exclusion assays; DNA synthesis was assessed with the bromodeoxyuridine pulse-labeling technique; apoptosis was studied by Giemsa staining, DNA fragmentation, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling reaction, flow cytometric determination of hypodiploid DNA content, and evaluation of caspase activity and cytochrome c (CytC) release; MYCN expression was determined by Northern and Western blotting. RESULTS: Flavopiridol caused dose- and time-dependent decreases in neuroblastoma viability by inducing apoptosis, as confirmed by morphologic and biochemical criteria. Cell death was preceded by DNA synthesis inhibition and G1-G2 arrest, reversed by the pancaspase inhibitor, zVAD-fmk, and associated with caspase-3 and -2 activation and CytC increase. Moreover, flavopiridol strongly down-regulated MYCN mRNA and protein expression. Exposure to hypoxia enhanced both the extent of apoptosis and flavopiridol effects on CytC, caspase 3, and MYCN. CONCLUSIONS: These results indicate that flavopiridol has growth-inhibitory and apoptotic activity against advanced-stage neuroblastomas in vitro and is worthy of further investigation for the treatment of this disease.     

Differential expression of N-myc and c-src proto-oncogenes during neuronal and schwannian differentiation of human neuroblastoma cells

Neuroblastoma (NB) cells can be induced to differentiate bidirectionally into either neuronal or schwannian cells by different inducers. However, the underlying mechanisms are poorly understood. We examined the expression of N-myc and c-src genes in 3 human NB-cell lines during either neuronal or schwannian differentiation in vitro, since proto-oncogenes are considered to play a pivotal role in regulating cell proliferation and differentiation. Decreased N-myc expression and increased c-src expression were observed during neuronal differentiation by retinoic acid, polyprenoic acid (E5166) and dibutyryl cyclic AMP, whereas the expression of N-myc and c-src genes was considerably reduced during schwannian differentiation by bromodeoxyuridine, demonstrating that the expression of N-myc and c-src genes was regulated independently in the bipolar differentiation processes of NB cells. Our results suggest that enhanced N-myc expression might be closely linked to the undifferentiated phenotypes of NB cells, that c-src expression might be related to the neuronal differentiation of NB cells, and that N-myc and c-src genes might be regulated independently in the determination of the bipolar differentiation of NB cells.     

Inhibition of proliferation and induction of apoptosis in human breast cancer cells by lauryl gallate

Lauryl gallate is an antioxidant food additive showing low toxicity to normal cells. Here, its antiproliferative effect has been studied on three human breast cancer cell lines: estrogen-dependent, wild-type p53, MCF7; estrogen-independent, non-functional p53, MDA-MB-231 and MCF7 ADR, which overexpresses P-glycoprotein (P-gp) and displays a multidrug-resistant phenotype. Lauryl gallate inhibited proliferation and induced cell cycle alterations in all three cell lines without altering P-gp functionality in the drug-resistant cells. A stable arrest in G(1) phase was observed in MCF7, while a slow-down of cell cycle progression was induced in the other two cell lines. Lauryl gallate increased p53 expression only in MCF7, and upregulated p21(Cip1) and reduced cyclin D1 levels in all three cell lines. The induction of apoptosis, demonstrated by annexin V-FITC labeling, PARP cleavage and mitochondrial membrane depolarization and morphological alterations, were clearly detected in MCF7 ADR and MDA-MB-231 and to a minor extent in MCF7. Overexpression of Bcl-2 in MCF7 ADR cells demonstrated its protective role against morphological alterations and apoptosis. Lauryl gallate induction of p21(Cip1) and apoptosis observed in all three cell lines was regulated by Erk1/2 activation. These findings suggest a potential use of lauryl gallate against tumors harboring p53 mutations and drug-resistant phenotypes.     

N-myc modulates expression of p73 in neuroblastoma

The human p73 gene is a homolog of p53, which has been localized to chromosome 1p36 in a region that is frequently deleted in neuroblastoma. Transfection of the p73 gene into neuroblastoma cells that lack detectable p73 protein has been shown to result in growth suppression and to induce neuronal differentiation. In this study, we have identified by means of restriction landmark genome scanning (RLGS) a genomic fragment that was frequently reduced in intensity in neuroblastomas. The cloned fragment contained exon 1 of p73 as well as intronic and promoter sequences. We investigated the genomic and expression status of p73 and N-myc in 34 neuroblastoma tumors and 12 neuroblastoma cell lines. Approximately a third of neuroblastomas in our series exhibited deletion of p73. Most tumors analyzed exhibited reduced expression of p73, as determined by quantitative RT-PCR, in the absence of detectable p73 gene deletion. The reduced expression of p73 correlated with overexpression of N-myc in a statistically significant manner. The N-myc gene was transfected into two neuroblastoma cell lines that lacked N-myc amplification to determine its effect on p73 RNA levels. p73 was detectable at low level by RT-PCR in untransfected SK-N-AS cells and became undetectable following N-myc transfection, whereas in SH-EP1 cells, p73 levels were substantially reduced following transfection but remained detectable. Our data suggest that the N-myc gene modulates expression of p73, allowing neuroblastoma cells to escape the growth suppressing properties of p73.     

Regulation of c- and N-myc expression during induced differentiation of murine neuroblastoma cells

Using clones N1E-115 and N1A-103 from mouse neuroblastoma C1300, a comparative analysis of c- and N-myc gene expression was undertaken both in proliferating cells and in cultures exposed to conditions which induce differentiation. Under the latter conditions, while N1E-115 cells extend abundant neurites and express many biochemical features of mature neurons, clone N1A-103 stops dividing and expresses certain neurospecific markers but is unable to differentiate morphologically. In both clones, chemical agents, i.e. 1-methyl cyclohexane carboxylic acid (CCA) or dimethyl sulfoxide (DMSO), induce a decrease in c-myc expression. Similar results were found for N-myc gene in N1E-115 cells, but in contrast, in clone N1A-103, N-myc expression is increased with CCA and not modified with DMSO. Globally, this study favours the hypothesis that changes in c-myc expression would correspond to cell division blockade and differentiation, while modulations in N-myc are more closely related to an early phase of terminal differentiation.     

Inhibition of invasion and induction of apoptosis by selenium in human malignant brain tumour cells in vitro

Selenium is considered to be one of the most promising micronutrients for cancer prevention and therapy, based on evidence from epidemiological studies, laboratory-based research and clinical trial intervention. There are ample reports of selenium methionine and sodium selenite's ability to induce apoptosis in various cancers in vitro. There are a few reports in the literature on the effects of selenium on established glioma cell lines but none on biopsy-derived short-term brain tumour cultures. In this in vitro study the effects of a range of concentrations (2-10 microg/ml) of sodium selenite were investigated in one low-passage culture of biopsy-derived glioma cells (IPSB-18, an anaplastic astrocytoma, P 18-22) and a normal human brain cell culture (CC2565, P11). Results from 2 viability assays, 3[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and sulphorodamine B (SRB) consistently showed that the IC50 for selenium in the astrocytoma was approximately 5 microg/ml whilst the normal brain cells were unaffected by selenium in the range of concentrations studied. Time-lapse video microscopy revealed that, while at 4 microg/ml selenium, the time taken to achieve 100% cell death was 17 h, with increasing concentrations of selenium from 6 to 8 microg/ml and finally at 10 microg/ml the IPSB-18 cells rounded up and died much more quickly. The time taken to achieve 100% cell death was 7 h, 7 h and 6 h, respectively, suggesting that the effect was similar at higher concentrations. Flow cytometry indicated that cell death was by apoptosis. RT-PCR results showed downregulation of the gene expression of 6 matrix metalloproteases (MMP2, 9, 14, 15, 16, 24), their inhibitors, TIMPs and epidermal growth factor receptor, in IPSB-18 cells treated with 2, 4 and 8 microg/ml of selenium. Collectively, the data in this study suggests that selenium, not only induces tumour cell-specific apoptosis but also has anti-invasive potential.     

Bovine seminal ribonuclease selectively kills human multidrug-resistant neuroblastoma cells via induction of apoptosis.

Bovine seminal ribonuclease (BS-RNase) is a homologue of RNase A with specific antitumor activity. The cytotoxic action of this agent was examined in human neuroblastoma (NB) cell lines (SK-N-SH and UKF-NB-4) possessing the multidrug resistance (MDR) phenotype and NB cell lines (IMR-32, UKF-NB-1, UKF-NB-2 and UKF-NB-3) without MDR. Although MDR cells expressed large amounts of mdr-1 mRNA, contained functional P-glycoprotein and had 20- to 105-fold lower sensitivities to doxorubicin and vincristine than cells with non-MDR phenotypes, BS-RNase was equally toxic to all NB cells at concentrations employed (0.2 to 100 microg/ml). BS-RNase showed high selectivity for NB cells and was non-toxic to normal fibroblasts and epithelial cells. Ultrastructural investigation and annexin V assay showed that BS-RNase is a powerful inductor of apoptosis. The antitumoral effects of BS-RNase were also demonstrated in vivo using established subcutaneous xenografts in athymic (nude) mice of the MDR-1-bearing UKF-NB-4 cell line. Intratumoral injections (12.5 mg/kg) of BS-RNase over four weeks resulted in complete tumor regression and absence of tumor regrowth over a two-week observation period after cessation of treatment. The results show that BS-RNase selectively kills NB cells by inducing apoptosis and that this agent is active against mdr-1 expressing cells both in vitro and in vivo. BS-RNase fulfills important criteria for a candidate antitumor agent in NB patients with advanced disease.     

Inhibition of the NF-kappaB pathway enhances TRAIL-mediated apoptosis in neuroblastoma cells

Neuroblastoma is the most common solid extracranial neoplasm in children and causes many deaths. Despite treatment advances, prognosis for neuroblastoma remains poor, and a critical need exists for the development of new treatment regimens. TNF-related apoptosis-inducing-ligand (TRAIL) induces cell death in a variety of tumors, but not in normal tissues. Moreover, TRAIL is nontoxic, making it a strong antitumor therapeutic candidate. We demonstrate that introduction of the TRAIL gene into neuroblastoma cell lines using an adenoviral vector leads to apoptotic cell death. RT-PCR and flow-cytometric analyses demonstrated that TRAIL's effect is mediated primarily via the TRAIL R2 receptor. As TRAIL can activate the nuclear factor-kappaB (NF-kappaB) signaling pathway, which can exert an antiapoptotic effect, we hypothesized that inhibition of NF-kappaB signaling may augment TRAIL's killing effects. TRAIL-mediated cell death was enhanced when neuroblastoma cells were simultaneously infected with a dominant-negative mutant of IkappaB kinase, a kinase essential for NF-kappaB activation. The combination of blockade of NF-kappaB signaling and expression of TRAIL induced apoptotic death in a greater proportion of SKNSH cells than did either treatment alone. Thus, concurrent inhibition of the NF-kappaB pathway and the induction of TRAIL-mediated apoptosis may become a useful approach for the treatment of neuroblastoma.     

短发夹RNA抑制喉癌细胞hTERT基因表达及诱导细胞凋亡

目的:探讨DNA载体途径的RNA干扰技术抑制人端粒酶逆转录酶(hTERT)基因表达及诱导喉癌细胞Hep-2凋亡的作用。方法:构建靶向hTERTmRNA的质粒pshRNA1、pshRNA2及对照质粒pshRNA3、pEGFP,并将其分别转染喉鳞癌Hep-2细胞。共聚焦荧光显微镜观察质粒转染及表达情况;RT-PCR测定hTERTmRNA表达;Western印迹测定hTERT蛋白表达;末端重复片断扩增-酶联免疫吸附(TRAP-ELISA)方法测定细胞端粒酶活性;MTT法研究细胞增殖活性变化;原位细胞凋亡(TUNEL)及透射电子显微镜研究细胞凋亡。结果:①共聚焦荧光显微镜下见大量的细胞呈现绿色荧光,与其他组比较,pshRNA1、2组呈现荧光的死亡细胞显著增加。②pshRNA1、2转染细胞后hTERTmRNA及hTERT蛋白表达显著降低;其端粒酶活性明显受到抑制:转染后2 d pshRNA1组活性为:0.159±0.039、pshRNA2组活性为0.163±0.028,与空白对照组1.523±0.076比较,差异有非常显著性(P<0.005)。③pshRNA1、2转染细胞后可显著抑制细胞增殖、诱导细胞凋亡。这两组细胞透射电镜可见典型细胞凋亡特征。结论:DNA载体途径的RNA干扰技术能有效抑制hTERT基因的表达及端粒酶活性并诱导癌细胞凋亡,此法可能成为抑制肿瘤细胞的新途径。     

Diverse responses to retinoid in morphological differentiation, tumorigenesis and N-myc expression in human neuroblastoma sublines

We established the subline RT-BMV-C6 from the parent human neuroblastoma cell line RT-BM by a process that required repeated subculture of cells, which were prone to disaggregation. RT-BMV-C6 and the parent cloned line, RT-BM-1, had an identical marker chromosome, confirming that both lines were derived from a common progenitor. In the analysis of surface antigen expression, RT-BMV-C6 did not react with UJ-127-11, Leu7 or KP-NAC2 MAbs to which RT-BM-1 showed positive binding. The levels of both N-myc amplification and expression in RT-BMV-C6 were twice as high as the level obtained in RT-BM-1. Colony-forming efficiency in soft agar was 2.0 +/- 0.8% for RT-BMV-C6 and 3 times greater than that for RT-BM-1 (0.6 +/- 0.1%). When 100 x 10(6) cells of RT-BM-1 and RT-BMV-C6 were inoculated into nude mice, tumor incidence was significantly higher for RT-BMV-C6 (6/6; 100%) than for RT-BM-1 (0/6; 0%). Our data show that N-myc is closely related to tumorigenicity in NB. When RT-BM-1 and RT-BMV-C6 were co-cultured with a new synthetic retinoid, polyprenoic acid (E5166), and dibutyryl cyclic AMP, RT-BM-1 was induced to neuronal differentiation, defined by the formation of neuronal processes and expression of neurofilaments, whereas RT-BMV-C6 was not. However, when exposed to E5166, N-myc expression of RT-BMV-C6 was more strongly reduced than that of RT-BM-1, and colony formation of RT-BMV-C6 was significantly inhibited as compared to RT-BM-1. These findings suggest that the reduction of N-myc expression might closely correlate with growth inhibition accompanying neuronal differentiation of neuroblastoma cells.