Receptor activator of nuclear factor-kappaB ligand (RANKL) directly modulates the gene expression profile of RANK-positive Saos-2 human osteosarcoma cells

Receptor activator of nuclear factor kappaB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) are the key regulators of bone metabolism. Recent findings demonstrated a crucial role of RANK in several bone-associated tumors. Indeed, we have recently demonstrated functional RANK expression both in a mouse and several human osteosarcoma cell lines. However, RANKL effects on osteosarcoma cells remain to be determined. In this study, we determined RANKL effects on RANK-positive Saos-2 human osteosarcoma cells. cDNA microarray and quantitative RT-PCR analyses clearly demonstrated that RANK-positive osteosarcoma cells were the target of RANKL as well as osteoclasts/osteoclast precursors. Thus, we present for the first time that RANKL can directly and significantly modulate gene expression of RANK-expressing Saos-2 cells. RANKL-modulated genes included genes that were implicated in protein metabolism, nucleic acid metabolism, intracellular transport, cytoskeleton organization and biogenesis, apoptosis and signaling cascade. Our results strengthen the involvement of the RANK/RANKL/OPG axis in osteosarcoma biology and capability to identify novel therapeutic approaches targeting RANK-positive osteosarcomas.     

MicroRNA profiling with correlation to gene expression revealed the oncogenic miR-17-92 cluster to be up-regulated in osteosarcoma

Osteosarcomas are genetically complex tumors with abundant structural and numerical alterations. The molecular pathogenesis of the disease is, however, still poorly understood. Aside from various oncogenes and tumor suppressor genes, deregulated microRNAs (miRNAs) are known to influence tumor development and biology. We therefore investigated six well-established osteosarcoma cell lines (HOS58, U2-OS, Saos-2, MNNG/HOS, SJSA-1, and MG-63) for genome-wide miRNA expression (miRBase Version 15.0, http://www.mirbase.org/) and correlated our findings with gene expression. Cultured osteoblasts (hFOB 1.19) and mesenchymal stem cells (L87/4) were used as normal references. Focusing only on miRNAs that were deregulated in the majority of osteosarcoma cell lines, we identified several miRNAs with oncogenic and tumor suppressor properties, including various members of the oncogenic miR-17-92 cluster. In addition, several genes involved in differentiation (RGMB, LRRC17), cell cycle control (CCNE1), and apoptosis (LIMA1, CAMK2N1) were found to be deregulated in osteosarcoma cell lines, most likely due to altered miRNA expression patterns. Our findings indicate a crucial impact of deregulated miRNAs with consecutive changes in gene expression in osteosarcomas, which strongly suggests pathogenetic and potentially therapeutic implications.     

转移抑制基因BRMS1mRNA在骨肉瘤细胞株中的表达及临床意义的研究

目的研究转移抑制基因BRMS1在人骨肉瘤细胞株中的体外转录水平,探讨其在侵袭转移过程中的作用。方法应用半定量RT-PCR技术检测BRMS1mRNA在3种人骨肉瘤细胞株(SaOS-2、HOS和MG-63)中的表达。结果半定量RT-PCR检测BRMS1mRNA在3株骨肉瘤细胞株中表达水平下降,在正常成骨细胞中则以较高水平表达。结论人骨肉瘤细胞株SaOS-2、HOS和MG-63细胞随侵袭转移能力的增强,BRMS1mRNA表达水平下降,为研究骨肉瘤的转移机制提供了理论基础。     

Mutational analysis of 9 different tumour-associated genes in human malignant mesothelioma cell lines

Seven tumour suppressor genes (Chk1, Chk2, Apaf1, Rb1, p53, p16(INK4a) and p14(ARF)) and two oncogenes (N-ras and BRAF) were screened in nine human malignant melanoma (HMM) cell lines for point mutations or small deletions/insertions by DGGE, TGGE and SCCP analysis. For the first time in human mesothelioma, Chk1 gene mutations were detected in two of the nine investigated HMM cell lines. P53 gene mutations were found in three cell lines and p16(INK4a) mutations in 5. Mutation of the Chk1 gene implies a novel disruption mechanism of the p53 pathway in HMM, without affecting p53 itself. According to our knowledge, this is the first mutation screening of Chk1, Chk2, Apaf1 and Rb1 in human malignant mesothelioma.     

Expression of neoplasia-related proteins of chemically transformed HuT fibroblasts in human osteosarcoma HOS fibroblasts and modulation of actin expression upon elevation of tumorigenic potential

Two sets of abundant cytoplasmic transformation-specific polypeptides, p788/p789 and p219/p220, have been identified by comparing in vitro-transformed human fibroblasts with diploid human fibroblasts. These polypeptides are also expressed by the human fibrosarcoma and osteosarcoma cell lines HT1080 the human fibrosarcoma and osteosarcoma cell lines HT1080 and HOS, respectively. HOS cells, however, synthesize only one of the two electrophoretic forms of each marker set, p789 and p219, at greatly reduced rates compared to the rates of synthesis found for HT1080 cells and the in vitro-transformed cell lines. Induction of expression of these neoplastic marker polypeptides is independent of the activation of a transforming gene that will induce focus formation in confluent mouse 3T3 cell monolayers. Activation of the met oncogene in MNNG-HOS cells and simultaneous elevation of tumorigenic potential did not lead to a significant change in the rate of the 600 most abundant polypeptide species with the exception of one of the two cytoplasmic actin polypeptides. While the normal ratio of beta-to gamma-actin which is approximately 2:1 was expressed in "untransformed" HOS cells, MNNG-HOS cells synthesized 50% less beta-actin resulting in a 1:1 ratio of beta-actin to gamma-actin. Our finding here, together with our previous characterization of the human beta-actin gene, leads us to predict that one of two functional beta-actin genes expressed in HOS cells has been inactivated in MNNG-HOS cells by either a regulatory or structural gene mutation.     

Analysis of p53 status in human cell lines using a functional assay in yeast: detection of new non-sense p53 mutation in codon 124

p53 tumor suppressor is a sequence-specific DNA-binding protein that controls the expression of many genes in response to diverse stress stimuli. p53 gene is often mutated in human cancer and in cancer cell lines. Several methods are available for identification of p53 mutations, including functional analysis of separated alleles in yeast (FASAY). FASAY distinguishes yeast colonies expressing functional p53 protein from colonies producing a dysfunctional p53 protein simply on the basis of color. We analyzed the p53 status of 26 human cell lines of different tissue origin using the functional assay in yeast. Wild-type p53 was found in six cell lines and various p53 aberrations in the remaining twenty. FASAY detected temperature-sensitive p53 mutations in breast cancer cell line, BT474, and leiomyosarcoma cell line, SK-LMS-1, and two independent p53 point mutations in SK-UT-1 and SK-LMS-1 leiomyosarcoma cell lines. In addition, the assay revealed that a recombination occurred between the two mutated p53 alleles producing a stable ratio of p53 wild-type alleles (11.7 or 2.7% respectively). In the case of acute myeloid leukemia cell line, ML-1, we detected both wild-type and heterozygous p53 status, depending on the source of the cell line. In Hs913T, HL60 and Saos-2 cell lines, FASAY failed to assess p53 status due to a large deletion/rearrangement of the p53 gene. In acute lymphoid leukemia HPB cell line, we disclosed unknown non-sense mutation in codon 124 of the p53 gene.     

Mutational analysis of Chk1, Chk2, Apaf1 and Rb1 in human malignant melanoma cell lines

Four tumour suppressor genes (Chk1, Chk2, Apaf1 and Rb1) in nine human malignant melanoma cell lines were screened for a loss of gene expression, point mutations and small deletions/insertions by cDNA-based DGGE/SCCP analysis. In two cell lines alterations of the investigated genes could be demonstrated. This result confirms our assumption of the participation of dysfunctional p53 inducer/effector genes in human melanoma aetiology. Furthermore, it points towards the probable principal role of diverse alternative p53-pathway disruption mechanisms in this highly therapy-resistant malignancy without affecting p53 itself. To our knowledge, this is the first CHK1/CHK2 mutation screening in human melanoma.     

miRNA-218 Inhibits Osteosarcoma Cell Migration and Invasion by Down-regulating of TIAM1, MMP2 and MMP9

Deregulated miRNAs participate in osteosarcoma genesis. In this study, the expression of miRNA-218 inhuman osteosarcomas, adjacent normal tissues and Saos-2 human osteosarcoma cells was first assessed. Then theprecise role of miRNA-218 in osteosarcoma cells was investigated. Upon transfection with a miR-218 expressionvector, the proliferation of Saos-2 human osteosarcoma cells determined using the ATPlite assay was significantlysuppressed, whilw migration of Saos-2 cells detected by wound healing and invasion determined using transwellswere dramatically inhibited. Potential target genes of miR-218 were predicted and T-cell lymphoma invasionand metastasis 1 (TIAM1) and matrix metalloproteinase 2 (MMP2) and 9 (MMP9) were identified. This wasconfirmed by western blotting, which showed that miR-218 expression inhibited TIAM1, MMP2 and MMP9protein expression. Collectively, these data suggest that miR-218 acts as a tumor suppressor in osteosarcomasby down-regulating TIAM1, MMP2 and MMP9 expression.     

MiR-129-5p靶向HMGB1抑制骨肉瘤细胞增殖和迁移

目的 探讨miR-129-5p对骨肉瘤(OS)细胞增殖和迁移的影响以及对HMGB1的调控作用.方法 RT-PCR和Western blot法分别检测骨肉瘤细胞株MG-63、Saos-2和成骨细胞hFOB1.19中miR-129-5p和HMGB1的表达.生物信息学预测miR-129-5p与HMGB1基因是否存在结合位点,...     

Dependence of induction of osteocalcin gene expression on the presence of wild-type p53 in a murine osteosarcoma cell line

The p53 gene undergoes rearrangement in a high percentage of osteosarcomas, resulting in loss of its expression. A p53-null murine osteosarcoma cell line F6 was transfected with either a wild-type or a mutant p53 gene. Stably transfected cell lines were obtained, and their differentiation capabilities were compared in vitro with the parental cell line. Alkaline phosphatase and osteocalcin expression were measured as early and late differentiation markers, respectively. Induction of alkaline phosphatase expression was not affected by the presence of either p53 gene, whereas osteocalcin expression was seen in cells containing the wild-type p53 gene but not in the parental p53-null or mutant-expressing cell lines. That the induction of osteocalcin was intrinsically dependent on the presence of wild-type p53 was also indicated by the use of a temperature-sensitive Val 135 p53 mutant at 32°C; predominant expression of p53 in the wild-type conformation resulted in osteocalcin expression. While the wild-type p53 gene could suppress tumor formation in vivo, the tumors expressing the mutant p53 gene grew two to three times as large as the tumors that did not express p53. Therefore, the absence of end-point differentiation in bone due to p53 rearrangements may contribute to the maintenance of the tumorigenic phenotype in osteosarcomas.     

基于IPA探析COPB2基因对人骨肉瘤细胞的分子调控机制CSCD

目的基于IPA(ingenuity pathway analysis)的生物信息学分析,探析COPB2对人骨肉瘤细胞的分子调控机制。方法RT-qPCR检测COPB2在人骨肉瘤细胞系U-2OS、MG-63、HOS和Saos-2中的表达水平。以U-2OS为研究对象,分为沉默组(KD)和对照组(NC)。绿色荧光蛋白标记的慢病毒感染U-2OS细胞,沉默组含COPB2沉默序列。人基因表达谱芯片检测COPB2沉默后U-2OS细胞基因表达谱变化差异,将检测信息输入IPA软件进行生物信息学分析,包括经典通路、上游调控、疾病功能、调控效应和互作网络等分析。结果COPB2在四种人骨肉瘤细胞系中表达丰度均高。人基因表达谱芯片显示,KD组与NC组相比,1393个基因表达出现显著差异,其中上调基因数831个、下调基因数562个。IPA经典通路分析显示COPB2敲除后,TSP1抑制血管形成通路、急性期反应通路被显著激活,细胞周期蛋白和细胞周期调控通路显著抑制。疾病功能和互作网路分析显示COPB2在骨肉瘤中发挥重要作用,主要影响细胞瘤变、肿瘤细胞侵袭和失巢凋亡等功能。结论COPB2在人骨肉瘤细胞系中高表达,可能通过细胞周期蛋白和细胞周期调控通路参与骨肉瘤的发生发展。COPB2与骨肉瘤细胞瘤变、侵袭力和失巢凋亡相关。     

导入Rb、p53、p16和H-ras反义RNA对人胃癌细胞恶性增殖的影响

构建Rb、p16、p53、H-ras反义RNA的逆转录病毒载体,利用脂质体介导和病毒感染的方法将其分别转入—存在Rb基因部分缺失、p16低表达、p53缺失和H-ras点突变的胃癌细胞系.观察这些基因对该细胞生长及致癌性的影响,探讨肿瘤相关基因在人胃癌发生、发展过程中的作用.结果表明,通过基因重组技术将多种基因分别重组至逆转录病毒载体PLXSN中,经鉴定获得正插及反插有该基因的重组质粒.将这些质粒分别用脂质体介导和病毒感染的方法转染BGC823细胞,G418筛选2～3周后,获得较高的转染效率.对转染后细胞DNA、RNA进行分析,证明外源性基因已整合人BGC823细胞并获稳定表达.表达有外源性p16、p53基因和H-ras反义RNA的裸鼠致癌性均有明显的抑制作用,而Rb基因对BGC823的恶性增殖能力没有明显抑制.这一结果表明p16、p53、H-ras几个基因的异常可能是导致这一个细胞癌变及最终发展成胃癌的主要原因.本实验进一步明确了胃癌的发生、发展是多基因变异累积的结果.     

Association of Loss of Heterozygosity at the p53 Locus with Chemoresistance in Osteosarcomas

Although the osteosarcoma is considered to be among the most chemosensitive malignancies and preoperative chemotherapy is commonly applied, an appreciable proportion of cases are in fact quite insensitive. Predictive markers for chemosensitivity are therefore desirable in order to develop effective treatment strategies. Thirty-two cases of conventional osteosarcomas treated at the Cancer Institute Hospital, Tokyo, were analyzed. The sensitivity to preoperative chemotherapy was investigated with reference to loss of heterozygosity (LOH) at the 17p13 ( p 53) and 13q14 ( Rb ) loci and expression of the cell-cycle associated proteins, p53, Rb, p21/Waf-1, mdm-2 and Ki-67, as detected immunohistochemically. LOH was detected by analyzing polymerase chain reaction products at marker microsatellite loci. The efficacy of chemotherapy was evaluated both radiologically and histologically. LOH at p 53 or Rb loci was seen in 54% (13/24) and 58% (14/24) of cases, respectively. Only 15% of osteosarcomas with LOH at the p 53 locus were sensitive to preoperative chemotherapy, as compared to 64% of tumors without such loss ( P <0.05). A similar but much less distinct tendency was observed with LOH at the Rb locus. No relationship was evident between chemosensitivity and immunohistochemical staining patterns for p53, Rb, p21/Waf-1, mdm-2 or Ki-67. The results suggest that p 53 gene deletion, but not the other parameters investigated, may be useful for predicting chemoresistance of osteosarcomas.     

Ras-mediated activation of ERK by cisplatin induces cell death independently of p53 in osteosarcoma and neuroblastoma cell lines

Activation of the mitogen-activated protein kinases ERK1/2 by the chemotherapeutic agent cisplatin has been shown to result in either survival or cell death. The downstream mediators of these opposing effects are unknown, as are the upstream signaling molecules. Activation of ERK is required for accumulation and phosphorylation of p53 following cisplatin treatment. We studied the role of ERK activation after cisplatin treatment under p53-negative and p53-positive conditions using a tetracycline-dependent expression vector in Saos-2 osteosarcoma cells. Dose-dependent activation of ERK first occurred 3-6 h after a 2-h cisplatin incubation and declined after 12-24 h in several tumor cell lines. Incubation of cell lines with the MEK1 inhibitors PD98059 or UO126 after, but not during, cisplatin treatment completely inhibited cisplatin-induced activation of ERK. The activation of ERK by cisplatin was inhibited by transient transfection with dominant-negative Ras-N17 in Saos-2 cells. Treatment of cells with PD98059 or UO126 after cisplatin incubation or inhibition of signaling through ERK by tetracycline-regulated expression of dominant-inhibitory ERK enhanced resistance to cisplatin in p53-negative osteosarcoma cells and reduced cisplatin-induced apoptosis. P53 was stabilized and phosphorylated in a MEK1-dependent manner after cisplatin incubation in Kelly neuroblastoma cells. Inhibition of signaling through ERK increased cell survival after cisplatin treatment in these cells as well. Expression of functional p53 did not change the proapoptotic effects of ERK activation in response to cisplatin in Saos-2 cells. Our results suggest that cisplatin-induced activation of ERK is mediated by Ras. ERK activation increased cisplatin-induced cell death independently of p53 in osteosarcoma and neuroblastoma cell lines.     

Effect of Vinorelbine on cell growth and apoptosis induction in human osteosarcoma in vitro

Vinorelbine (VNR) is a semi-synthetic vinca alkaloid known to exert its antitumour activity by interfering with the polymerisation of tubulin. It has shown a broad spectrum of activity in some advanced carcinomas of lung, breast and ovary. This report demonstrates for the first time the antiproliferative effect of VNR and its molecular mechanism in human osteosarcoma in vitro. TP53 wild-type HOS cells and TP53 mutated MG-63 cells were chosen for this study. In each cell line, VNR caused a significant dose- and time-dependent growth inhibition and induced apoptotic death independent of TP53 status. Phosphorylation and/or alteration of Bcl-2 were not induced by VNR, thereby indicating a new pathway utilised by the drug to induce apoptosis in this tumour in vitro. VNR produced a down-regulation of cyclin D1 and an up-regulation of p53 expression in TP53 wild-type HOS cells, whereas no alteration in cyclin D1 expression was evident in the TP53 negative MG-63 cells. These data suggest a new potential use for Vinorelbine as a therapeutic agent against human osteosarcoma.     

Adenovirus-mediated p53 gene therapy in osteosarcoma cell lines: sensitization to cisplatin and doxorubicin

The poor prognosis for patients with metastatic osteosarcoma (OS) indicates that new therapeutic options should be explored. Studies with adenoviral-mediated p53 gene transfer have been conducted in many cancer types including cervical, ovarian, prostatic and head and neck tumors. However, limited work has been carried out with pediatric cancers, including OS. Using three viral constructs containing cDNA for wild-type p53, mutant p53 (Cys135Ser) and lacZ, we studied the effect of adenoviral-mediated gene therapy in four OS cell lines: Saos-2 (p53-/-), HOS (R156P), KHOS/NP (R156P) and MNNG (R156P, F270L). We demonstrated that the virus efficiently enters the cells using the beta-galactosidase assay. Using the MTT assay, we have shown a dose-dependent decrease in cell viability 72 h post-treatment that occurs with Ad-wtp53 but not with Ad-mutp53. We have also shown that treatment with Ad-wtp53 significantly increases sensitivity of the cell lines to cisplatin and doxorubicin, chemotherapeutic agents commonly used in the treatment of OS. Our results indicate that restoration of wt p53 function in OS cells provides a basis for novel approaches to treatment of this disease.