Molecular-cloning of the human cycg1 gene encoding a g-type cyclin - overexpression in human osteosarcoma cells

Cyclins constitute a growing family of regulatory proteins that complex with, and activate, protein kinases involved in cell cycle control. Dysregulation of cyclin expression and/or cyclin-dependent kinase (cdk) activities may play a pivotal role in oncogenesis. In this report, we characterize a novel human cyclin gene by molecular cloning. This gene, designated CYCG1, encodes a human homologue of the rat G-type cyclin, exhibiting structural features and conserved sequence motifs of identified G(1) cyclins. The CYCG1 gene is expressed constitutively in synchronized human WI-38 fibroblasts and MG-63 osteosarcoma cells, which is reminiscent of CLN3 in Saccharomyces cerevisiae. Marked overexpression of CYCG1 is observed in a subset of human osteosarcoma cells, providing a potential link to cancer.     

MicroRNAs in osteosarcoma: diagnostic and therapeutic aspects

MicroRNAs (miRNAs) are small RNA molecules, which can interfere with the expression of several genes and act as gene regulator. miRNAs have been proved as a successful diagnostic and therapeutic tool in several cancers. In this review, the differential expression of miRNAs in osteosarcoma and their possibility to be used as diagnostic and therapeutic tools have been discussed. Osteosarcoma is the most common primary bone tumor that mainly affects children and adolescents. The current treatment of osteosarcoma remains difficult, and osteosarcoma causes many deaths because of its complex pathogenesis and resistance to conventional treatments. Several studies demonstrated that the differential expression patterns of miRNAs are a promising tool for the diagnosis and treatment of osteosarcoma. Although some aspect of the mechanism of action of miRNAs in controlling osteosarcoma has been identified (e.g., targeting the Notch signaling pathway), it is far beyond to the clear understanding of miRNA targets in osteosarcoma. Identification of the specific target of miRNAs may aid molecular targets for drug development and future relief of osteosarcoma.     

c-Kit expression in human normal and malignant stem cells prognostic and therapeutic implications

The human stem cell factor/c-Kit signaling pathway is pivotal for the survival of embryonic, foetal and adult stem cells and for their fundamental role in generating healthy functioning cell and tissue types during embryonic, foetal and adult life. Common biological features between human stem cells and cancer cells include (A) self-renewal, (B) extensive capacity of proliferation, (C) migration to and homing at distant sites and (D) resistance to toxic agents. Given these shared attributes, cancer was proposed to originate from transforming mutation(s) in normal stem cells that dysregulate their physiological programs. This theory has been recently supported by the findings that among all malignant cells within a particular tumour, only cell fraction expressing stem cell markers such as c-Kit named 'cancer stem cells' has the exclusive potential to generate tumour cell population. The involvement of c-Kit and its mutation in various haematological malignancies and solid tumours are reviewed. The impacts of dysregulated c-Kit as oncogenic tyrosine kinase on autocrine stimulation and resistance to chemotherapy of cancer stem cells are evaluated. The significance and efficacy of molecular therapeutic targeting of c-Kit signaling pathway in the management of patients with c-Kit-positive malignancies are appraised.     

Molecular pathways: vasculogenic mimicry in tumor cells: diagnostic and therapeutic implications

Tumor cell vasculogenic mimicry (VM) describes the functional plasticity of aggressive cancer cells forming de novo vascular networks, thereby providing a perfusion pathway for rapidly growing tumors, transporting fluid from leaky vessels, and/or connecting with endothelial-lined vasculature. The underlying induction of VM seems to be related to hypoxia, which may also promote the plastic, transendothelial phenotype of tumor cells capable of VM. Since its introduction in 1999 as a novel paradigm for melanoma tumor perfusion, many studies have contributed new insights into the underlying molecular pathways supporting VM in a variety of tumors, including melanoma, glioblastoma, carcinomas, and sarcomas. In particular, critical VM-modulating genes are associated with vascular (VE-cadherin, EphA2, VEGF receptor 1), embryonic and/or stem cell (Nodal, Notch4), and hypoxia-related (hypoxia-inducible factor, Twist1) signaling pathways. Each of these pathways warrants serious scrutiny as potential therapeutic, vascular targets, and diagnostic indicators of plasticity, drug resistance, and the aggressive metastatic phenotype.     

Retroperitoneal schwannomas: diagnostic and therapeutic implications

AIMS AND BACKGROUND: Schwannomas are a rare group of soft-tissue tumors that are derived from the peripheral nerve sheath and rarely develop in the retroperitoneum. METHODS AND STUDY DESIGN: We reviewed the clinicopathological features of 4 patients referred to our unit between October 1999 and March 2004 who on radiological examination were diagnosed with pancreatic, adrenal, psoas and retroperitoneal fat tissue tumors and subsequently underwent surgical treatment. RESULTS: The preoperative diagnosis was incorrect in all cases. At time of surgery, we found a mass probably arising from the adrenal gland in 2 patients, a lesion originating from the femoral nerve in 1 patient, and a retroperitoneal mass without a clear site of origin in 1 patient. Pathological evaluation revealed schwannomas in all cases, with no signs of malignancy. Complete surgical excision was performed in all patients without any major postoperative complications. At the time of writing all patients are alive with no evidence of local or distant recurrence. CONCLUSIONS: Radical surgical excision is considered the best treatment for these neoplasms, resulting in a very good longterm prognosis.     

c-Abl expression in chronic lymphocytic leukemia cells: clinical and therapeutic implications

c-Abl is important for normal B-cell development, but little is known about the function of this nonreceptor tyrosine kinase in chronic lymphocytic leukemia (CLL). Therefore, the aim of the present study was to examine the clinical, therapeutic, and pathogenetic importance of c-Abl in this disease. We show that the malignant cells of CLL predominantly express the type 1b splice variant of c-Abl and that the expression of c-Abl protein is higher in CLL cells than in normal peripheral blood B cells. Moreover, we show that the levels of c-Abl protein expression correlate positively with tumor burden and disease stage, and negatively with IgV(H) mutation. We also show that STI-571, an inhibitor of c-Abl kinase activity, induces apoptosis of CLL cells with high c-Abl expression levels through a mechanism involving inhibition of nuclear factor kappaB. We conclude that overexpression of c-Abl is likely to play a pathogenetic role in CLL and that STI-571 may be of potential use in the treatment of this disease.     

骨肉瘤组织中Survivin基因表达及其与细胞周期蛋白D1表达的关系

目的 :探讨骨肉瘤中抗凋亡基因Survivin的表达状态及其与细胞周期蛋白D1(cyclinD1)表达的关系。方法 :采用原位杂交、免疫组织化学技术、DNA原位末端标记 (TUNEL)法检测 3 8例骨肉瘤、15例骨软骨瘤、5例正常骨组织中SurvivinmRNA表达及骨肉瘤组织中cyclinD1表达和细胞凋亡情况 ,比较SurvivinmRNA表达与骨肉瘤临床病理参数及cyclinD1表达的关系。结果 :SurvivinmRNA在正常骨和骨软骨瘤组织无明显表达 ,但显著表达于骨肉瘤中 65 8% ( 2 5 /3 8) ,其表达率与骨肉瘤组织学分级无关 ,与WHO分型及转移有关 ,P <0 0 5 ;骨肉瘤细胞凋亡指数(AI)均值为 8 3 % ( 0～ 17% ) ,Survivin阴性组AI值显著高于阳性组 ,P <0 0 1;骨肉瘤中cyclinD1过度表达 ( 71 1% ,2 7/3 8) ,其与Survivin表达正相关 ,rs=0 3 70 ,P <0 0 5。结论 :Survivin选择性表达于骨肉瘤组织 ,通过抑制细胞凋亡 ,并与cyclinD1协同作用参与骨肉瘤的发生发展 ,Sur vivin可能是评价骨肉瘤预后的重要参考指标     

Hypoxia increases the expression of stem cell markers in human osteosarcoma cells

Osteosarcoma (OS) is the most common primary malignant tumor of bone. It is a common phenomenon that osteosarcoma cells have a hypoxic microenvironment. Hypoxia can dedifferentiate cells of several malignant tumor types into stem cell-like phenotypes. However, the role of hypoxia in stemness induction and the expression of cancer stem cell (CSC) markers in human osteosarcoma cells has not been reported. The present study examined the effects of hypoxia on stem-like cells in the human osteosarcoma MNNG/HOS cells. Under the incubation with 1% oxygen, the expression of CSCs markers (Oct-4, Nanog and CD133) in MNNG/HOS cells were increased. Moreover, MNNG/HOS cells cultured under hypoxic conditions were more likely to proliferate into spheres and resulted in larger xenograft tumor. Hypoxia also increased the mRNA and protein levels of hypoxia-inducible factor (HIF)-1α. Then rapamycin was used, which has been shown to lower HIF-1α protein level, to inhibit the hypoxic response. Rapamycin suppressed the expression of HIF-1α protein and CSCs markers (Oct4, Nanog and CD133) in MNNG/HOS cells. In addition, pretreatment with rapamycin reduced the efficiency of MNNG/HOS cells in forming spheres and xenograft tumors. The results demonstrated that hypoxia (1% oxygen) can dedifferentiate some of the MNNG/HOS cells into stem cell-like phenotypes, and that the mTOR signaling pathway participates in this process via regulating the expression of HIF-1α protein.     

miR-132 targeting cyclin E1 suppresses cell proliferation in osteosarcoma cells

In this study, we investigated the roles of miR-132 in tumor growth of osteosarcoma. We found that overexpression of miR-132 significantly suppressed in vitro cell proliferation and in vivo tumor growth. In addition, miR-132 overexpression induced G1/S cell cycle arrest of osteosarcoma cells. Further study showed that miR-132 could interact with the 3′-untranslated region of cyclin E1 (CCNE1) gene and repress its expression. Re-expression of CCNE1 (without the 3′UTR) could partially abrogate the miR-132-induced cell proliferation inhibition. Of significance, contrary to CCNE1, expression level of miR-132 was significantly lower in osteosarcoma tissues than in the adjacent normal tissues. Taken together, these results indicate that miR-132 functions as a tumor suppressor in osteosarcoma and that its suppressive effects are mediated chiefly by repressing CCNE1 expression.     

Cyclin D1 and cyclin E expression in malignant thyroid cells and in human thyroid carcinomas

Evidence of the involvement of cyclin gene alterations in human cancer is growing. In this study, we sought to determine the pattern of expression of cyclin D1 and cyclin E in normal and malignant thyroid cells. Quiescent rat thyroid cells in culture, induced to synthesize DNA by thyrotropin (TSH), expressed cyclin D1 gene after 6 hr and cyclin E gene with a peak at 18 hr from the stimulus; K-ras-transformed rat thyroid cells, which grew without addition of hormones necessary for normal cell proliferation, expressed elevated levels of cyclin D1 and cyclin E, compared with normal differentiated thyroid cells. Human benign and malignant thyroid tumors and their relative normal tissues were then analyzed. Neither major genetic alterations nor amplifications for cyclin D1 and cyclin E genes were found by Southern blot analysis in genomic DNAs extracted from all types of thyroid tumors. Moreover, statistical analyses of densitometric values from Northern blots did not show increased levels of cyclin D1 and E mRNAs in the tumor samples, compared with normal thyroid. Immunohistochemical analyses of formalin-fixed, paraffin-embedded sections of tissues with specific antibodies revealed a prevalent cytoplasmic cyclin E staining in the thyroid tissues analyzed. Cyclin D1, instead, was present in the cytoplasm of normal thyroids and adenomas, but in 31% of thyroid papillary carcinomas analysed, it was overexpressed, with a localization in the nucleus. Our in vivo observations suggest that unlike cyclin E, elevated nuclear cyclin D1 expression defines a subset of thyroid papillary carcinomas, and might be a contributory factor to thyroid tumorigenesis. Int. J. Cancer 76:806–811, 1998.© 1998 Wiley-Liss, Inc.     

Interleukin (IL)-12 and IL-12 gene transfer up-regulate Fas expression in human osteosarcoma and breast cancer cells

Expression of Fas (CD95, APO-1), a cell surface receptor capable of inducing ligand-mediated apoptosis, is involved in tissue homeostasis and elimination of targeted cells by natural killer and T cells. Corruption of this pathway, such as reduced Fas expression, can allow tumor cells to escape elimination and promote metastatic potential. In this study, the status of Fas expression has been examined in the parental SAOS human osteosarcoma cells that do not metastasize and in selected variants that cause lung metastases in 16 weeks (LM2) or 8 weeks (LM6) after i.v. injection into nude mice. Fas expression correlated with the metastatic potentials of the three cell lines. Northern and fluorescence-activated cell-sorting analyses indicated that LM6 cells expressed Fas at a lower level than seen in the parental cells. Infection of the LM6 cells with an adenoviral vector containing the murine interleukin (IL)-12 gene (AD:mIL-12) or treatment with recombinant murine IL-12 resulted in a dose-dependent up-regulation of FAS: The up-regulation of Fas by IL-12 was also demonstrated in human etoposide-resistant MDA-MB-231 breast cancer cells. [(3)H]Thymidine growth inhibition studies indicated that the cell surface Fas induced after IL-12 exposure was functional and able to mediate cell death on cross-linking with anti-FAS: We also demonstrate that this effect is independent of IFN-gamma. Whereas these cell lines are sensitive to IFN-gamma, incubation with IFN-gamma does not increase susceptibility to Fas-mediated cell death, nor do these cells produce IFN-gamma with or without IL-12 treatment. We hypothesize that expression of Fas may play a role in the elimination of metastatic tumor cells in the lung, an organ in which Fas ligand is expressed. The antitumor activity of IL-12 may be secondary in part to its ability to up-regulate Fas expression on tumor cells, which subsequently increases immune-mediated destruction of osteosarcoma cells.     

Chemokine receptors in advanced breast cancer: differential expression in metastatic disease sites with diagnostic and therapeutic implications

Background: We investigated the expression of CXCR4, CCR7, estrogen receptor (ER), progesterone receptor (PR) and HER2-neu in human metastatic breast cancers to determine whether these biological biomarkers were preferentially expressed in any organ-specific metastases.Materials and methods: CXCR4, CCR7, ER, PR and HER2-neu expression levels were evaluated by immunohistochemical staining using paraffin-embedded tissue sections of metastatic breast cancers (n = 41) obtained by either diagnostic biopsy or surgical resection.Results: The metastatic sites included the following: bone (n = 15), brain (n = 14), lung (n = 6), liver (n = 2), and omental metastases (n = 2). CXCR4 was expressed in 41% of cases, CCR7 expression was demonstrated in 10%, and HER2-neu overexpression was present in 27%. CXCR4 was more likely to be expressed in bone metastases than visceral metastases (67% versus 26%, P = 0.020). Visceral sites demonstrated a lower rate of CXCR4 positivity (33% and 23%, respectively, for lung and brain metastases). Similarly, CCR7 was more likely to be found in bone metastases than visceral sites (27% versus 0%, P = 0.037).Conclusions: These results indicate that CXCR4 can contribute to the homing of breast cancer cells to the bone. This finding might have important clinical implications since patients with metastatic bone disease may achieve the highest benefit from a CXCR4-targeted therapy.     

MicroRNA in pancreatic cancer: Pathological,diagnostic and therapeutic implications

MicroRNAs (miRNAs) are a group of small non-coding RNA molecules of 17–25 nucleotides (nt) in length, predicted to control the activity of about 30% of all protein-coding genes in mammals. Altered expressions of miRNAs are reported in various cancers and may associate with cancer pathogenesis, apoptosis, and cell growth, thereby functioning as either tumor suppressors or oncogenes. Recent reports showed that deregulation of miRNA contribute to tumor development and progression and hence, have diagnostic and prognostic value in several human malignancies. This review discusses the current status of miRNA in pancreatic cancer development, progression, diagnosis, and therapy.     

雌激素受体在骨肉瘤中的表达及治疗中的作用的研究进展

近年来,雌激素受体在部分生殖及非生殖系统肿瘤中发现有高表达,因成骨细胞中同样发现有雌激素受体的表达,拟雌激素类药物已广泛应用于抗骨质疏松治疗。在骨肉瘤组织及细胞系中,多数研究认为雌激素受体有表达,且在细胞系中应用抗激素及激素刺激均有效,同时发现应用于乳腺癌内分泌治疗的雌激素受体调节类抗癌药物在其他肿瘤中具有化疗增敏效应。这些发现可能是对于骨肉瘤患者,尤其是对于发现有肺部转移且化疗反应差的患者是一种新的有效的治疗手段。雌激素相关抗癌疗法在非骨肉瘤类的许多肿瘤研究中已有大量报道,本文主要介绍雌激素受体在骨肉瘤中的表达及治疗中的作用的研究进展。     

Metabolic alterations in cancer cells and therapeutic implications

Cancer metabolism has emerged as an important area of research in recent years. Elucidation of the metabolic differences between cancer and normal cells and the underlying mechanisms will not only advance our understanding of fundamental cancer cell biology but also provide an important basis for the development of new therapeutic strategies and novel compounds to selectively eliminate cancer cells by targeting their unique metabolism. This article reviews several important metabolic alterations in cancer cells, with an emphasis on increased aerobic glycolysis (the Warburg effect) and glutamine addiction, and discusses the mechanisms that may contribute to such metabolic changes. In addition, metabolic alterations in cancer stem cells, mitochondrial metabolism and its influence on drug sensitivity, and potential therapeutic strategies and agents that target cancer metabolism are also discussed.     

Increased Fas expression reduces the metastatic potential of human osteosarcoma cells.

PURPOSE: The process of metastasis requires the single tumor cell that seeds the metastatic clone to complete a complex series of steps. Identifying factors responsible for these steps is essential in developing and improving targeted therapy for metastasis. Resistance to receptor-mediated cell death, such as the Fas/Fas ligand pathway, is one mechanism commonly exploited by metastatic cell populations. EXPERIMENTAL DESIGN AND RESULTS: LM7, a subline of the SAOS human osteosarcoma cell line with low Fas expression, was selected for its high metastatic potential in an experimental nude mouse model. When transfected with the full-length Fas gene (LM7-Fas), these cells expressed higher levels of Fas than the parental LM7 cells or LM7-neo control-transfected cells. These cells were also more sensitive to Fas-induced cell death than controls. When injected intravenously into nude mice, the LM7-Fas cell line produced a significantly lower incidence of tumor nodules than control cell lines. Lung weight and tumor nodule size were also decreased in those mice injected with LM7-Fas. Levels of Fas were quantified in osteosarcoma lung nodules from 17 patients. Eight samples were Fas negative, whereas the remaining 9 were only weakly positive compared with normal human liver (positive control). CONCLUSIONS: Our results demonstrate that altering Fas expression can impact the metastatic potential of osteosarcoma cells. We conclude that the increase of Fas on the surface of the LM7 osteosarcoma cells increased their sensitivity to Fas-induced cell death in the microenvironment of the lung, where Fas ligand is constitutively expressed. Thus, loss of Fas expression is one mechanism by which osteosarcoma cells may evade host resistance mechanisms in the lung, increasing metastatic potential. Fas may therefore be a new therapeutic target for osteosarcoma.     

GAS7在人骨肉瘤中的表达及其意义

Objective： To detect the expression of GAS7 in osteosarcoma and discuss its significance. Methods： Immunohistochemistry SABC method was applied to detect GAS7 expression in specimens of 54 osteosarcoma and 15 osteochondroma cases. Results： The positive expression rate of GAS7 was 74.7% （40/54） in the group of osteosarcoma and 0% （0/15） in the group of osteochondroma. There was a significant difference in the comparison of GAS expression in these two groups （P 〈 0.05）. GAS7 was higher expressed in the samples which complicated with relapse or pulmonary metastasis than the control group （P 〈 0.05）. There was no significant association between GAS7 expression and the size of tumor, the gender or the age of the patients （P 〉 0.05）. Conclusion： The hyper-expression ot GAST may play an important role in the initiation and development of human osteosarcoma.     

Prognostic and therapeutic implications of increased ATP citrate lyase expression in human epithelial ovarian cancer

Altered metabolism is one of the most significant features of cancer cells. ATP citrate lyase (ACL), a key enzyme in de novo lipid synthesis, has been reported to be overexpressed or activated in several cancer types. To determine the role of ACL in ovarian cancer progression, we detected ACL expression in human epithelial ovarian cancer tissues. qRT-PCR and western blotting showed higher ACL expression in malignant tissues compared to normal ovarian tissues. Immunohistochemical analysis showed that phosphorylated ACL was increased in ovarian cancer tissues and that its expression correlated well with tumor grade, FIGO stage and poorer prognosis. To explore the therapeutic potential of ACL, we assessed the effect of ACL-siRNA on cellular proliferation and cell cycle distribution. ACL knockdown inhibited cellular proliferation and induced cell cycle arrest in A2780 cells. Taken together, our findings suggest that ACL may contribute to the pathogenesis of human epithelial ovarian cancer, and may serve as a novel therapeutic target.     

Prognostic and therapeutic relevance of HER2 expression in osteosarcoma and Ewing's sarcoma

Expression of HER2 was evaluated by immunohistochemical techniques in 84 osteosarcoma (OS) and 113 Ewing's sarcoma (ES) paraffin-embedded tumour biopsies. HER2 gene status was also assessed in a panel of cell lines as well as in vitro efficacy of trastuzumab (a humanised antibody directed against HER2) as single agent or in combination with the insulin-like growth factor I receptor (IGF-IR) IR3 antibody. Overexpression of HER2 was present in 32% of OS and 16% of ES and was significantly associated with the increased expression of P-glycoprotein, a surface molecule responsible for multidrug resistance. Event-free survival analyses revealed a prognostic value for HER2 and/or P-glycoprotein expression in OS, but not in ES. However, despite its prognostic relevance, no therapeutic effectiveness was observed pre-clinically for trastuzumab-driven therapy, in both OS or ES cell lines, unless the antibody was associated with anti-IGF-IR targeting strategies. Therefore, the therapeutic potential of trastuzumab in these neoplasms may be better exploited in combined treatments with anti-IGF-IR approaches.