Increased tumor cell proliferation in mantle cell lymphoma is associated with elevated insulin-like growth factor 2 mRNA-binding protein 3 expression

Mantle cell lymphoma is an aggressive, non-curable B-cell lymphoma, characterized by the translocation t(11;14)(q13;q32) involving CCND1 and a high number of additional genetic alterations. Chromosomal gains of 7p are frequent in mantle cell lymphoma, with insulin-like growth factor II mRNA-binding protein 3 (IGF2BP3 aka IMP3) being the most upregulated gene in this region. IGF2BP3 is a member of the IGF II mRNA-BP family, and increased IGF2BP3 expression is associated with an aggressive behavior in many malignant tumors. We here analyze selected genes related to IGF signaling in gene expression and genomic array data of 8 mantle cell lymphoma cell lines and 12 primary mantle cell lymphomas and study IGF2BP3 protein expression in 172 well-characterized primary mantle cell lymphomas by immunohistochemistry. The majority of mantle cell lymphoma cell lines and primary cases showed elevated IGF2BP3 mRNA expression and a subset also expressed the IGF1 and IGF2 receptors. On the protein level, 66 of 172 primary mantle cell lymphomas showed IGF2BP3 expression in >50% of tumor cells, and strong IGF2BP3 protein expression was highly associated with increased proliferation as measured by the Ki-67 index, but not with overall survival of mantle cell lymphoma patients. Only a subset of mantle cell lymphomas with marked IGF2BP3 expression had an underlying chromosomal gain in 7p, suggesting that additional mechanisms are involved in the upregulation of IGF2BP3 in mantle cell lymphoma. In seven paired mantle cell lymphoma samples, IGF2BP3 protein expression remained constant between primary diagnosis and relapse. Increased IGF2BP3 expression and, potentially, enhanced IGF signaling may contribute proproliferative stimuli in the evolution of mantle cell lymphoma tumor cells.     

Proteoglycans and WT1 as markers for distinguishing adenocarcinoma,epithelioid mesothelioma,and benign mesothelium

Malignant mesothelioma is a tumour frequently accompanied by an effusion with elevated hyaluronan levels. To distinguish malignant mesothelioma, adenocarcinoma, and reactive benign mesothelium with cytological and histological methods including immunocytochemistry is a major diagnostic challenge. The Wilms' tumour susceptibility gene 1 (WT1), expressed during transition of mesenchyme to epithelial tissues, is regarded as a marker for the mesothelial lineage. Its effect on the cell phenotype may be regulated via the syndecans, i.e. proteoglycans on the cell surface. To determine how WT1, proteoglycans, and hyaluronan synthase are expressed in mesothelioma, adenocarcinoma, and reactive benign mesothelium, we studied these molecules at the mRNA and protein levels, with the additional purpose of finding diagnostic parameters. Adenocarcinoma cells produced more mRNA for syndecan-1, but cells derived from mesothelium expressed WT1, biglycan, and larger amounts of syndecan-2. The difference in gene expression of these two syndecans was best monitored by the ratio between them. Syndecan-4 was highly expressed in all malignant cell lines, this expression being 1.7-5 times greater than in benign mesothelial cells. Although hyaluronan synthase-1 and versican could not distinguish between the three conditions, versican expression was associated with a high rate of proliferation. These findings suggest that syndecan-1 and syndecan-2 together may be useful diagnostic markers, with stronger staining for the latter epitope in mesothelial tissues. The alternating appearance of these two syndecans can be shown not only by RT-PCR and FACS in vitro, but also by immunohistochemistry on clinical material.     

Expression of insulin-like growth factor mitogenic signals in adult soft-tissue sarcomas: significant correlation with malignant potential

The insulin-like growth factor (IGF) signal transduction system involves receptors, ligands and binding proteins (IGFBPs) that have been shown to have mitogenic and distinct anti-apoptotic effects on malignant cell lines of both epithelial and mesenchymal origin. Expression of the IGF signal system might be a mechanism by which human soft-tissue sarcomas (STS) obtain a proliferative advantage over normal adjacent tissues. IGFBP2, one of at least six different binding proteins identified to date, is secreted by most sarcoma cell lines and appears to be involved in cell proliferation and transformation. Circulating levels of this protein are markedly increased in malignancy. We have assessed 46 adult STS specimens of low, intermediate and high pathological grade of malignancy for the immunohistochemical expression of IGFBP2, IGF1, IGF2, IGF1 receptor- and - (IGF1R/). The protein expression was measured by quantitative color video image analysis and semi-quantitative evaluation, and the measurements correlated well (Spearman, P<0.001). Using both methods, significant differences in expression of IGFBP2 among each of the three grades, expression of IGF2 between intermediate and high grade, and expression of IGF1R between low-intermediate and low-high grade were observed (Dunnett test, P<0.05). Multiple regression analysis for both quantitative and semi-quantitative data confirmed the significance of the relationship and independence of the proteins, except IGF2. We concluded that IGFBP2 and IGF1R are independent predictors of the malignant potential of adult STS.     

Immunohistochemical distribution patterns of epidermal growth factor receptor in malignant mesothelioma and non-neoplastic mesothelium

Summary An immunohistochemical study of the epidermal growth factor (EGF) receptor in non-neoplastic pleural mesothelium (35 cases) and in human malignant mesothelioma (36 cases) was made, using a murine monoclonal antibody OM-11-951. All malignant mesotheliomas and non-neoplastic pleural biopsies exhibited a strong cytoplasmic immunoreactivity in mesothelial cells. Nuclear immunoreactivity was detected in mesothelial cells of all specimens of both malignant and non-neoplastic pleura. No statistically significant differences were found between malignant mesothelioma and non-neoplastic pleural mesothelium. There were differences, between the three subtypes of mesothelioma, in the number of cells that exhibited nuclear staining. Statistically significant differences were noted between the epithelial subtype and the mesenchymal subtype (P< 0.005), epithelial subtype versus the mixed cell type (P< 0.005) and between the mesenchymal component of the mixed cell type and the mesenchymal type (P<0.0005). We conclude that there is strong expression of EGF receptor in both malignant mesothelioma and in non-neoplastic pleural mesothelium. Different staining patterns are seen when comparing the different subtypes of mesotheliomas with each other. EGF receptor expression cannot be used to distinguish between malignant and benign mesothelium.     

Effect of hepatocyte growth factor on the expression of E- and P-cadherin in gastric carcinoma cell lines

Hepatocyte growth factor (HGF), identical to scatter factor, (SF) is a secretory glycoprotein from fibroblasts which dissociates and increases the motility of various types of epithelial cells. After treatment of three gastric carcinoma cell lines (MKN-28, MKN-45 and TMK-1) with HGF (10 ng/ml), TMK-1 cells lost their tight cell to cell contact and showed marked scattering, while the two other cell lines remained unaffected. To learn about the underlying mechanism of the HGF induced scattering, we examined the expression of adhesion molecules and growth factor/receptor systems at the mRNA and protein level. The observed scattering of treated TMK-1 cells was associated with a reduction in the expression of E- and P-cadherin protein. The respective mRNA levels remained unchanged after HGF/SF treatment. In the two other cell lines, which showed no scattering, there were no changes in the expression of E-and P-cadherin. All other growth factors and their receptors examined (TGF-, EGFR, c-met and c-erbB₂) remained constant and were not affected by HGF treatment. The results suggest that HGF/SF may regulate cell adhesion in gastric carcinomas via E-and P-cadherin expression at the protein level.     

RNAi介导的IGF1R基因沉默对肝癌细胞生长、迁移与侵袭的影响

 目的: 研究RNA干扰(RNAi)介导的胰岛素样生长因子1受体 (IGF1R) 基因沉默对肝癌细胞生长、迁移与侵袭的影响。方法: 设计并筛选抑制IGF1R mRNA表达效率最高的siRNA序列,构建该序列的慢病毒表达载体,转染293T细胞进行病毒包装。将包装好的慢病毒感染Huh7和Hep3B肝癌细胞,筛选沉默 IGF1R 基因表达的稳定细胞株。将上述稳定细胞株扩大培养,检测细胞IGF1R mRNA表达变化,细胞生长、迁移与侵袭能力变化,以及Ki-67、p-AKT、p-ERK1、Gli1、β-catenin、cyclin D1、p21、BCL-XL的蛋白表达水平变化。结果: 与空白及阴性对照组比较,感染携带 IGF1R 干扰序列慢病毒的Huh7和Hep3B肝癌细胞IGF1R mRNA表达水平显著下调,细胞增殖活性明显降低,细胞凋亡显著增加,细胞迁移和侵袭能力明显受到抑制,细胞中IGF1R、Ki-67、p-AKT、p-ERK1、Gli1、β-catenin、cyclin D1、p21及BCL-XL蛋白表达水平均显著降低。结论: RNAi介导的 IGF1R 基因沉默可明显抑制Huh7和Hep3B肝癌细胞生长及恶性生物学特征,这可能与IGF1R表达水平显著下调而引起上述调控细胞增殖、抗凋亡基因以及相关信号通路基因的蛋白表达水平显著降低有关。     

Comparative Phenotypic Studies of Duct Epithelial Cell Lines Derived from Normal Human Pancreas and Pancreatic Carcinoma

We have investigated the mRNA/protein expression of several tyrosine kinase receptors, growth factors, and p16^INK4A cyclin inhibitor in cell lines derived from normal human pancreatic duct epithelium (HPDE) and compared them with those of five pancreatic ductal carcinoma cell lines. Cultured HPDE cells express low levels of epidermal growth factor receptor (EGFR), erbB2, transforming growth factor (TGF)-α, Met/hepatocyte growth factor receptor (HGFR), vascular endothelial growth factor (VEGF), and keratinocyte growth factor (KGF). They also expressed high levels of amphiregulin but did not express EGF and cripto. The expression levels were similar in primary normal HPDE cells and those expressing transfected E6E7 genes of human papilloma virus-16, but their immortalization appeared to enhance the expression of EGFR and Met/HGFR. In comparison, pancreatic carcinoma cell lines commonly demonstrated overexpression of EGFR, erbB2, TGF-α, Met/HGFR, VEGF, and KGF, but they consistently showed marked down-regulation of amphiregulin mRNA expression. In contrast to all carcinoma cell lines that showed deletions of the p16 gene, HPDE cells consistently demonstrated normal p16 genotype and its mRNA expression. This is the first report that compares the phenotypic expression of cultured pancreatic ductal carcinoma cells with epithelial cell lines derived from normal human pancreatic ducts. The findings confirm that malignant transformation of human pancreatic duct cells commonly results in a deregulation of expression of various growth factors and receptors.     

The expression and role of insulin-like growth factor II in malignant hemangiopericytomas

Hemangiopericytoma is a rare soft tissue tumor originating from contractile pericapillary pericytes. To address the issue of molecular genetic events that participate in genesis and progression of hemangiopericytoma we analyzed insulin-like growth factor (IGF) II and IGF I receptor in 29 tumors collected from a human tumor bank network. Seven of these tumors were associated with severe hypoglycemia; six were retroperitoneal and one was located in the leg. Of 22 tumors tested 12 (54.5%) exhibited IGF II mRNA, while almost 90% (17 of 19) of hemangiopericytomas exhibited IGF I receptor mRNA. Sera from some patients whose tumors expressed IGF II mRNA contained elevated levels of IGF II. Removal of the tumor eliminated most of the IGF II immunoreactivity from the sera. The potential role of IGF II as a growth-promoting factor was examined on three malignant primary hemangiopericytoma cell cultures. Extracellular addition of IGF II significantly enhanced cell proliferation in a dose-dependent manner. Antisense oligodeoxynucleotides that specifically inhibit IGF II mRNA, at a concentration of 40 or 80 micrograms/ml, inhibited the growth of hemangiopericytoma cells significantly, by 40%. Simultaneous administration of antisense deoxyoligonucleotides to both IGF II and IGF I receptor inhibited tumor cell proliferation by even 80%. Our data suggest that tumor cells produce IGF II, and that this in turn stimulates their proliferation by autocrine mechanisms.     

Insulin-like growth factor family in malignant haemangiopericytomas: the expression and role of insulin-like growth factor I receptor

Haemangiopericytoma is a rare soft tissue tumour originating from the contractile pericapillary cells. Relatively little is known about its molecular pathogenesis. To address this issue, the insulin-like growth factor family (IGFs) was analysed in 19 tumours collected from a human tumour bank network. Seven of the tumours were associated with severe hypoglycaemia. Of these, six were retroperitoneal and one was located in the leg. 3 out of the 19 tumours (15·8 per cent) were positive for insulin-like growth factor I (IGF I) mRNA and 11 were positive for IGF II mRNA (57·9 per cent). Almost 90 per cent of haemangiopericytomas expressed IGF I receptor (IGF IR) mRNA (17 out of 19), five (26·3 per cent) expressed IGF binding protein 1 (IGF BP1), three (15·8 per cent) expressed IGF BP2, and four (21 per cent) exhibited IGF BP3 mRNA. All of the 14 haemangiopericytomas examined with regard to specific receptor binding were IGF IR positive, ranging from 1·2 to 16·2 per cent. Binding was much higher in IGF I/IGF IR positive tumours (15·3±0·7) than in IGF I negative/IGF IR positive tumours (5·1±3·3). The potential role of IGF IR as a growth promoting factor in malignant haemangiopericytoma was studied using antisense oligonucleotides and monoclonal antibody αIR3 that specifically inhibit IGF IR synthesis or activity. 10 µM IGF IR antisense oligonucleotides significantly inhibited the growth of haemangiopericytoma cells in culture, by around 50 per cent; monoclonal antibody against IGF IR (αIR3) also significantly inhibited proliferation. The data suggest that IGF IR may play an important role in the genesis and progression of malignant haemangiopericytomas. Copyright © 1999 John Wiley & Sons, Ltd.     

Altered expression of insulin-like growth factor -1 and insulin like growth factor binding proteins-2 and 5 in the mouse mutant Hypodactyly (Hd) correlates with sites of apoptotic activity

Insulin-like growth factor-I (IGF-I) mediated signalling has been implicated to be of significant importance during vertebrate embryonic development. IGF-I signalling has also been shown to be modulated by a number of IGF binding proteins that are thought to act as either agonists or antagonists of IGF activity. IGF-I has been implicated in a number of cellular processes, including cell division and programmed cell death (apoptosis). We have used the mouse mutant Hypodactyly (Hd) as a tool to determine the role of IGF-I and two key IGF binding proteins (IGFBP-2 and IGFBP-5) during embryonic development. The Hd mutant is a good model with which to study developmental cascades, since it has a distinct phenotype in the limb where cellular and molecular circuits have been thoroughly investigated. The distinctive pointed limb buds observed in Hd mutant embryos have been shown to be the result of a massive increase in apoptosis. We show that all three genes, IGF-1, IGFBP-2 and IGFBP-5, display restricted expression patterns during limb development. Indeed, IGFBP-5 shows a remarkable similarity to the expression of Engrailed-1, which is the vertebrate homologue of the Drosophila selector gene Engrailed. We show that there is downregulation in the expression of IGFBP-2 in the entire apical ectodermal ridge (AER) in homozygous Hd/Hd limb buds, whereas IGFBP-5 is downregulated in specific regions in the mutant AER. IGF-I expression is downregulated in Hd limb buds in regions undergoing high levels of cell death, consistent with its proposed role as an anti-apoptotic factor, while IGFBP-5 is found at higher levels in regions of cell death, consistent with reports of its association with apoptosis in adult tissues. We propose that these three components of the IGF axis could be involved in the manifestation of the mutant phenotype in Hypodactyly, and that this is probably a result of their ability to regulate cell survival and cell death. Accepted: 1 February 2000     

Vascular endothelial growth factor is an autocrine growth factor in human malignant mesothelioma

Vascular endothelial growth factor (VEGF), a potent mitogen for vascular endothelium, is expressed in malignant pleural mesothelioma (MM). The present report examines the effect of VEGF on MM growth. Four MM cell lines produced significantly higher VEGF levels than normal mesothelial cells (1946+/-14 pg/ml vs. 180+/-17 pg/ml; p<0.001). In addition, MM cells expressed the tyrosine kinase-related VEGF receptors Flt-1 and KDR. Recombinant human VEGF phosphorylated both Flt-1 and KDR and increased proliferation of all four MM cell lines in a dose-dependent fashion. Neutralizing antibodies against either VEGF, Flt-1 or KDR significantly reduced MM cellular proliferation. In addition, expression of VEGF, Flt-1, and KDR was observed in MM biopsies. Moreover, higher VEGF levels were found in the pleural effusions of MM patients than in the effusions of patients with non-malignant pleural disease (1885.7+/-894.9 pg/ml vs. 266.9+/-180.5 pg/ml; p<0.001). Linear regression analysis showed a significant inverse correlation between serum VEGF levels and MM patient survival (r=0.72; p<0.01). No correlation was found between tumour vessel density and either serum (r=0.26; p=0.42) or pleural effusion (r=0.35; p=0.26) VEGF levels. These results indicate that VEGF, via activation of its tyrosine kinase receptors, may be a key regulator of MM growth. In addition, VEGF production could have an impact on patient survival, not only by promoting tumour angiogenesis but also by directly stimulating tumour growth.     

Insulin-like growth factors and insulin-like growth factor binding proteins in the endometrium. Effect of intrauterine levonorgestrel delivery

Insulin-like growth factor (IGF) system is one of the growth factor systems that are believed to modulate steroid hormone actions in the endometrium through autocrine/paracrine mechanisms. IGF-I and IGF-II stimulate proliferation and differentiation, and maintain differentiated cell functions in several cell types in vitro. Endometrial stromal cells produce IGF-I and IGF-II as well as the high affinity IGF-binding proteins (IGFBP), whereas epithelial cells and, in a lesser amount, stromal cells contain cell membrane receptors for IGF. Oestrogen stimulates IGF-I gene expression, and IGF-II gene expression is associated with endometrial differentiation. The mRNA of six high affinity IGFBPs, which can modulate IGF actions, are expressed in human endometrium. The most abundant IGFBP in human endometrium is IGFBP-1, which is secreted by predecidualized/decidualized endometrial stromal cells in late secretory phase and during pregnancy. The primary negative regulator of IGFBP-1 production is insulin. IGFBP-1 competes with type I IGF receptor for binding of IGF in the endometrium and in cultured human trophoblastic cells. IGF-I mRNA is suppressed and mRNA encoding IGF-II and IGFBP-1 are consistently up-regulated in decidualized endometrium in women treated with the intrauterine levonorgestrel system (LNG-IUS). Strong cytoplasmic staining for IGFBP-1 was detected in decidualized endometrium in women using LNG-IUS for contraception or for endometrial protection during post-menopausal oestrogen replacement therapy. Simultaneously, oestrogen receptors were present, while progesterone receptors were hardly detectable in the endometrium by immunohistochemistry. The latter findings suggest that suppression of IGF-I action by IGFBP-1 may be one of the molecular mechanisms accounting for progestagenic and anti-oestrogenic effects of LNG-IUS in the endometrium. Consequently, examination of local IGF-I, IGF-II and IGFBP-1 expression might provide additional information when evaluating the effect of different progestins on the endometrium at the molecular level.     

人胰岛素样生长因子1基因转染对大鼠骨骼肌成肌细胞缺血再灌注损伤的影响

目的: 探讨人胰岛素样生长因子1(hIGF-1)基因转染对大鼠骨骼肌成肌细胞体外缺血再灌注损伤的影响及机制。方法: 分别用pLghIGF-1SN质粒(IGF组)和对照质粒pLgGFPSN(GFP 组)转染大鼠成肌细胞。未转染的成肌细胞(control组)作为细胞对照。转染后用免疫细胞化学、RT-PCR及ELISA检测基因表达。转染后3~14 d检测各组细胞的扩增率。转染的细胞接受缺血再灌注损伤后7 d,TUNEL法检测各组细胞的凋亡百分数,RT-PCR 检测各组细胞的bax和bcl-2 mRNA的表达,Western blotting检测各组细胞caspase-3的表达。结果: 基因转染后免疫细胞化学和RT-PCR 检测发现IGF组细胞有hIGF-1表达,GFP组和control组细胞未见hIGF-1表达;IGF组细胞上清中可检测到hIGF-1的蛋白表达,control组和GFP组细胞上清中未检测到hIGF-1蛋白表达。转染后14 d,IGF组细胞的扩增率显著大于control组和GFP组(P< 0.05);细胞经缺血再灌注损伤后,TUNEL染色检测结果显示:与GFP组相比,IGF组细胞的凋亡百分比显著降低(P< 0.05);RT-PCR 检测结果显示:与GFP组相比,IGF组细胞的bax mRNA表达显著降低,bcl-2 mRNA的表达显著增加(P< 0.05);Western blotting检测结果显示:与GFP组相比,IGF组细胞caspase-3蛋白表达显著降低。结论: IGF-1基因转染可增加成肌细胞的抗凋亡能力,其机制可能和降低Bax和caspase-3表达,增加Bcl-2的表达有关。     

Antibody to type I insulinlike growth factor receptor inhibits growth of Wilms'' tumor in culture and in athymic mice.

The role of the type I insulinlike growth factor (IGF) receptor in regulating growth of Wilms' tumor (WT) was evaluated by examining the effect of antibody-mediated inhibition of this receptor on tumor growth in cell cultures and as heterotransplants in athymic mice. An antibody to the human type I IGF receptor (alpha IR-3) inhibited 125I-IGF-1 binding and prevented stimulation of thymidine incorporation by IGF-1 in vitro. Intraperitoneal administration of alpha IR-3 to nude mice bearing WT heterotransplants prevented tumor growth for 4 weeks and resulted in partial regression of established tumors. These data indicate the importance of IGF action in control of WT growth in vivo, and suggest potential therapeutic application using antigrowth factor receptor antibodies to block growth factor action.     

Progranulin (granulin-epithelin precursor,PC-cell-derived growth factor,acrogranin) mediates tissue repair and tumorigenesis

Progranulin (Pgrn) is a pluripotent secreted growth factor that mediates cell cycle progression and cell motility. It activates the extracellular regulated kinases and phosphatidyl inositol-3 kinase signal cascades, among others, and increases expression of cyclins D and B. Structurally, it belongs to none of the well-established growth factor families. It regulates developmental events as diverse as the onset of cavitation in the preimplantation embryo and male-specific brain differentiation. During wound repair it promotes granulation and neovascularization. It regulates inflammation through a tripartite loop with secretory leukocyte protease inhibitor (SLPI) which protects pgrn from proteolysis, and elastase, which digests it to smaller peptides. Intact pgrn is anti-inflammatory through the inhibition of some of the actions of tumor necrosis factor, while the proteolytic peptides may stimulate the production of proinflammatory cytokines such as interleukin 8. Pgrn is highly expressed in aggressive cancer cell lines and clinical specimens including breast, ovarian, and renal cancers as well as gliomas. In experimental systems it confers an aggressive phenotype on poorly tumorigenic epithelial cancer cells. The malignancy of highly tumorigenic progranulin-expressing cell lines depends on the expression level of the pgrn gene since attenuating pgrn mRNA levels in pgrn-responsive cells greatly inhibits tumor progression. Given its actions in wound repair and tumorigenesis pgrn may prove a useful clinical target, both for prognosis and for therapy.Abbreviations EGF Epidermal growth factor - FAK Focal adhesion kinase - FGF Fibroblast growth factor - grn/epi Granulin-epithelin - IGF Insulin-like growth factor - MAPK Mitogen-activated protein kinase - MEF Mouse embryo fibroblast - PDGF Platelet-derived growth factor - Pgrn Progranulin - PI-3K Phosphatidylinositol-3 kinase - SLPI Secretory leukocyte protease inhibitor - TGF- Transforming growth factor- - TGFe Epithelial transforming growth factor - TNF Tumor necrosis factor - VEGF Vascular endothelial growth factor