Expression of transforming growth factor alpha and epidermal growth factor receptor messenger RNA in neoplastic and nonneoplastic human kidney tissue

Using Northern blot analysis, we have demonstrated that mRNA for transforming growth factor alpha (TGF-alpha) was expressed in five malignant kidney tissue specimens but was not detected in their autologous nonneoplastic homologues. In addition, the expression of epidermal growth factor (EGF) receptor mRNA in these malignant tissues was 2- to 3-fold greater than in nontransformed tissues. In two cases examined using immunohistochemistry, we were able to correlate the increased expression of the mRNA with an increase in protein expression. Since TGF-alpha is known to bind to the EGF receptor, the finding of an increased expression of both TGF-alpha and EGF receptor mRNA in kidney tumor tissue suggests that interaction between TGF-alpha and the EGF receptor may play a role in promoting transformation and/or proliferation of kidney neoplasms, perhaps by an autocrine mechanism.     

Platelet-derived growth factor receptor alpha in glioma: a bad seed

Recent collaborative, large-scale genomic profiling of the most common and aggressive brain tumor glioblastoma multiforme(GBM) has significantly advanced our understanding of this disease. The gene encoding platelet-derived growth factor receptor alpha(PDGFRα) was identified as the third of the top 11 amplified genes in clinical GBM specimens. The important roles of PDGFRα signaling during normal brain development also implicate the possible pathologic consequences of PDGFRα over-activation in glioma. Although the initial clinical trials using PDGFR kinase inhibitors have been predominantly disappointing, diagnostic and treatment modalities involving genomic profiling and personalized medicine are expected to improve the therapy targeting PDGFRα signaling. In this review, we discuss the roles of PDGFRαsignaling during development of the normal central nervous system(CNS) and in pathologic conditions such as malignant glioma. We further compare various animal models of PDGF-induced gliomagenesis and their potential as a novel platform of pre-clinical drug testing. We then summarize our recent publication and how these findings will likely impact treatments for gliomas driven by PDGFRα overexpression. A better understanding of PDGFRα signaling in glioma and their microenvironment, through the use of human or mouse models, is necessary to design a more effective therapeutic strategy against gliomas harboring the aberrant PDGFRα signaling.     

Expression of messenger RNAs for platelet-derived growth factor and its receptors in human sarcoma cell lines

Growth factors of the platelet-derived growth factor (PDGF) family have been thought to possess autocrine functions in certain neoplasms of mesenchymal and glial origin. This notion has been based on observations that these tumors express PDGF genes and produce PDGF-like growth factors. Corresponding data on PDGF receptor expression in sarcoma cell lines is essentially lacking. The cloning of cDNA for 2 distinct PDGF receptors with different abilities to recognize the members of the PDGF family and availability of recombinant PDGF for binding studies have recently made it possible to study the expression of both receptor types in tumor cell lines. We present here a study on 8 human sarcoma cell lines, and show a large variability and independency in the expression of the 2 PDGF receptor types as well as of the genes encoding the corresponding ligands.     

Platelet-derived growth factor-AA is an essential and autocrine regulator of vascular endothelial growth factor expression in non-small cell lung carcinomas

It is widely accepted that angiogenesis is required for tumor progression. Vascular endothelial growth factor (VEGF) is a key molecule for tumor angiogenesis; however, its expressional regulation is not well understood during all stages of tumorigenesis. Using cell lines and surgical specimens of human non-small cell lung cancers (NSCLCs), we here show that platelet-derived growth factor-AA (PDGF-AA) is an essential autocrine regulator for VEGF expression. To directly assess the expression of PDGF-AA-dependent VEGF and its roles in tumorigenesis, we stably transfected established cell lines with their antisense genes. In addition, the levels of PDGF-AA and VEGF expression in surgical sections were measured and compared with clinicopathologic findings such as tumor size and patient prognosis. PDGF-AA tightly regulated VEGF expression and had a greater effect on tumor size and patient prognosis than did VEGF in both cell lines and surgical sections. PDGF-AA expression was not seen in the atypical adenomatous hyperplasia at all, whereas VEGF was occasionally seen. Furthermore, the frequency of VEGF expression was higher in advanced NSCLCs than in precancerous lesions, which was tightly correspondent to the results for PDGF-AA. These results indicate that PDGF-AA is an important regulator of the frequency and level of VEGF expression during the transition from a precancerous lesion to advanced cancer. The PDGF-AA/VEGF axis, therefore, may be a ubiquitous autocrine system for enhancing angiogenic signals, and PDGF-AA, and its related pathways could be a more efficient target of antiangiogenic therapy for cancers than VEGF and its pathways.     

Antitumor and antimetastatic activity of ribozymes targeting the messenger RNA of vascular endothelial growth factor receptors.

Chemically stabilized hammerhead ribozymes are nuclease-resistant, RNA-based oligonucleotides that selectively bind and cleave specific target RNAs. Due to their potential for specifically inhibiting gene expression, ribozymes are being investigated for therapeutic applications as well as for the elucidation of gene function. In particular, we have investigated ribozymes that target the mRNA of the vascular endothelial growth factor (VEGF) receptors because VEGF signaling is an important mediator of tumor angiogenesis and metastasis. Here we report pharmacodynamic studies testing anti-Flt-1 (VEGFR-1) and anti-KDR (VEGFR-2) ribozymes in animal models of solid tumor growth and metastasis. Ribozymes targeting either Flt-1 or KDR significantly inhibited primary tumor growth in a highly metastatic variant of Lewis lung carcinoma. However, only treatment with the anti-Flt-1 ribozyme resulted in a statistically significant and dose-dependent inhibition of lung metastasis in this model. The anti-Flt-1 ribozyme was then tested in a xenograft model of human metastatic colorectal cancer in which significant inhibition of liver metastasis was observed. Taken together, these data represent the first demonstration that synthetic ribozymes targeting VEGF receptor mRNA reduced the growth and metastasis of solid tumors in vivo.     

Expression of leukemia inhibitory factor and its receptor in breast cancer: A potential autocrine and paracrine growth regulatory mechanism

Leukemia inhibitory factor (LIF) is a pluripotent cytokine which has a diverse array of effects on hematopoietic and epithelial cells. Depending on the nature of the target cells, these effects can be growth-stimulatory or growth-inhibitory. Receptors for leukemia inhibitory factor (LIFR) have been identified on a variety of hematopoietic and epithelial cells. We have recently demonstrated in vitro growth stimulation of human breast cancer cells, both primary tumors and cultured cell lines, by LIF. To begin to understand the in vivo relevance of these observations, we investigated the expression of LIF and LIFR in human breast cancer specimens. Specimens from 50 cases were immunostained with mouse monoclonal antibodies D62.3 and M1 (to stain for LIF and LIFR, respectively). LIF expression was observed in 78% of the specimens and correlated with favorable biological features, i.e. low S-phase fraction (SPF) (P=0.001) and diploidy (P = 0.08). LIFR expression was observed in 80% of the tumors and correlated with the presence of estrogen receptor (ER) (P=0.04) and diploidy (P=0.07). Coexpression of LIF and LIFR was associated with diploidy (P=0.02) and low SPF (P=0.05). LIF staining was primarily cytoplasmic whereas LIFR staining was cytoplasmic in the majority of cases and membranous in a minority of cases. The presence of LIFR in the primary tumor specimens correlated with the growth stimulation of tumor cells (derived from the same specimens) by exogenous LIF in methylcellulose colony assays. The findings support a widespread but probably complex role for LIF and LIFR in breast tumor growth regulation which should be investigated in greater detail in larger cohorts of tumors.     

FGF5 as an oncogenic factor in human glioblastoma multiforme: autocrine and paracrine activities

Fibroblast growth factor 5 (FGF5) is widely expressed in embryonic but scarcely in adult tissues. Here we report simultaneous overexpression of FGF5 and its predominant high-affinity receptor (FGFR1 IIIc) in astrocytic brain tumour specimens (N=49) and cell cultures (N=49). The levels of both ligand and receptor increased with enhanced malignancy in vivo and in vitro. Furthermore, secreted FGF5 protein was generally present in the supernatants of glioblastoma (GBM) cells. siRNA-mediated FGF5 downmodulation reduced moderately but significantly GBM cell proliferation while recombinant FGF5 (rFGF5) increased this parameter preferentially in cell lines with low endogenous expression levels. Apoptosis induction by prolonged serum starvation was significantly prevented by rFGF5. Moreover, tumour cell migration was distinctly stimulated by rFGF5 but attenuated by FGF5 siRNA. Blockade of FGFR1-mediated signals by pharmacological FGFR inhibitors or a dominant-negative FGFR1 IIIc protein inhibited GBM cell proliferation and/or induced apoptotic cell death. Moreover, rFGF5 and supernatants of highly FGF5-positive GBM cell lines specifically stimulated proliferation, migration and tube formation of human umbilical vein endothelial cells. In summary, we demonstrate for the first time that FGF5 contributes to the malignant progression of human astrocytic brain tumours by both autocrine and paracrine effects.     

Inhibition of both the autocrine and the paracrine growth of human leukemia with a fully human antibody directed against vascular endothelial growth factor receptor 2

Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. Here we show that certain "liquid" tumors such as acute myeloid leukemia not only produce VEGF but also express functional VEGFR, resulting in an autocrine loop for tumor growth and propagation. In addition, the leukemia-derived VEGF can also stimulate the production of growth factors, including interleukin 6 (IL6) and granulocyte-macrophage colony stimulating factor (GM-CSF), by human endothelial cells, which in turn further promotes the growth of leukemia cells (the paracrine loop). A fully human anti-VEGFR2 (or kinase insert domain-containing receptor, KDR) antibody, IMC-2C6, strongly blocks KDR/VEGF interaction and neutralizes VEGF-stimulated activation of KDR in endothelial cells. In a system where leukemia cells are co-cultured with endothelial cells, IMC-2C6 inhibits both the production of IL6 and GM-CSF by endothelial cells and the growth of leukemia cells. Finally, IMC-2C6 effectively blocks VEGF-induced migration of KDR+ human leukemia cells, and when administered in vivo, significantly prolonged survival of mice inoculated with KDR+ human leukemia cells. Taken together, our data suggest that anti-KDR antibodies may have broad applications in the treatment of both solid tumors and certain types of leukemia.     

Human glial fibrillary acidic protein: complementary DNA cloning, chromosome localization, and messenger RNA expression in human glioma cell lines of various phenotypes.

Glial fibrillary acidic protein (GFAP) is a constituent of intermediate filaments of glial cells of the astrocyte lineage. We cloned a human GFAP complementary DNA, deduced the amino acid sequence, and established the chromosomal location (17q21) of the GFAP gene by Southern blot hybridization of somatic cell hybrids and by in situ hybridization. The authenticity of the complementary DNA was proven by expressing it in glioma cells lacking endogenous GFAP; after microinjection of the complementary DNA, such cells became positive for staining with GFAP antibodies. The levels of fibronectin (FN) and GFAP mRNA of ten human glioblastoma cell lines, determined by Northern blot hybridization of RNA, were related to other phenotypic characteristics [cell morphology and expression of the genes encoding platelet-derived growth factor (PDGF) receptors]. A high expression of GFAP mRNA was found only in cells lacking fibronectin mRNA and protein. Glioma cells with a fibroblastic phenotype (bipolar, FN+/GFAP-) were found to express both types of PDGF receptors (alpha and beta). Relatively high levels of PDGF alpha-receptor mRNA, in the absence of beta-receptor expression, were found in cell lines that express GFAP and lack detectable levels of fibronectin mRNA. The findings are compatible with the idea that the genes encoding PDGF receptors in glioma cells are regulated in concert with other genes, the expression of which may reflect the developmental program of normal glia cell lineages.     

Overexpression of epidermal growth factor and insulin-like growth factor-I receptors and autocrine stimulation in human esophageal carcinoma cells

The growth-stimulatory effects of epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and insulin-like growth factor-I (IGF-I) on the human esophageal carcinoma cell line CE48T/VGH were evaluated. Under serum-free conditions, EGF, TGF-alpha, and IGF-I promoted 3.6- to 4.1-fold increased cell proliferation. Scatchard analyses and Northern blot hybridization revealed that both the EGF/TGF-alpha receptor and the IGF-I receptor were overexpressed in CE48T/VGH cells. Furthermore, ligand-dependent autophosphorylation of the EGF receptor and the IGF-I receptor was clearly detected using antireceptor and antiphosphotyrosine antibodies. Autocrine regulation was strongly indicated by the following evidence: (a) CE48T/VGH cells were found to express TGF-alpha and IGF-I genes, (b) serum-free conditioned medium promoted the growth of CE48T/VGH cells and stimulated the autophosphorylation of the EGF/TGF-alpha receptor and the IGF-I receptor, and (c) the addition of IGF-I receptor antibodies significantly suppressed CE48T/VGH cell growth under serum-free conditions. Our studies suggest that the overexpression of EGF and IGF-I receptors and autocrine growth regulation may concertedly control the proliferation of esophageal carcinoma cells.     

胰腺导管癌神经生长因子mRNA表达及意义

目的研究胰腺导管癌和慢性胰腺炎组织中神经生长因子(NGF mRNA)的表达及临床病理意义.方法 51例胰腺癌和10例慢性胰腺炎手术切除标本经40 g/L中性甲醛固定后常规制作石蜡包埋切片,NGF mRNA染色为原位杂交法.结果胰腺癌组织NGF mRNA阳性率(54.9%)明显高于慢性胰腺炎(10.0%),差异有高度显著性(χ2=6.76,P＜0.01);高分化腺癌、未转移胰腺癌NGF mRNA表达阳性率明显低于低分化腺癌(25.0%对84.2%;χ2=13.74,P＜0.01)和转移病例(31.3%对65.7%;χ2=5.27,P＜0.05), NGF mRNA与胰腺癌其他临床病理特征无明显相关.结论 NGF mRNA表达可能与胰腺癌发生、进展、转移及预后有较密切的关系,可能是胰腺癌发生、发展及预后的重要生物学标记物.     

血管内皮生长因子在人脑胶质瘤中的表达及意义

目的研究人脑胶质瘤中血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)的表达及临床病理意义.方法应用SABC免疫组化技术检测67例人脑胶质瘤、8例正常脑组织中VEGF表达.结果VEGF仅在肿瘤组织中表达,阳性表达率为83.6%,而在正常脑组织中无表达.VEGF与胶质瘤恶性程度(P<0.01)及肿瘤复发(P<0.01)相关.结论胶质瘤细胞能分泌VEGF,VEGF表达与肿瘤复发及预后有关.     

Amplification and expression of the epidermal growth factor receptor gene in human glioma xenografts

Xenografts from eight malignant human gliomas were established in athymic mice and were used to study amplification and expression of the epidermal growth factor receptor (EGFR) gene. Tissue identity between biopsy and xenografts was confirmed by karyotypic profiles, which showed that each glioma xenograft retained structural abnormalities, including double minute chromosomes, present in the parent glioma. EGFR gene amplification was found in six of the eight glioma biopsies and their corresponding xenografts. Expression of the EGFR gene was measured by Scatchard analysis, affinity reactions, immunoprecipitations, Western immunoblots, and immunocytochemistry; significant expression of the EGFR gene was only detectable in xenografts with EGFR gene amplification. Moreover, five of the six xenografts with EGFR gene amplification demonstrated structural alterations of the EGFR gene, which was associated with low-molecular-weight EGFR proteins. These xenografts represent an excellent tissue source and in vivo model system for characterizing the epidermal growth factor receptor in malignant human gliomas.