Expression Of Int-2 Research Articles

Inhibitory effects of int-2 gene on the invasion and metastasis of oral cancer cells.

The purpose of this study was to investigate the effects of int-2 transfection on the invasiveness and metastasis of oral cancer BcaCD885 cells, and to determine the relevant mechanisms of action. High-purity int-2 eukaryotic expression plasmids were prepared and transfected using a modified cationic liposome-mediated transfection protocol. Nucleoside diphosphate kinase A (NDPKA) expression before and after transfection was examined, as well as changes in cell invasiveness and metastasis capabilities. Int-2 was confirmed to be stably expressed post-transfection into oral cancer cells. Expression of int-2 in BcaCD885 cells was significantly different before and after transfection. The proportion of invasive cells were 70.3%±8.2% and 46.5%±5.7%, and the proportion of chemotaxis cells were 78.5%±7.9% and 49.6%±7.5%, in the in the control and experimental groups respectively. The adhesion capability of cells in the experimental group was also significantly reduced. Upregulation of int-2 expression can significantly inhibit the invasion and metastasis of BcaCD885 cells.

Inhibitory effects of Gleditsia sinensis fruit extract on telomerase activity and oncogenic expression in human esophageal squamous cell carcinoma

Previous studies have shown that the anomalous fruit extract of Gleditsia sinensis (GSE) exhibited apoptotic properties in various solid and non-solid tumors in vitro. However, the inhibitory actions of GSE on oncogenic expression and telomerase activity in esophageal squamous cell carcinoma (ESCC) have not been studied before. In the present study, the anti-cancer effects of GSE were demonstrated in three ESCC cell lines (HKESC-1, HKESC-2 and SLMT-1) by MTS and anchorage-independent clongen-icity assays, expression studies on oncogenes at 11q13 (CCND1, INT2, FGF4 and EMS1) and real-time quantitative telomeric repeat amplification protocol assay to show the inhibitory effect of GSE on telomerase in ESCC. The means of MTS50 of GSE for the ESCC cell lines and non-tumor NIH 3T3 cells were 21 and 163 microg/ml respectively. The anchorage-independent clongenicity assay showed that SLMT-1 cells lost their colony-forming potential which was dose-dependent to GSE. Moreover, GSE demonstrated dose-dependent suppression on the expression of INT2, EMS1 and FGF4, and inhibition of telomerase activity in the ESCC cell lines. Our overall results thus provide the first evidence that the anti-cancer effects of GSE on ESCC involve the suppression of oncogenic expression and inhibition of telomerase activity. Our findings also offer a new opportunity for the future development of GSE as a novel anti-cancer agent for ESCC and possibly for other cancers.

Amplification of the Int-2 gene in head and neck squamous cell carcinoma

Cellular oncogenes have been implicated in head and neck cancer development since 1986. More recently interest has focused on chromosome 11q13; oncogenes therein undergoing ongoing investigation include Bcl-1/Prad-1, Hst-1 and Int-2. Our laboratory has studied the Int-2 oncogene for several years, primarily in the breast. This paper presents our investigations of Int-2 in the head and neck. Thirty-four paraffin-embedded primary squamous cell carcinomas were studied for Int-2 gene amplification using a carefully controlled method of sequence quantification by DNA dot blots. Amplification, mostly low level, was identified in 62 per cent of samples studied. No clinical correlation to amplification could be found. Further studies are underway looking for evidence of expression of Int-2 in fresh tissues and for amplification and expression of other oncogenes on this amplicon.

Targeted disruption of int‐2 (fgf‐3) causes developmental defects in the tail and inner ear

The int-2 gene (also designated fgf-3) was originally identified because its transcription is activated by the nearby integration of mouse mammary tumor virus in virus-induced tumors. Molecular analyses have revealed that the int-2 gene produces at least four mRNAs, all of which encode a protein that has 40-50% amino acid sequence similarity with the fibroblast growth factors (FGFs). Int-2 gene expression is localized to a small number of discrete sites in the developing mouse, but has not been detected in any nonneoplastic adult tissue. The expression data and the amino acid sequence similarity with the FGFs suggested several potential roles for int-2 during normal development. To evaluate these possibilities, we initiated a genetic analysis of int-2. A gene-targeting protocol was used to generate embryonic stem (ES) cells that are heterozygous for an insertion of the neo(r) gene into the first protein-coding exon of int-2. These cells were used to establish a line of mice that carry the gene disruption. Animals that are heterozygous for the int-2neo allele are normal and fertile. Homozygous mutants survive embryonic development and can be visually identified after 12.5 days of gestation by a short, initially dorsally curled tail. This defect is potentially due to the disruption of int-2 expression in the primitive streak/tail bud. Most of the homozygous mutants die at or soon after birth, but several have survived to adulthood. In addition to the tail phenotype, the surviving homozygotes show, to varying extents, symptoms characteristic of inner ear abnormalities.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of int-2 oncogene in Kaposi's sarcoma lesions.

Fibroblast growth factors (FGFs), such as basic FGF, have been implicated in the growth of Kaposi's sarcoma (KS) cells in vitro. In the evaluation of the expression of the various genes of the different members of the FGF family and their receptors in fresh KS tissue specimens, int-2 was found to be expressed in more than half of the KS tumors examined. Using reverse transcription PCR, the expression of int-2 was detected in 21 of 38 (55.2%) fresh KS biopsy specimens. In contrast, int-2 mRNA transcripts were not found in normal appearing skin from the same patients except in one sample which was obtained from an AIDS patient with disseminated KS lesions. Sequence data confirmed that the amplified sequences were derived from int-2 mRNA with proper splicing. In addition, 12 nucleic acid alterations were identified in eight out of nine KS tumor samples sequenced. Using immunohistochemical methods, int-2 protein was detected in some of the spindle-shaped tumor cells surrounding the abnormal endothelial-lined vascular slits histologically characteristic of KS. Int-2 specific immunostaining was shown to be present in both the nuclei and cytoplasm of these spindle cells but was more pronounced in the nuclei. Neither amplification nor gross rearrangement of the int-2 gene was detected in KS lesions by Southern blot analysis. These results suggest that the expression of int-2 may play a role in the pathogenesis KS by stimulating local angiogenesis and cell proliferation.

Mouse mammary tumor virus infection accelerates mammary carcinogenesis in Wnt-1 transgenic mice by insertional activation of int-2/Fgf-3 and hst/Fgf-4.

Transgenic mice carrying the Wnt-1 protooncogene modified for expression in mammary epithelial cells exhibit hyperplastic mammary glands and stochastically develop mammary carcinomas, suggesting that additional events are necessary for tumorigenesis. To induce such events and to identify the genes involved, we have infected Wnt-1 transgenic mice with mouse mammary tumor virus (MMTV), intending to insertionally activate, and thereby molecularly tag, cooperating protooncogenes. Infection of breeding female Wnt-1 transgenics decreased the average age at which tumors appeared from approximately 4 months to approximately 2.5 months and increased the average number of primary tumors per mouse from 1-2 to > 5. A smaller effect was observed in virgin females, and infection of transgenic males showed no significant effect on tumor latency. More than half of the tumors from the infected breeding group contained one or more newly acquired MMTV proviruses in a pattern suggesting that most cells in tumors arose from a single infected cell. Analyses of provirus-containing tumors for induced or altered expression of int-2/Fgf-3, hst/Fgf-4, int-3, and Wnt-3 showed activation of int-2 in 39% of tumors, hst in 3%, and both int-2 and hst in 3%. DNA analyses with probes for protooncogenes and MMTV confirmed that the activations resulted from proviral insertions. There was no evidence for proviral insertions at the int-3, Wnt-3, or Wnt-1 loci. These findings provide further evidence that fibroblast growth factors Int-2 and Hst can cooperate with Wnt-1, another secreted factor, in mammary tumorigenesis, and they illustrate the capacity of this system to identify cooperating oncogenes.

Capture-RT-PCR assay of mRNA in breast tumors:int-2 and HERV-Kenv mRNA

Gene expression at the mRNA level is likely to be a rapidly responding intermediate biomarker for use as a surrogate endpoint in Phase II clinical trials of breast cancer. Using a technology called “capture-RT-PCR,” we have been able to show ectopic int-2 mRNA expression in 7/9 breast tumors, including two fibroadenomas. The level of expression varied, high expression being observed in three tumors. Frequent expression of int-2 in breast tumors is contrary to published reports, possibly reflecting differences in technology. int-2 expression in fibroadenomas suggests that the marker may precede malignancy. Conversely, if fibroadenomas are not premalignant, int-2 expression may be gratuitous. These alternatives need to be tested in a clinical trial. Human endogenous retrovirus K (HERV-K) env gene expression was detected in only 1/7 breast tumors. Expression of this retroviral gene is more restricted than int-2 expression. c-myc was expressed in all 10 tumors studied, albeit at widely varying levels. Expression of prad1 and erbB-2 are under study. Gene expression will be compared with gene amplification. It is anticipated that a capture-RT-PCR assay simultaneously signifying levels of expression of several oncogenes/anti-oncogenes will be a most informative surrogate endpoint biomarker. In particular, chaotropic salt methods that simplify sample preparation and mRNA isolation and reduce these combined steps to a 10 minute procedure will be presented.

Mice homozygous for a targeted disruption of the proto-oncogeneint-2have developmental defects in the tail and inner ear

We derived mice that carry a targeted insertion of a neor gene in the int-2 (Fgf-3) proto-oncogene coding sequences. The mutation was found to be recessive and mice that were homozygous for the insertion did not often survive to adulthood. The mutant mice had defects in the development of the tail and inner ear that could be correlated with disruption of int-2 expression in the posterior primitive streak and hindbrain or otic vesicle. While the tail phenotype was 100% penetrant, we found that the inner ear phenotype had reduced penetrance and variable expressivity. The variable expressivity could not be attributed to variability in the genetic background of the mutant allele or to leaky expression from the mutant allele. Thus, we conclude that even in a uniform genetic background, stochastic variation in the expression of a developmental circuit can result in dramatic differences in phenotypic consequences.

Acquired expression of hst-1 in an autonomous subline (Chiba subline 2) derived from androgen-responsive mouse mammary tumor (Shionogi carcinoma 115).

Since growth of Shionogi Carcinoma 115 (SC 115) and its autonomous subline (CS 2) were regulated by fibroblast growth factor-like peptide, expression of int-2 and hst-1 was examined in these cell lines. Hybridization of genomic DNA with long terminal repeat of mouse mammary tumor virus (MMTV) revealed the same pattern of restriction fragments, showing the same integration of MMTV. Although weak expression of int-2 was noticed in the two cells, clear expression of hst-1 was seen only in CS 2 cultured with/without testosterone. It is suggested that autonomous growth of androgen-unresponsive CS 2 is connected with expression of hst-1.

Characterization of INT-2: A member of the fibroblast growth factor family

int-2 was discovered as a proto-oncogene transcriptionally activated by MMTV proviral insertion during mammary tumorigenesis in the mouse. Sequence analysis showed int-2 to be a member of the fibroblast growth factor family of genes. In normal breast and most other adult mouse tissues, int-2 expression was not detected except for low levels in brain and testis. However, using in situ hybridization, expression was found at a number of sites during embryonic development, from day 7 until birth. An analysis of the int-2 transcripts found in embryonal carcinoma cells revealed six major classes of RNA initiating at three promoters and terminating at either of two polyadenylation sites. Despite the transcriptional complexities, all size classes of RNA encompass the same open reading frame. Using an SV40 early promoter to drive transcription of an int-2 cDNA in COS-1 cells, several proteins were observed. These were shown to be generated by initiation from either of two codons: One, a CUG, leads to a product which localizes extensively to the cell nucleus and partially to the secretory pathway. In contrast, initiation at a downstream AUG codon results in quantitative translocation across the endoplasmic reticulum and the accumulation of products ranging in size from 27.5 x 10(3) Mr to 31.5 x 10(3) Mr in organelles of the secretory pathway. These proteins represented glycosylated and non-glycosylated forms of the same primary product with or without the signal peptide removed. These findings suggest the potential for a dual role of int-2; an autocrine function acting at the cell nucleus, and a possible paracrine action through a secreted product.

The structure and function of the int-2 oncogene

The classification of int-2 as a growth factor is based primarily on the similarities between the predicted amino acid sequence and that of basic fibroblast growth factor (bFGF), as well as other members of this expanding family of related proteins. In this review, we summarise the background to the identification of int-2 as a proto-oncogene in virally induced mouse mammary tumours and describe key features of the structure and expression of both the mouse and human homologues. The normal sites of int-2 expression include specific embryonic cell types suggesting multiple inductive or morphogenetic roles. Recent progress in the characterisation of the int-2 product will be discussed in relation to the similarities and differences between int-2 and other FGFs.

Expression of the FGF-related proto-oncogene int-2 during gastrulation and neurulation in the mouse.

The proto-oncogene int-2 has been implicated in the formation of mouse mammary-tumour-virus-induced mammary tumours. Analysis of the predicted coding sequence indicates that int-2 is a member of the fibroblast growth factor family. Previous studies using Northern blot analysis suggested that normal expression of int-2 may be confined to extra-embryonic endoderm lineages of embryonic stages of mouse development. We have used in situ hybridization and Northern blot analysis to examine directly int-2 expression in embryo stem cells and in the developing embryo from early gastrulation to midsomite stages. Complex patterns of accumulation of int-2 RNA were observed in embryonic and extra-embryonic tissues. The data suggest multiple roles for int-2 in development which may include migration of early mesoderm cells and induction of the otocyst.

Oncogene expression during progression of mouse mammary tumor cells; activity of a proviral enhancer and the resulting expression of int-2 is influenced by the state of differentiation.

The RAC cell lines are derived from a mammary tumor induced by the mouse mammary tumor virus (MMTV). Polygonal cells have retained many characteristics of epithelial cells and induce adenocarcinomas; cuboidal cells are poorly tumorigenic; and elongated cells produce highly malignant sarcoma-like tumors. MMTV proviral integrations, one of which has cis-activated the int-2 gene, demonstrated that the lines are clonal descendents from a single tumor cell. Polygonal cells have a high constitutive expression of MMTV and contain int-2 RNA. In contrast, the cuboidal and the elongated cells show no detectable expression of int-2, even in the presence of glucocorticoid hormone. Transcription of MMTV in these cells is also low but can be stimulated by dexamethasone, albeit to levels lower than in polygonal cells. Thus, expression of int-2 seems to be caused by an enhancing activity on the MMTV provirus which is not dependent on steroid hormone and is specific for mammary tumor cells with epithelial characteristics. Progression in this cell system does not require sustained expression of int-2.