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At least 217 records · Page 12Linked to original sources
PubMed2005

Regulatory sequences of H19 and IGF2 genes in DNA-based therapy of colorectal rat liver metastases.

BACKGROUND: Malignant tumors of the liver are among the most common causes of cancer-related death throughout the world. Current therapeutic approaches fail to control the disease in most cases. This study seeks to explore the potential utility of transcriptional regulatory sequences of the H19 and insulin growth factor 2 (IGF2) genes for directing tumor-selective expression of a toxin gene (A fragment of diphtheria toxin), delivered by non-viral vectors. METHODS: The therapeutic potential of the toxin vectors driven by the H19 and the IGF2-P3 regulatory sequences was tested in a metastatic model of rat CC531 colon carcinoma in liver. RESULTS: Intratumoral injection of these vectors into colon tumors implanted in the liver of rats induced an 88% and a 50% decrease respectively in the median tumor volume as compared with the control groups. This therapeutic action was accompanied by increased necrosis of the tumor. Importantly, no signs of toxicity were detected in healthy animals after their treatment by the toxin expression vectors. CONCLUSIONS: DT-A was preferentially expressed in liver metastases after being transfected with H19 or IGF2-P3 promoter-driven DT-A expression plasmids, causing a very significant inhibition of tumor growth as a result of its cytotoxic effect. Our findings strongly support the feasibility of our proposed therapeutic strategy, which may contribute to open new gene therapeutic options for human liver metastases.

Animals↗
PubMed2002

Possible physiological role of H19 RNA.

The product of the imprinted oncofetal H19 gene is an untranslated RNA of unknown function. With the human cDNA Atlas microarray, we detected differentially expressed genes modulated by the presence of H19 RNA. Many of the genes that are upregulated by H19 RNA are known to contribute to the invasive, migratory, and angiogenic capacities of cells. Moreover, we provided experimental data indicating that whereas H19 RNA did not have any growth advantage for the cells when cultured in 10% fetal calf serum, it did confer an advantage when cells were cultured in serum-poor medium. This observation can be explained in part by the inability of the H19-expressing cells to induce the cyclin-dependent kinase inhibitor p57(kip2) in response to serum stress. Our results favor the possible role of the H19 gene in promoting cancer progression, angiogenesis, and metastasis.

Bromodeoxyuridine↗
PubMed2003

Biallelic expression of the H19 gene during spontaneous hepatocarcinogenesis in the albumin SV40 T antigen transgenic rat.

Previous studies in this laboratory have demonstrated that the earliest cytogenetic alteration in the development of hepatic neoplasms in a transgenic strain of rats bearing the albumin Simian virus 40 T antigen (Alb SV40 T Ag) construct was a duplication of the chromosome 1q4.1-1q4.2 band. In this region, in the rat genome a cluster of linked imprinted genes occurs. One of these imprinted genes, H19, which is expressed in fetal liver but not in adult liver, was found to be expressed in virtually all neoplasms investigated. A single-nucleotide polymorphic marker in the H19 coding sequence was identified in two rat strains and utilized for the investigation of H19 imprinting. Our results reveal monoallelic expression of the maternal gene in fetal liver, but biallelic expression of the H19 gene in liver neoplasms, thus demonstrating the basis for the deregulation of the imprinted gene expression during hepatocarcinogenesis. These results suggest that the loss of genomic imprinting of the H19 gene found in the liver neoplasms of the Alb SV40 T Ag rat may result not from allelic loss, but from adverse changes in the epigenetic imprints present in the 5'-upstream region of the H19 promoter of the parental alleles.

Albumins↗
PubMed2001

Frequent loss of imprinting of IGF2 and MEST in lung adenocarcinoma.

Genomic imprinting is a parental origin-specific chromosomal modification that causes differential expression of maternal and paternal alleles of a gene. Accumulating evidence suggests that deregulation of imprinted genes, including loss of imprinting (LOI), plays a role in oncogenesis. In the present study, we investigated allelic expression of six imprinted genes in human lung adenocarcinomas as well as in matched normal lung tissue. Informative cases showing heterozygosity for the gene of interest were selected from 35 patients. LOI of the insulin-like growth factor 2 gene (IGF2) and mesoderm-specific transcript (MEST, also known as paternally expressed gene 1) was noted in 47% (seven of 15) and 85% (11 of 13) of informative cases, respectively. Monoallelic expression was maintained in all the matched normal tissues examined. LOI of IGF2 was seen more frequently in moderately to poorly differentiated adenocarcinomas. In contrast, H19, small nuclear ribonucleoprotein-associated polypeptide N gene (SNRPN), necdin gene (NDN), and long QT intronic transcript 1 (LIT1) exhibited consistent monoallelic expression in all the informative samples. These findings indicated that independent deregulation took place in imprinted genes and suggested that aberrant imprinting of IGF2 and MEST was involved in the development of lung adenocarcinoma.

Adenocarcinoma↗
PubMed2004

Identification of novel differentially expressed genes by the effect of a high-fat n-6 diet in experimental breast cancer.

In previous studies, we demonstrated that high corn oil diets promote the development of 7,12-dimethylbenz(alpha)anthracene (DMBA)-induced mammary tumors. In this study, we have investigated whether modulation of gene expression is one of the mechanisms by which this high-fat diet exerts such effects. Female Sprague-Dawley rats were induced with DMBA and fed normolipidic (3% corn oil) or high-fat (20% corn oil) diet. Screening of genes differentially expressed in adenocarcinomas from the high corn oil diet group compared to the control diet group was performed with cDNA microarrays. The resulting six upregulated and nine downregulated genes were validated by Northern blot and/or reverse transcription (RT)-polymerase chain reaction (PCR). Further investigation in a higher number of adenocarcinomas showed that in the high-fat n-6 diet group, where the tumor phenotype was verified to be more aggressive, the expression of submaxillary gland alpha-2u globulin, vitamin D(3)-upregulated protein 1 (VDUP1), H19, and the unknown function gene that codifies the expressed sequence tag (EST)-Rn.32385 was significantly decreased in comparison with the control group (C). These results, together with the fact that VDUP1, H19, and this globulin have been associated with cell proliferation and differentiation, open a new line of research about how the underexpression of these genes contributes to the stimulating effect of a high corn oil diet on experimental mammary carcinogenesis.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2004

Changes in the DNA methylation profile of the rat H19 gene upstream region during development and transgenic hepatocarcinogenesis and its role in the imprinted transcriptional regulation of the H19 gene.

Monoallelic expression of the imprinted H19 and insulin-like growth factor-2 (Igf2) genes depends on the hypomethylation of the maternal allele and hypermethylation of the paternal allele of the H19 upstream region. Previous studies from our laboratory on liver carcinogenesis in the F1 hybrid of Fischer 344 (F344) and Sprague-Dawley Alb SV40 T Ag transgenic rat (SD) strains revealed the biallelic expression of H19 in hepatomas. We undertook a comparative study of the DNA methylation status of the upstream region of H19 in fetal, adult, and neoplastic liver. Bisulfite DNA sequencing analysis of a 3.745-kb DNA segment extending from 2950 to 6695 bp of the H19 upstream region revealed marked variations in the methylation patterns in fetal, adult, and neoplastic liver. In the fetal liver, equal proportions of hyper- and hypomethylated strands revealed the differentially methylated status of the parental alleles, but in neoplastic liver a pronounced change in the pattern of methylation was observed with a distinct change to hypomethylation in the short segments between 2984 and 3301 bp, 6033-6123 bp, and 6518-6548 bp. These results indicated that methylation of all cytosines in this region may contribute to the imprinting status of the rat H19 gene. This phenomenon of differential methylation-related epigenetic alteration in the key cis-regulatory domains of the H19 promoter influences switching to biallelic expression in hepatocellular carcinogenesis. Similar to mouse and human, we showed that the zinc-finger CCTCC binding factor (CTCF) binds to the unmethylated CTCF binding site in the upstream region to influence monoallelic imprinted expression in fetal liver. CTCF does not appear to be rate limiting in fetal, normal, and neoplastic liver. 3' to the CTCF binding sites, another DNA region exhibits methylation of CpG's in both DNA strands in adult liver, retention of the imprint in fetal liver, and complete demethylation in neoplastic liver. In this region is also a putative binding site for a basic helix-loop-helix leucine-zipper transcription factor, TFEB. The differential CpG methylation seen in the adult that involves the TFEB binding site may explain the lack of expression of the H19 gene in adult normal liver. Furthermore, these findings demonstrate that the loss of imprinting of the H19 gene in hepatic neoplasms of the SD Alb SV40 T Ag transgenic rat is directly correlated with and probably the result of differential methylation of CpG dinucleotides in two distinct regions of the gene that are within 4 kb 5' of the transcription start site. Cytogenetic analysis of hepatocytes in the transgenic animal prior to the appearance of nodules or neoplasms indicates a role of such loss of imprinting in the very early period of neoplastic development, possibly the transition from the stage of promotion to that of progression.

5' Untranslated Regions↗
PubMed2004

Gene expression in the bladder carcinoma rat model.

We investigated gene expression in N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced rat bladder carcinoma in order to test its applicability as a model for the study of novel therapeutic modalities, particularly gene therapy. We administered BBN in the drinking water to Wistar rats for up to 30 wk and induced papillary transitional cell carcinoma (TCC), which is similar to the most prevalent type of human bladder cancer. Tumor evolution was similar to that found in previous studies. However, we described the morphological stages according to modern human bladder carcinoma terminology. Our main goal was to examine the expression levels of the H19 gene, of the insulin-like growth factor 2 (Igf2) transcripts expressed from promoters P2 and P3 and of the telomerase subunits that we had previously investigated as tools for targeted gene therapy of bladder cancer. We detected at 30 wk of BBN exposure significant upregulation of these sequences in the rat bladder tumors, similar to our previous findings in human bladder cancer. To reinforce the similarity of this model to the corresponding human disease, we searched for additional tumor-specific genes documented as having altered expression in human bladder carcinoma, using cDNA expression arrays (Clontech). We suggest that BBN-induced rat bladder cancer has morphological, biological, and molecular parallels to human bladder cancer and is an attractive model for studying novel alternatives of therapeutic intervention.

Animals↗
PubMed2005

DNA methylation in the CTCF-binding site I and the expression pattern of the H19 gene: does positive expression predict poor prognosis in early stage head and neck carcinomas?

Loss of allele-specific expression by the imprinted genes IGF2 and H19 has been correlated with a differentially methylated region (DMR) upstream to the H19 gene. The H19-DMR contains seven potential CCCTC-binding factor (CTCF) binding sites. CTCF is a chromatin insulator and a multifunctional transcription factor whose binding to the H19-DMR is suppressed by DNA methylation. Our study included a group of 41 head and neck squamous cell carcinoma (HNSCC) samples. The imprinting status of the H19 gene was analyzed in 11 out of 35 positive cases for H19 gene expression, and only 1 of them showed loss of imprinting. We detected a significant correlation (P = 0.041, Fisher's exact test) between H19 expression and tumor recurrence. Among H19 positive cases, six were T2, in which five developed recurrence and/or metastasis. Inversely, in the group of tumors that showed no H19 gene expression, 5 out of 24 were T2 and only 1 presented regional recurrence. These data support the hypothesis that H19 expression could be used as a prognostic marker to indicate recurrence in early stage tumors. We also examined the methylation of the CTCF binding site 1 in a subgroup of these samples. The H19 gene silencing and loss of imprinting were not correlated with the methylation pattern of the CTCF binding site 1. However, the significant correlation between H19 expression and tumor recurrence suggest that this transcript could be a marker for the progression of HNSCC.

Adult↗
PubMed2002

Developmentally-regulated changes of Xist RNA levels in single preimplantation mouse embryos, as revealed by quantitative real-time PCR.

Xist RNA localizes to the inactive X chromosome in cells of late cleavage stage female mouse embryos (Sheardown et al., 1997: Cell 91:99-107). Fluorescence in situ hybridization (FISH), however, does not quantify the number of Xist transcripts per nucleus. We have used real-time reverse transcription-polymerase chain reaction (RT-PCR) to measure Xist RNA levels in single preimplantation embryos and to establish developmental profiles in both female and male samples. The gender of each embryo was readily established based on Xist RNA levels, by counting Xist gene copies per cell, and by independent detection of the presence/absence of Sry, a Y chromosome-specific gene. Xist expression in males was found to be very low at all stages, as suggested by FISH. In contrast, female embryos contained measurable levels of Xist mRNA starting at the late 2-cell stage and rapidly accumulated Xist transcripts until morula stage. Xist RNA accumulation per embryo then reached a plateau, while cell division continued. We propose that during early cleavage high enough levels of Xist mRNA are transcribed to generate a pool of unbound molecules. This pool would serve to temporarily maintain X chromosome inactivation without additional transcription while the trophectoderm and inner cell mass (ICM) differentiate. The ICM would then loose the paternally imprinted pattern of X inactivation originally present in all embryonic cells.

Animals↗
PubMed2003

Differential pattern of Xist RNA accumulation in single blastomeres isolated from 8-cell stage mouse embryos following laser zona drilling.

Xist gene expression begins at the late 2-cell stage in female mouse embryos and by the third division results in the accumulation of an average 100 copies of Xist RNA per cell, as measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). In the blastocyst, the trophectoderm maintains the paternally imprinted pattern of Xist expression present during early development, while either the maternal or the paternal X chromosome can express Xist among cells of the inner mass. Fluorescent in situ hybridization (FISH) has previously established that Xist transcripts are localized on the silenced X chromosome, forming aggregates of variable dimensions in blastomeres of 8-cell embryos. This observation and the fact that Xist RNA accumulation per cell sharply decreases after morula stage raise the possibility that cells of cleaving embryos contain different levels of Xist RNA, perhaps linked to their subsequent developmental fates. We show here that Xist RNA is efficiently recovered from single blastomeres isolated from 8-cell embryos following laser zona drilling. Sexing of the samples and simultaneous quantification of Xist RNA in individual cells is achieved with a multiplex Xist/Sry real-time RT-PCR assay sensitive to the single-copy level. This analysis reveals that Xist RNA is indeed accumulated to substantially different levels in individual blastomeres of the same 8-cell embryo and that two blastomeres contain most of the molecules per embryo. These results support the conclusion that cells of the early mammalian embryo are not all functionally equivalent. Differential Xist gene expression could arise from differences in DNA methylation, or the order in which cells divide.

Animals↗
PubMed2002

Mouse Xist expression begins at zygotic genome activation and is timed by a zygotic clock.

The imprinted mouse Xist (X-inactive specific transcript) gene is involved in the initiation of X-chromosome inactivation. Only the paternal Xist is expressed in preimplantation development beginning from the 4-cell stage, preceding and in correlation with paternal X-inactivation in the extraembryonic lineage of the blastocyst. To better understand the mechanisms regulating Xist expression in early development, we investigated the precise timing of its onset. We set up a single-cell RT-PCR for the simultaneous analysis on single embryos of Xist and Hprt (internal control) cDNAs and a Y-chromosome specific DNA sequence, Zfy (for embryo sexing). Applying this procedure, we demonstrate that Xist expression begins at the G2-phase of 2-cell female embryos, earlier than previously reported and at the same time of the major wave of zygotic genome activation (ZGA). We then examined, if Xist expression at the 2-cell stage is dependent on the lapse of time spent since fertilization, as previously reported for zygotic genes. One-cell embryos at the G2-phase of the first cell-cycle were cultured with cytochalasin D (inhibitor of cytokinesis but not of DNA synthesis or nuclear progression) for a time equivalent to the 4-cell stage in control, untreated embryos. We show that Xist activation occurs at a scheduled time following fertilization despite the embryos being blocked at the 1-cell stage, suggesting the existence of a zygotic clock involved in the regulation of the transcription of this imprinted gene.

Animals↗
PubMed2005

Expression of imprinted genes is aberrant in deceased newborn cloned calves and relatively normal in surviving adult clones.

Cattle are the species used most frequently for the development of assisted reproductive technologies, such as nuclear transfer. Cattle cloning can be performed by a large number of laboratories around the world, and the efficiency of nuclear transfer in cattle is the highest among all species in which successful cloning has been achieved. However, an understanding of the expression of imprinted genes in this important species is lacking. In the present study, real time reverse transcription polymerase chain reaction (RT-PCR) was utilized to quantify the expression of the bovine Igf2, Igf2r, and H19 genes in eight major organs (brain, bladder, heart, kidney, liver, lung, spleen, and thymus) of somatic cell cloned calves that died shortly after birth, in three tissues (skin, muscle, and liver) of healthy clones that survived to adulthood, and in corresponding tissues of control animals from natural reproduction. We found that, deceased bovine cloned calves exhibited abnormal expression of all three genes studied in various organs. Large variations in the expression levels of imprinted genes were also seen among these clones, which were produced from the same genetic donor. In surviving adult clones, however, the expression of these imprinted genes was largely normal, except for the expression of the Igf2 gene in muscle, which was highly variable. Our data showed disruptions of expression of imprinted genes in bovine clones, which is possibly due to incomplete reprogramming of donor cell nuclei during nuclear transfer, and these abnormalities may be associated with the high neonatal mortality in cloned animals; clones that survived to adulthood, however, are not only physically healthy but also relatively normal at the molecular level of those three imprinted genes.

Animals↗
PubMed2004

Sonographic findings in Beckwith-Wiedemann syndrome related to H19 hypermethylation.

Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome associated with congenital malformations and tumour predisposition. BWS results from variable mutations or epigenetic modifications of imprinted genes in the 11p15 chromosomal region. We present a fetus with mild general overgrowth and bilateral enlarged echogenic kidneys with loss of the corticomedullary differentiation in which prenatal diagnosis of BWS was suspected. The rest of the fetal anatomy and the amniotic fluid volume appeared normal. After termination of the pregnancy, molecular analysis confirmed the diagnosis of BWS by showing an isolated hypermethylation of the H19 gene.

Adult↗
PubMed2003

Expression-patterns of the RNA component (hTR)and the catalytic subunit (hTERT) of human telomerase in nonneoplastic prostate tissue, prostatic intraepithelial neoplasia, and prostate cancer.

BACKGROUND: Telomerase, a ribonucleoprotein, is composed of a RNA component (hTR) and two protein subunits. One of these subunits, the catalytic subunit (human telomerase reverse transcriptase, hTERT), represents a reverse transcriptase. hTERT-expression is closely correlated with telomerase activity. The telomerase is thought to be involved in immortalization process. By adding hexamic repeats to the end of chromosomal DNA, the telomeres, the enzyme is able to stop progresssive telomeric DNA loss that occurs during cell division due to the end replication problem that stops the mitotic clock. METHODS: Expression-patterns of hTR using radioactive in situ hybridization with (35)S-labelled RNA probes were compared with immunhistochemical staining for hTERT in 14 cases of archival paraffin-embedded samples of normal prostatic tissue, high-grade prostatic intraepithelial neoplasia (PIN), prostatic cancer, and one atypical adenomatous hyperplasia (AAH). Beside the expression-patterns each telomerase component was evaluated semiquantitatively. RESULTS: hTERT and hTR can be found in nonneoplstic tissue and are upregulated in premalignant transformated lesions. AAH showed no hTERT-expression and low hTR-expression. There is a heterogenous expression within prostatic carcinomas. CONCLUSIONS: No association was observed between the grade of the tumour differentiation and semiquantitative levels of hTR- or hTERT-expression. Telomerase is of limited value for the diagnostic of malignant or benign lesions in prostate.

Carcinoma↗
PubMed2001

Development of two bacterial artificial chromosome shuttle vectors for a recombination-based cloning and regulated expression of large genes in mammalian cells.

Most conditional expression vectors designed for mammalian cells have been valuable systems for studying genes of interest by regulating their expressions. The available vectors, however, are reliable for the short-length cDNA clones and not optimal for relatively long fragments of genomic DNA or long cDNAs. Here, we report the construction of two bacterial artificial chromosome (BAC) vectors, capable of harboring large inserts and shuttling among Escherichia coli, yeast, and mammalian cells. These two vectors, pEYMT and pEYMI, contain conditional expression systems which are designed to be regulated by tetracycline and mouse interferons, respectively. To test the properties of the vectors, we cloned in both vectors the green fluorescence protein (GFP) through an in vitro ligation reaction and the 17.8-kb-long X-inactive-specific transcript (Xist) cDNA through homologous recombination in yeast. Subsequently, we characterized their regulated expression properties using real-time quantitative RT-PCR (TaqMan) and RNA-fluorescent in situ hybridization (FISH). We demonstrate that these two BAC vectors are good systems for recombination-based cloning and regulated expression of large genes in mammalian cells.

3T3 Cells↗
PubMed1994

Expression of the Xist gene in urogenital ridges of midgestation male embryos.

We show by RT-PCR analysis that transcripts from the Xist gene, which is normally expressed from the inactive X chromosome of female somatic cells and postnatal male germ cells, are transiently expressed also in male embryos around the time of testis differentiation, mainly in somatic cells of urogenital ridges. In the postnatal testis, we find that Xist transcripts are mainly localized within the nucleus of haploid spermatids. These findings suggest that inactivation of the X-chromosome might occur not only in adult male germ cells, but also, transiently, in somatic cells of the male urogenital ridge. Both in the embryonal gonad and in differentiating germ cells Xist expression in males overlaps the pattern of expression of the testis determining gene Sry.

Animals↗