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S G Gray

Publications and source records attributed to S G Gray.

At least 19 recordsLinked to original sources
PubMed2001

Methylation changes in the human IGF2 p3 promoter parallel IGF2 expression in the primary tumor, established cell line, and xenograft of a human hepatoblastoma.

Hepatoblastoma (HB) is a rare malignant embryonal liver tumor. Its pathogenesis has been associated with altered regulation of the IGF2 and H19 genes, and previous studies have suggested a correlation between abnormal methylation and altered expression of these genes in hepatoblastoma. Upregulation of the activity of the IGF2 promoter P3 has previously been shown to be tightly correlated with demethylation in hepatoblastoma. Here, we have used bisulfite genomic sequencing to characterize the methylation pattern of the IGF2 promoter P3 in the hepatoblastoma-derived cell line Hep T1, in the original tumor from which Hep T1 is derived, and in nude mouse xenografts of the Hep T1 cell line. The results show a clear difference in methylation pattern of the most proximal region of the IGF2 P3 promoter between the primary tumor, the cell line, and the xenografts. RNase protection and mRNA in situ hybridization revealed that variations in methylation patterns was paralleled by the levels of IGF2 P3 mRNA, which was detectable in the primary tumor and xenografts, but not in the cell line. Furthermore, it was demonstrated that H19 was reactivated and demethylated in the HepT1 cell line by 5-azaCytidine, in contrast to IGF2 P3, which was not demethylated or reactivated. We suggest that methylation of the proximal IGF2 P3 is important for its regulation.

Animals↗
PubMed2001

Expression levels of insulin-like growth factor binding proteins and insulin receptor isoforms in hepatoblastomas.

Hepatoblastoma, a rare pediatric liver tumour, is a poorly understood disease. While expression studies for some members of the Insulin-like growth factor axis have been studied in hepatoblastoma, a systematic analysis of the IGF-axis has not been carried out. We have examined a series of hepatoblastomas with matched normal liver tissue for gene expression differences with emphasis on members of the insulin-like growth factor binding proteins. The expression profiles obtained reveal that the expression of these genes are altered in these tumors. The results indicate that the IGF-axis is seriously disturbed in the tumors.

Adolescent↗
PubMed2001

The human histone deacetylase family.

Since the identification of the first histone deacetylase (Taunton et al., Science 272, 408-411), several new members have been isolated. They can loosely be separated into entities on the basis of their similarity to various yeast histone deacetylases. The first class is represented by its closeness to the yeast Rpd3-like proteins, and the second most recently discovered class has similarities to yeast Hda1-like proteins. However, due to the fact that several different research groups isolated the Hda1-like histone deacetylases independently, there have been various different nomenclatures used to describe the various members, which can lead to confusion in the interpretation of this family's functions and interactions. With the discovery of another novel murine histone deacetylase, homologous to yeast Sir2, the number of members of this family is set to increase, as 7 human homologues of this gene have been isolated. In the light of these recent discoveries, we have examined the literature data and conducted a database analysis of the isolated histone deacetylases and potential candidates. The results obtained suggest that the number of histone deacetylases within the human genome may be as high as 17 and are discussed in relation to their homology to the yeast histone deacetylases.

Amino Acid Sequence↗
PubMed2001

Promoter-specific transcription of the IGF2 gene: a novel rapid, non-radioactive and highly sensitive protocol for mRNA analysis.

The human insulin-like growth factor-II (IGF2) is a regulatory peptide which is critical in normal fetal growth. IGF2 gene transcription is controlled by the usage of four promoters P1-P4 of which promoters P2-P4 are genomically imprinted. Disruption of imprinting and the resulting increase of gene dosage have been shown to be implicated in tumor progression in a variety of human tumors. Due to the need for high amounts of tissue material for conventional methods such as Northern blotting or ribonuclease protection assay (RPA), studies on IGF2 expression have most often been limited to the detection of total IGF2 transcript, though different dysregulatory events can be responsible for the abundance of IGF2 mRNA found in many tumors. We established a highly sensitive competitive RT-PCR assay for the four different transcripts of the IGF2 gene with transcript-specific external RNA competitors in which we take advantage of fluorescence-based quantification on a semiautomated sequencer. The amount of total RNA needed is approximately 100 times lower than the amounts required for Northern blotting or RPA, so that even cytological samples can be analyzed. We applied the assay to a series of eleven hepatoblastomas (HB) in which normal adjacent liver tissue could also be analyzed.

Child, Preschool↗
PubMed2001

Histone acetylation/deacetylation and cancer: an "open" and "shut" case?

DNA in eukaryotic cells is packaged into chromatin. The main packaging component of chromatin is the nucleosome, and this is composed of proteins known as histones. Histones can be reversibly modified in several ways, and the best characterized of these modifications is histone acetylation. This is a reversible modification, which is carried out by two families of enzymes, the histone acetyltransferases (HATs), and the histone deacetylases (HDACs). These enzymes have important activities in many cellular processes including transcription, DNA replication and cell cycle progression. The mechanisms underlying tumor formation are multifaceted, and often involve mutations or alterations of genes involved with the regulation and control of the cell cycle or cell death. Because of their important roles in the regulation of such events, enzymes that affect histone acetylation status are increasingly being associated with tumors. This article describes some of the current knowledge about histone acetyltransferases and histone deacetylases, and how their multitudinal roles in cellular events may have important roles in tumorigensis.

Acetylation↗
PubMed2001

Differential expression of class I HDACs: roles of cell density and cell cycle.

Enzymes which affect histone acetylation status have been shown to play an important role in determining transcriptional activity in chromatin through conformational modification of its structure. Since the timely presence of such enzymes may be of critical importance, our experiments were designed to determine whether the level of expression of HDAC1 is cell cycle dependent and/or affected by a high cell density. Our results show that in mouse fibroblasts the expression of mHDAC1 is neither affected by cell cycle phases nor by cell density. In contrast, the expression of several hHDACs including hHDAC1 were affected in a cell density dependent fashion in the human prostate adenocarcinoma cell line PC3, paralleling our previously published findings in the hepatocellular carcinoma derived cell line Hep3B. Differential recruitment of HDAC mRNAs suggests that these enzymes may play unique roles in different cell types and under different environmental conditions (i.e., exposure to various cell densities and cell-cell contacts). Our study has implications for the proposed use of HDAC inhibitors in the treatment of human malignancy, highlighting issues of drug action selectivity in tissues and potential secondary effects.

Adenocarcinoma↗
PubMed2000

IGF-II and IL-2 act synergistically to alter HDAC1 expression following treatments with trichostatin a.

Histone deacetylases play key roles in the regulation of gene transcription. Studies have shown that expression of interleukins IL-2 and IL-8, and insulin-like growth factor 2 (IGF2) are affected by treatment with histone deacetylase inhibitors. We have previously shown that the gene for histone deacetylase 1 (HDAC1) is upregulated following treatment with TSA. The murine homologue of this gene has been reported to be inducible by IL-2. In this study, we have examined the effects IL-2, IGF-II and TSA have on HDAC1 expression in the human hepatocellular carcinoma derived cell line Hep3B. Our results indicate that in contrast to the mouse, HDAC1 is not inducible by IL-2. However, in TSA treated cells, IL-2 and IGF-II were found to act synergistically to reduce TSA induced HDAC1 mRNA levels almost to normal.

Acetylation↗
PubMed2000

Altered expression of members of the IGF-axis in hepatoblastomas.

Previous reports have demonstrated that expression of insulin-like growth factor 2 (IGF2) is altered in hepatoblastoma. Using RNAase protection analysis (RPA), we examined the gene expression for IGF1, IGF2, IGF1R, M6P/IGF2R, IGFBP-1 and IGFBP-2 in a series of hepatoblastomas with corresponding normal liver from the same individuals. The results show that the expression of the IGF-axis members included in the present study are altered between tumour and normal, and indicate that the IGF-axis may be involved in hepatoblastoma development.

Genes, Tumor Suppressor↗
PubMed2000

Comparative genomic hybridization reveals population-based genetic alterations in hepatoblastomas.

Hepatoblastoma is a malignant paediatric liver tumour. In order to approach the genetic background of this malignancy we have screened a panel of eighteen cases from Europe and Japan for chromosomal imbalances using comparative genomic hybridization (CGH). The most frequent losses included chromosomal regions 13q21-q22 (28%) and 9p22-pter (22%), while the most frequent gains occurred on 2q23-q24 (33%), 20q (28%) and 1q24-q25 (28%). A significant difference in CGH alterations between the tumours from patients of Caucasian and Japanese was revealed where loss of 13q was found only in the Japanese samples. In conclusion, the findings indicate several candidate regions for suppressor genes and oncogenes potentially involved in the hepatoblastomas of different ethnic origin.

Adolescent↗
PubMed2000

Modulating IGFBP-3 expression by trichostatin A: potential therapeutic role in the treatment of hepatocellular carcinoma.

The Hep3B cell line analyzed in the present study is a widely used in vitro model in studies characterizing pathogenetic, functional, and therapeutic aspects of human hepatocellular carcinoma (HCC). Here we have determined the chromosomal composition using a combination of cytogenetic techniques. In agreement with the original description for this cell line, Hep3B was found to have a hypotriploid chromosome content carrying 59-63 chromosomes and no cytogenetic differences were demonstrated between early and late passages suggesting that this cell line has remained stable after repeated subculturing. Mutations and alterations of the IGF-axis as well as of chromosome 1p34, where the genes for histone deacetylase 1 (HDAC1) and transforming growth factor beta receptor interacting protein-1 (TRIP-1) map, are frequent events in hepatocarcinogenesis. This study characterizes the Hep3B cell line in detail at the karyotypic level, using comparative genomic hybridization (CGH), spectral karyotyping (SKY), G-banding and FISH techniques. We have also examined the effects of the histone deacetylase inhibitor trichostatin A (TSA) on members of the IGF-axis, and analysed them with regard to the karyotype. The results show that expression of one member of the IGF-axis, IGFBP-3, is greatly upregulated by treatment of Hep3B cells with TSA. As IGFBP-3 has been shown to induce apoptosis, these results suggest a possible use for histone deacetylase inhibitors and/or IGFBP-3 in the treatment of HCC.

Carcinoma, Hepatocellular↗
PubMed2000

Expression of genes involved with cell cycle control, cell growth and chromatin modification are altered in hepatoblastomas.

Hepatoblastoma is a rare pediatric liver tumor. While much progress has been made in the treatment of the disease, very little is known about the moleculer events underlying the pathogenesis of this disease. We sought to investigate a series of hepatoblastomas for alterations in gene expression patterns with emphasis on important cell regulatory genes, including chromatin modifying enzymes, cyclin dependent kinase inhibitors, growth factors, oncogenes and cell cycle regulators. Total RNA was extracted from a series of sporadic hepatoblastomas with matched normal liver, some unmatched tumors and fetal livers, and gene expression was measured for various genes using RNase Protection Analysis (RPA). The results of this analysis show that the expression of many important regulatory genes are distinctly altered in these tumors, and a subset of tumors can be distinguished on the basis of these gene expression differences and histopathological features. Because the molecular events underlying the pathogenesis of this rare tumor are so poorly understood, this study represents a first step in determining some of the possible mechanisms involved which may provide future avenues of research.

Case-Control Studies↗
PubMed1999

IGF-II enhances trichostatin A-induced TGFbeta1 and p21(Waf1,Cip1, sdi1) expression in Hep3B cells.

Cell growth and division are controlled through the actions of cyclin-dependent kinases (CDKs) and cyclin dependent kinase inhibitors (CKIs). Treatment of cell lines with Trichostatin A leads to induction of one of these CKIs, p21, and growth arrest. Induction of p21 can also occur through the actions of TGFbeta1. Latent TGFbeta1 can be activated by the M6P/IGF2R. In the present study we have examined the effect of TSA on members of the IGF axis, the CKIs p21 and p27, and also TGFbeta1 in Hep3B cells. The only member of the IGF axis to be affected by treatments was IGF2. Expression of another gene from the same chromosomal location, H19, was also affected. TGFbeta1 expression was greatly enhanced by TSA. In addition, both CKIs, p21 and p27, were upregulated by TSA. Effects of adding IGF-II or TGFbeta1 to TSA-treated cells on p21 induction were examined. The results show that the induction of p21 by TSA can be modulated by additions of IGF-II whereas addition of TGFbeta1 affects its own expression but not p21. In conclusion, the results indicate that the induction of p21 and cell growth arrest caused by Trichostatin A may involve multiple signaling pathways.

Apoptosis↗
PubMed1999

Methylation, gene expression and the chromatin connection in cancer (review)

The mechanisms by which cells regulate gene expression are often altered in tumors. Modulating aspects of the components responsible for the nuclear packaging of DNA is one means by which the cell can control transcription, either by packaging the DNA such that access to specific sites of transcription is blocked or by modifying the DNA itself to prevent transcription factor binding. One such DNA modification is the methylation of cytosines. In addition, histone acetylation status has been linked recently through a large number of studies to the regulation of gene expression. Expressed genes are located in highly acetylated chromatin. The acetylation status of nucleosomes (the basic packaging unit of chromatin), is regulated by a group of enzymes, histone acetyltransferases (HATs), and histone deacetylases (HDACs). These two elements, methylation and histone acetylation have also been linked together, whereby methylation is used to direct gene repression through a histone deacetylase complex. Methylation, HATs, and HDACs have been shown to be altered in tumors. We present an overview of the current knowledge surrounding these elements in cancer, and in the final sections describe the likelihood of alterations of the histone modifying apparatus in hepatocellular carcinoma.

Animals↗
PubMed1998

Effects of cell density and trichostatin A on the expression of HDAC1 and p57Kip2 in Hep 3B cells.

The interplay between the acetylation and deacetylation activities within the cell has been postulated to be a mechanism by which the cell regulates expression from genes at the level of chromatin. We have examined the expression pattern of the human histone deacetylase gene HDAC1 and the cyclin dependent kinase inhibitor p57Kip2 in the hepatocellular carcinoma cell line Hep 3B. HDAC1 expression was elevated at low cell densities, but once a critical threshold point in cell density was attained, expression was reduced to very low levels. Treatment of the cells with trichostatin A (TSA), a potent inhibitor of histone deacetylases, was found to affect expression. p57Kip2 was found to be downregulated by TSA, whereas HDAC1 was upregulated. These effects were found to be cell density dependent. The results suggest that HDAC1 plays a role in its own regulation, and that investigations using TSA should be carried out when cells grow exponentially.

Cell Count↗
PubMed1998

Developmental-dependent DNA methylation of the IGF2 and H19 promoters is correlated to the promoter activities in human liver development.

We have previously shown that the four promoters of the IGF2 gene are under a tight but dynamic control during human liver development, whereby P3 and P1 are reciprocally active before and after birth respectively while the P2 and P4 promoters are constitutively active at a relatively lower level. In this study, we investigated the methylation status of the promoters P1 and P3 of IGF2 and the promoter region of the H19 gene in developing human livers ranging from fetal to late adult. A region of about 300 bp immediately upstream of the IGF2 exon 5 was found to be subjected to a developmental-specific methylation and this may correlate to the P3 promoter activity. The P1 domain of IGF2 was also found to be methylated in a developmentally-specific pattern. The promoter region of the H19 gene displayed different methylation patterns in different development stages showing decreased general methylation with increase of age. Therefore, regional- and developmental-specific DNA methylation is displayed in the promoter regions of the IGF2 and H19 genes. This may be an important factor involved in gene regulation in the developing human liver.

Adult↗