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W A Held

Publications and source records attributed to W A Held.

At least 19 recordsLinked to original sources
PubMed2001

Excessive CpG island hypermethylation in cancer cell lines versus primary human malignancies.

Cancer cell lines are widely used in many types of cancer research, including studies aimed at understanding DNA hypermethylation of gene promoters in cancer. Hypermethylation of promoters is capable of repressing the expression of tumor suppressor genes and may play a role in the development and/or progression of cancer. Although both primary malignancies and cancer cell lines exhibit this epigenetic phenomenon, there has been no direct comparison between them. In order to address this question, we have utilized restriction landmark genomic scanning to measure the hypermethylation phenotypes of cancer cell lines and compared these data with the same analysis performed on primary malignancies. In all cases, cancer cell lines exhibit significantly higher levels of CpG island hypermethylation than the primary malignancies they represent. Colon cancer cell lines are most similar to their respective tumors, with only a 5-fold increase in hypermethylation, while head and neck squamous cell carcinoma cell lines show a 93-fold increase in hypermethylation. Furthermore, >57% of the loci methylated in cell lines are never methylated in 114 primary malignancies studied. Seventy percent of loci hypermethylated in cell lines are hypermethylated in lines from more than one type of cancer. These data indicate that most CpG island hypermethylation observed in cancer cell lines is due to an intrinsic property of cell lines as opposed to the malignant tissue from which they originated.

CpG Islands↗
PubMed2001

Novel methylation targets in de novo acute myeloid leukemia with prevalence of chromosome 11 loci.

Aberrant DNA methylation is believed to be important in tumorigenesis by causing either transcriptional inactivation of genes or chromosomal instability. Several laboratories have identified promoter hypermethylation of tumor suppressor genes in acute myeloid leukemia (AML). However, these studies do not provide a global assessment of overall methylation changes and do not allow the identification of novel methylated sequences. Previously, nonrandom CpG island methylation was reported in 17 adult de novo AML diagnostic samples when compared with the corresponding remission samples by means of restriction landmark genomic scanning (RLGS). That study has been expanded on by an analysis of a larger set of CpG islands (1740 vs 1184), which now provides details of 33 cloned methylated loci, including 21 known genes or expressed sequence tags. Five of these cloned loci appear to be methylated only in AML and not in the 6 solid tumors studied in this study (more than 98 samples analyzed). Chromosomal location was available for 30 of the 33 loci, and 5 of these 30 (17%) are localized to chromosome 11, suggesting a trend toward overrepresentation of methylation events on this chromosome. These results provide evidence for widespread aberrant methylation in AML, with identification of novel methylation targets, epigenetic changes that appear unique to AML, and apparent preferential methylation on chromosome 11.

Adult↗
PubMed2001

Identification of a novel member of the snail/Gfi-1 repressor family, mlt 1, which is methylated and silenced in liver tumors of SV40 T antigen transgenic mice.

DNA methylation is the only known mechanism for an epigenetic genomic DNA modification that is capable of altering gene expression. A recent study reveals that the pattern of CpG island methylation is largely characteristic of tumor type, suggesting that distinct sets of genes are inactivated by methylation during development of each tumor type. We compared previously the methylation status between normal liver and liver tumors in SV40 T/t antigen transgenic mice (MT-D2 mice) using Restriction Landmark Genomic Scanning for Methylation (RLGS-M) and identified several loci/spots that appeared to be methylated frequently in liver tumors. One of these spots, B236, identified a locus on chromosome 12 (D12Ncvs7) syntenic with human 14q12-q21 that is frequently lost in certain human cancers. Shotgun sequencing of a bacterial artificial chro mosome clone containing this spot/locus was performed to identify genes within this region. The Genescan program predicted an open reading frame of a novel, intron-less gene adjacent to the B236 spot that encodes a putative 493-amino acid protein containing the SNAG repressor motif in the NH2-terminal region and five C2H2-type zinc finger motifs in the COOH-terminal half. This putative gene, methylated in liver tumor (mlt 1), is a novel member of the SNAG transcriptional repressor family with 43% amino acid identity to insulinoma-associated protein 1. An open reading frame encoding a protein quite similar to mouse mlt 1 (56% amino acid identity) was located in the syntenic region of the human genome, indi cating that mlt 1 is evolutionarily conserved in human. Northern blot analysis revealed that mlt 1 is normally expressed in brain, spleen, stom ach, and liver. However, mlt 1 expression was silenced in the liver tumors of MT-D2 mice. The putative promoter region of mlt 1 is unmethylated in normal tissues but methylated in all liver tumors from 11 MT-D2 mice We also found that mlt 1 was methylated and not expressed in N18TG-22 cells, a mouse neuroblastoma cell line. Treatment of N18TG-2 cells with a demethylating agent, 5-aza-deoxycytidine, resulted in an expression of mlt 1, indicating that the repression of mlt 1 is attributable to methylation Thus, mlt 1 is a novel target gene that is silenced by methylation during liver tumorigenesis initiated by SV40 T antigen.

Amino Acid Sequence↗
PubMed2000

Restriction landmark genomic scanning of mouse liver tumors for gene amplification: overexpression of cyclin A2.

SV40 T/t antigen-induced liver tumors from transgenic mice were analyzed by Restriction Landmark Genomic Scanning (RLGS). Using NotI as the restriction landmark, RLGS targets CpG islands found in gene-rich regions of the genome. Since many RLGS landmarks are mapped, the candidate gene approach can be used to help determine which genes are altered in tumors. RLGS analysis revealed one tumor-specific amplification mapping close to CcnA2 (cyclin A2) and Fgf2 (fibroblast growth factor 2). Southern analysis confirmed that both oncogenes are amplified in this tumor and in a second, independent liver tumor. Whereas Fgf2 RNA is undetectable in tumors, CcnA2 RNA and cyclin A2 protein was overexpressed in 25 and 50% of tumors, respectively. Combining RLGS with the candidate gene approach indicates that cyclin A2 amplification and overexpression is a likely selected event in transgenic mouse liver tumors. Our results also indicate that our mouse model for liver tumorigenesis in mice accurately recapitulates events observed in human hepatocellular carcinoma.

Age Factors↗
PubMed2000

Methylation and downregulated expression of mac25/insulin-like growth factor binding protein-7 is associated with liver tumorigenesis in SV40T/t antigen transgenic mice, screened by restriction landmark genomic scanning for methylation (RLGS-M).

Restriction landmark genomic scanning for methylation (RLGS-M) was used to detect alterations in DNA methylation associated with murine SV40 T/t antigen-induced hepatocarcinogenesis. An altered locus/spot (S130) was cloned and found to correspond to sequences in the 5' flanking region and 5' portion of the cDNA for the murine mac25/insulin-like growth factor binding protein-7 (Igfbp-7) gene. IGFBPs are believed to be capable of binding insulin, Igf1, and Igf2 and modulating mitogenic effects. Previous studies have shown that Igf2 has an important role in promoting liver tumorigenesis. Quantitative PCR was used to access the methylation status of the NotI site just 5' to the coding region and the expression level of the mac25/igfbp-7 gene. The results indicated that the degree of methylation was inversely related to the expression level and is consistent with a role for DNA methylation in silencing mac25/Igfbp-7 gene expression and function for mac25/Igfbp-7 as a tumor suppressor gene.

Animals↗
PubMed2000

Aberrant CpG-island methylation has non-random and tumour-type-specific patterns.

CpG islands frequently contain gene promoters or exons and are usually unmethylated in normal cells. Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation. The investigation of aberrant CpG-island methylation in human cancer has primarily taken a candidate gene approach, and has focused on less than 15 of the estimated 45,000 CpG islands in the genome. Here we report a global analysis of the methylation status of 1,184 unselected CpG islands in each of 98 primary human tumours using restriction landmark genomic scanning (RLGS). We estimate that an average of 600 CpG islands (range of 0 to 4,500) of the 45,000 in the genome were aberrantly methylated in the tumours, including early stage tumours. We identified patterns of CpG-island methylation that were shared within each tumour type, together with patterns and targets that displayed distinct tumour-type specificity. The expression of many of these genes was reactivated by experimental demethylation in cultured tumour cells. Thus, the methylation of particular subsets of CpG islands may have consequences for specific tumour types.

Adenocarcinoma↗
PubMed1999

Restriction landmark genome scanning for aberrant methylation in primary refractory and relapsed acute myeloid leukemia; involvement of the WIT-1 gene.

There is substantial evidence to suggest that aberrant DNA methylation in the regulatory regions of expressed genes may play a role in hematologic malignancy. In the current report, the Restriction Landmark Genomic Scanning (RLGS) method was used to detect aberrant DNA methylation (M) in acute myeloid leukemia (AML). RLGS-M profiles were initially performed using DNA from diagnostic, remission, and relapse samples from a patient with AML. Rp18, one of the eight spots found that was absent in the relapse sample, was cloned. Sequence analysis showed that the spot represented a portion of the WIT-1 gene on human chromosome 11p13. Rp18 was missing in the relapse sample due to a distinct DNA methylation pattern of the WIT-1 gene. Twenty-seven AML patients that entered CR after therapy (i.e., chemosensitive) were studied and only 10 (37%) of the diagnostic bone marrow (BM) samples showed methylation of WIT-1. However, seven of eight (87.5%) diagnostic BM samples from primary refractory AML (chemosensitive) showed methylation of WIT-1. The incidence of WIT-1 methylation in primary refractory AML was significantly higher than that noted in chemosensitive AML (P=0.018). Together, these results indicate that RLGS-M can be used to find novel epigenetic alterations in human cancer that are undetectable by standard methods. In addition, these results underline the potential importance of WIT-1 methylation in chemoresistant AML.

Acute Disease↗
PubMed1999

A direct repeat sequence at the Rasgrf1 locus and imprinted expression.

Genomic imprinting is an epigenetic modification that can lead to parental-specific monoallelic expression of specific autosomal genes. While methylation of CpG dinucleotides is thought to be a strong candidate for this epigenetic modification, little is known about the establishment or maintenance of parental origin-specific methylation patterns. We have recently identified a portion of mouse chromosome 9 containing a paternally methylated region associated with a paternally expressed imprinted gene, Ras protein-specific guanine nucleotide-releasing factor 1 (Rasgrf1). This area of chromosome 9 also contains a short, direct tandem repeat in close proximity to a paternally methylated NotI site 30 kb upstream of Rasgrf1. Short, direct tandem repeats have been found associated with other imprinted genes and may act as important regulatory structures. Here we demonstrate that two rodent species (Mus and Rattus) contain a similar direct repeat structure associated with a region of paternal-specific methylation. In both species, the Rasgrf1 gene shows paternal-specific monoallelic expression in neonatal brain. A more divergent rodent species (Peromyscus) appears to lack a similar repeat structure based on Southern Blot analysis. Peromyscus animals show biallelic expression of Rasgrf1 in neonatal brain. These results suggest that direct repeat elements may play an important role in the imprinting process.

Animals↗
PubMed1998

A methylation imprint mark in the mouse imprinted gene Grf1/Cdc25Mm locus shares a common feature with the U2afbp-rs gene: an association with a short tandem repeat and a hypermethylated region.

We identified a sperm-specific methylation imprint mark (Site II) associated with a short tandem repeat sequence and a site/region methylated in both gametes (Site I) in the Grf1 locus on mouse chromosome 9, which shared a common feature with the U2afbp-rs gene. Sites or regions of gamete-specific methylation in imprinted genes are strong candidates for carrying information regarding the parental origin of alleles. The gamete-specific methylation pattern of Sites I and II was conserved after fertilization, but attained the somatic cell pattern by the blastocyst stage. In primordial germ cells, Site I was methylated, but Site II was unmethylated in both male and female embryos, suggesting that the sperm-specific methylation imprint mark in Site II was established during spermatogenesis. These common features in methylation imprint regions may be a clue to identifying regions carrying primary information for the imprinting regulation.

Animals↗
PubMed1997

Genomic imprinting and Igf2 influence liver tumorigenesis and loss of heterozygosity in SV40 T antigen transgenic mice.

Maternal-specific loss of heterozygosity (LOH) and allelic imbalances [i.e., partial LOH (pLOH)] observed in SV40 T/t antigen-induced liver tumors suggests that an imprinted gene on chromosome 7 is involved in liver tumorigenesis. Maternal-specific LOH/pLOH may reflect the loss of a maternally expressed tumor suppressor gene or the acquisition of paternally active alleles of a growth promoter. In addition, two oppositely imprinted genes on distal chromosome 7, Igf2 and H19, are re-expressed in most liver tumors from an SV40 T/t antigen transgenic line (M11T-G). Igf2 is a paternally expressed growth promoter, and H19 is a maternally expressed gene that can suppress growth in some tumor cell lines. We studied the role of Igf2 during liver tumorigenesis by creating Igf2 (+/-) M11T-G mice. These mice are essentially null for Igf2 expression because imprinting normally precludes maternal Igf2 expression. M11T-G, Igf2 (+/-) males exhibit a 15-fold reduction in the frequency of large tumors. Igf2 (+/-) tumors do not express maternal Igf2, indicating rigid imprinting control in the liver. LOH/pLOH analysis was performed on the tumors and indicates that acquisition of paternally active Igf2 alleles is a major selective event for M11T-G liver tumorigenesis. This also implies the existence of an imprinted, maternally expressed tumor suppressor gene on chromosome 7 that is unlikely to be H19.

Alleles↗
PubMed1997

An oocyte-specific methylation imprint center in the mouse U2afbp-rs/U2af1-rs1 gene marks the establishment of allele-specific methylation during preimplantation development.

An oocyte-specific methylation imprint mark region, consisting of approximately 200 bp from the mouse imprinted gene U2afbp-rs, was identified within an area containing a CpG island and a short tandem repeat sequence. The oocyte-specific methylation was preserved in fertilized eggs and then expanded on the repressed maternal allele during preimplantation development until the adult methylation pattern was achieved by 12.5 days of embryonic development. These results indicate that the oocyte-specific imprinting mark region acts as a center in establishing the hypermethylated region on the repressed maternal allele. Furthermore, a region that is hypermethylated in both gametes was identified but its hypermethylation was conserved only on the maternal allele during preimplantation development, suggesting that some factor(s) inherited from oocytes may act to maintain hypermethylation on the maternal allele.

Alleles↗
PubMed1997

Restriction landmark genomic scanning (RLGS-M)-based genome-wide scanning of mouse liver tumors for alterations in DNA methylation status.

Restriction landmark genomic scanning for methylation (RLGS-M) was used to detect, and subsequently clone, genomic regions with alterations in DNA methylation associated with tumorigenesis. Use of a methylation-sensitive enzyme for the landmark cleavage allows analysis of changes in methylation patterns. In this study, we used RLGS-M to analyze SV40 T antigen-induced mouse liver tumors derived from interspecific F1 hybrids between Mus spretus (S) and C57BL/6 (B6). Because 575 S- and B6-specific RLGS loci/spots have been mapped, tumor-related alterations in the RLGS profile could be immediately localized to specific chromosomal regions. We previously found that the loss of contiguous loci/spots could be attributed primarily to DNA loss, whereas loss of solitary loci/spots could be attributed primarily to DNA methylation. In this study, we examined 30 mouse liver tumor samples for loss of the 507 mapped loci/spots. Fourteen solitary loci/spots found to be absent or reduced in more than 75% of tumor samples were cloned and subjected to DNA sequence analyses. Two loci were identified as alpha4 integrin and p16/CDKN2, genes reported to be involved in tumorigenesis. Thus, RLGS-M can detect alterations in the methylation status of known tumor suppressor genes and provide a method for detecting and subsequently cloning novel genomic regions that undergo alterations in methylation during tumorigenesis.

Animals↗
PubMed1997

Cyclin-dependent kinase 6 (CDK6) amplification in human gliomas identified using two-dimensional separation of genomic DNA.

DNA amplification is a common mechanism invoked by many human tumors to elicit overexpression of genes whose products are involved in drug resistance or cell proliferation. Although amplified regions in tumor DNA may exceed several megabases in size, segments of amplicons with a high probability of containing gene sequences may be amenable to detection by restriction landmark genomic scanning (RLGS), a high-resolution DNA analysis that separates labeled NotI fragments in two dimensions. Here, we tested this by applying RLGS to matched samples of glioma and normal brain DNA and found tumor-specific amplification of the gene encoding cyclin-dependent kinase 6 (CDK6), an observation not previously reported in human tumors. The CDK6 gene has been localized to chromosome 7q21-22, but in the gliomas studied here, it was not coamplified with either the syntenic MET (7q31) or epidermal growth factor receptor (7p11-p12) genes, suggesting that this may be part of a novel amplicon in gliomas. We then corroborated this finding by identifying both amplification-associated and amplification-independent increases in CDK6 protein levels in gliomas relative to matched normal brain samples. These data implicate the CDK6 gene in genomic amplification and illustrate the potential of RLGS for the more general identification and cloning of novel genes that are amplified in human cancer.

Base Sequence↗
PubMed1996

Absence of imprinting in U2AFBPL, a human homologue of the imprinted mouse gene U2afbp-rs.

The mouse gene U2 auxiliary factor binding protein related sequence (U2afbp-rs) has previously been shown to be genomically imprinted with monoallelic expression from the paternal allele. To determine if the human homologue is imprinted and contains conserved structural features which regulate imprinting, we isolated genomic clones from a human P1-derived artificial chromosome (PAC) library that map to human chromosome 5q22-31, a region syntenic to the proximal portion of mouse chromosome 11 where U2afbp-rs resides. A genomic subclone was isolated which contained an open reading frame with high homology to the mouse gene. This subclone also maintained the intronless character of the mouse gene. A KpnI polymorphism within the open reading frame of the gene was found to occur in 21% (8/38) of the alleles tested from human placental tissue samples. RT-PCR analysis of human placentas using the KpnI polymorphism to determine the parental origin of the alleles indicates biallelic expression of the human chromosome 5 U2AFBPL gene.

Alleles↗
PubMed1996

Identification of Grf1 on mouse chromosome 9 as an imprinted gene by RLGS-M.

Normal mammalian development requires a diploid combination of both haploid parental genomes. Uniparental disomy for certain segments of specific chromosomes results in aberrant development or prenatal lethality, indicating that the parental genomes have undergone modifications during gametogenesis. These modifications result in parent-of-origin specific expression for some genes, a phenomenon called genomic imprinting. Recent work with DNA methyltransferase deficient mice showed that differential methylation is the probable basis of the imprinted character of several genes. Screening for endogenous imprinted loci using restriction landmark genomic scanning with methylation sensitive enzymes (RLGS-M) identified eight imprinted RLGS (Irigs) candidate loci. Molecular analysis of the genomic region of one of the loci (Irigs2) resulted in the discovery of the paternally imprinted U2afbp-rs gene within a previously identified imprinted region on mouse chromosome 11 (refs 5, 7). This paper describes the characterisation of a novel imprinted RLGS-M locus, Irigs3, on mouse chromosome 9 (ref. 6). Within this locus we identified the Grf1 (also called Cdc25Mm) gene, which is homologous to the RAS-specific guanine nucleotide exchange factor gene, CDC25, in Saccharomyces cerevisiae. Grf1 is located about 30 kb downstream of the methylation imprinted site, identified by RLGS-M, and shows paternal allele specific expression in mouse brain, stomach and heart. Our results indicate that imprinting may have a role in regulating mitogenic signal transduction pathways during growth and development.

Animals↗
PubMed1994

Genetic analysis of liver tumorigenesis in SV40 T antigen transgenic mice implies a role for imprinted genes.

Liver tumors from interspecific hybrid, transgenic mice containing the SV40 early region linked to a mouse major urinary protein enhancer/promoter were analyzed for loss of heterozygosity to identify chromosomal regions which potentially contain genetic loci involved in multistep tumorigenesis. A broad pattern of complete and partial loss of heterozygosity or allelic imbalance was observed with frequent loss of heterozygosity/partial loss of heterozygosity of loci on chromosomes 1, 5, 7, 8, and 12. In tumors from Mus domesticus x Mus spretus F1 mice a strong preference for loss of the domesticus allele of H19 on chromosome 7 was observed, whereas loss of heterozygosity/partial loss of heterozygosity on chromosome 8 involved preferential loss of spretus alleles. In tumors from reciprocal crosses with Mus castaneus, the maternal chromosome 7 H19 allele was preferentially lost irrespective of whether it was domesticus or castaneus, strongly suggesting the involvement of an imprinted gene(s) in tumor progression.

Alleles↗