Biomedical subjects

E Meese

Publications and source records attributed to E Meese.

65 records · Page 4Linked to original sources

PubMed1989

Application of natural partial digests to pulsed-field gel analysis of the amplified MDR locus.

The analysis by pulsed-field gel electrophoresis of partial digestion products visualized by probes for the human multidrug resistance (MDR) locus has been used to further establish the restriction map of this region. Results place the MDR1 and MDR2 genes on a single SfiI fragment, with partial digestion products establishing the distance between these genes to be 230-250 kb. The feasibility and potential advantages of using "natural" partials to generate detailed restriction maps within an amplified DNA domain are discussed.

Blotting, Southern↗

PubMed1988

Rapid step-gradient purification of mitochondrial DNA.

A convenient modification of the step gradient (CsCl/ethidium bomide) procedure is described. This rapid method allows isolation of covalently closed circular DNA separated from contaminating proteins, RNA and chromosomal DNA in ca. 5 h. Large scale preparations can be performed for circular DNA from eukaryotic organelles (mitochondria). The protocol uses organelle pelleting/NaCl-sarcosyl incubation steps for mitochondria followed by a CsCl step gradient and exhibits yields equal to the conventional procedures. It results in DNA sufficiently pure to be used for restriction endonuclease analysis, subcloning, 5'-end labeling, gel retention assays, and various types of hybridization.

Animals↗

PubMed1987

Loss of heterozygosity and the origin of meningioma.

In some human tumors, loss of particular genes manifested indirectly by loss of heterozygosity for specific RFLPs seems to uncover either heterozygous deletions leading to a gene doses effect or homozygous deletions due to a silent allele at the corresponding locus, both causing the loss of regulatory functions (antioncogenes, suppressor genes). Meningioma, a benign human tumor derived from the coverings of brain and spinal cord, is associated with complete loss, rarely deletion, of one chromosome 22. About 60% of meningiomas exhibit monosomy 22 in all or part of cells; however, about 40% display a normal karyotype. Comparison of constitutional and tumor genomes from 12 patients showed loss of heterozygosity on 22 in three cases, suggesting the involvement of events at the DNA level.

Adult↗

PubMed1987

Enhanced expression of four cellular oncogenes in a human glioblastoma cell line.

Examination of a human glioblastoma cell line displaying a relatively stable karyotype and absence of both copies of chromosome #13 (HeRo) as well as of a SV-40 transformed subline (HeRo-SV) using analysis on the DNA and RNA level showed that both cell lines express high levels of abl, erb B, myc, and Ha-ras mRNA. Neither gene amplification nor gene rearrangement at the loci concerned nor abnormal transcription account for this activation of expression. The possible influence of the deleted sequences in the context of a suppressor gene hypothesis is discussed.

Cell Line↗

PubMedDate not supplied

Enrichment of chromosome specific hncDNAs by magnetic bead coupled Alu sequences.

We have employed a strategy for the rapid enrichment of cDNA clones from human chromosome 22 utilizing magnetic beads. Starting from a somatic cell hybrid which retains chromosome 22 in rodent background, heteronuclear (hn) RNA was transcribed into hncDNA using poly dT-primers. Using linker specific primers hncDNA was amplified by PCR. To identify chromosome 22 specific hncDNAs a highly human specific Alu consensus sequence (PD39) was biotinylated and hybridized to the PCR product of the hncDNAs in solution. Hybridized hncDNA-PD39 complexes are captured using streptavidin-coated magnetic beads. Hybridized hncDNAs are selectively amplified by PCR. To verify the chromosome specificity the hncDNA was used as probe for in situ hybridization. Following two rounds of selection with magnetic beads there was an increasingly strong hybridization signal on chromosome 22. The capturing of hncDNAs by magnetic beads as described in this study is faster and more efficient than previously described methods for the isolation of chromosome specific hncDNAs. The novel approach has been employed to generate hncDNAs highly enriched for chromosome 22 specific sequences.

Animals↗

PubMedDate not supplied

Simultaneous isolation of high molecular weight RNA and DNA from limited amounts of tissues and cells.

A simple method is described for the simultaneous isolation of both DNA and RNA from tissues and cultured cells obtainable in limited quantities only. The method is based on a suitable combination of steps designed for preparations of high molecular weight nucleic acids in cases when restricted amounts of tissues like small-sized and unique biopsies of tumors are available for studies of gene organization and expression. Using this protocol, undegraded total RNA suitable for Northern blot analysis and high molecular weight DNA for Southern blots was obtained from various sources (mammary and colon carcinomas, meningiomas, colonic and placental tissue, and several cell cultures).

Blotting, Southern↗

PubMedDate not supplied

Assessing chromosome 22 microdeletions in meningioma with new polymorphic DNA markers.

In an approach to identify new candidates for tumor related genes, a set of established and new polymorphic DNA markers (simple sequence repeats) was used in menigioma samples to test for deletions within human chromosome 22. For this purpose, cytogenetically well characterized tumors were applied and those with monosomy #22 were omitted; only disomic material was analyzed. When comparing DNA from peripheral blood and tumor tissues using markers spaced throughout the long arm (22q) no allelic loss was observed indicating that, in disomic samples, deletions are present as microdeletions undetectable by the marker spacing chosen in this study, or that they do not occur frequently.

Biomarkers, Tumor↗

PubMedDate not supplied

Genetic imbalances in preinvasive tissue of hypopharynx provide evidence for cytogenetic heterogeneity.

Multiple chromosomal aberrations have been reported in head and neck squamous cell carcinoma (HNSCC). But less information is available on specific patterns of chromosomal amplifications which distinguish different areas of head and neck tumors. To elucidate genetic mechanisms causing the aggressive growth and high proliferation of hypopharyngeal squamous cell carcinoma (SCC), we performed reverse chromosome painting (RCP) on a total of eight hypopharyngeal SCC including invasive carcinoma and preinvasive tissue. Five hypopharyngeal invasive carcinomas showed amplifications on chromosome 3q. Furthermore, we detected gains on chromosomes 11q and 6p. Compared to the histologically classified preinvasive tissues, we found amplified alterations on chromosome 6p, 11q and 12q, but none of them showed gains on chromosome 3q. This observed heterogeneity in hypopharyngeal SCC might reflect a specific role of chromosome 3q as a late event in the highly invasive capacity of these SCC.

Carcinoma, Squamous Cell↗

PubMedDate not supplied

Characterization of a continuous cell line (MHH-NB-11) derived from advanced neuroblastoma.

A continuous cell line was established from an explanted tumor biopsy obtained from a patient with advanced neuroblastoma, which showed no response to chemotherapy. This cell line MHH-NB-11 retained most properties of the original immature tumor, even after xenotransplantation into nude mice. The cell line consisted of small dense cells with scant cytoplasm and thin; long processes and expressed neuron-specific enolase and synaptophysin, but neither GFAP nor S-100 protein. Karyotyping showed karyograms with 49 to 54 chromosomes, with a modal at 52. Most cells had trisomy 2,7,8,20, but only few structural aberrations were observed. Two of four chromosomes 1 showed a rearrangement of the terminal 1p segment, and all cells had a long HSR on the long arm of one of the chromosomes 13. This region hybridized in situ with the N-myc probe pNB-1. N-myc was amplified 20-fold in this neuroblastoma cell line as determined in Southern blot analysis. This cell line should be a useful tool in vitro or as a xenograft model for neuroblastoma research.

Animals↗

PubMedDate not supplied

Two independent amplification events on chromosome 7 in glioma: amplification of the epidermal growth factor receptor gene and amplification of the oncogene MET.

Amplifications of cellular oncogenes and growth factor genes have previously been reported in gliomas. Here we have evaluated 21 gliomas for amplification of tumor related genes including NMYC, EGFR, TGFalpha, MET, CMYC, SRC, HRAS, NRAS, SEC, ROS1, JUN, and WNT1. Five amplifications were observed. The epidermal growth factor receptor (EGFR) gene was amplified in 4 glioblastomas. The oncogene MET was amplified in a glioblastoma which showed no EGFR gene amplification. Importantly, both genes are located on chromosome 7 and belong to a family with tyrosine kinase activity. There was no amplification found for TGFalpha which was previously reported to be amplified in gliomas. The finding of MET and EGFR independently amplified in glioma lends further support to a crucial role of chromosome 7 in the development of gliomas.

Chromosomes, Human, Pair 7↗

PubMedDate not supplied

Comparative amplification analysis of human glioma tissue and glioma derived fragment spheroids using reverse chromosome painting (RCP).

Tumor fragment spheroids (TFS) represent an organotypic in vitro model with preserved cytoarchitecture and matrix components of the native tumor in situ. In order to determine whether DNA amplifications within gliomas remain stable in spheroid culture, tumor fragment spheroids were established from 15 human gliomas including 14 glioblastoma and one anaplastic astrocytoma. Native tumor tissue, monolayers as well as TFS were evaluated for DNA amplification using reverse chromosome painting (RCP). A modified protocol for DNA isolation from TFS was established. Amplifications in the original tumor tissue were found on chromosomes 12q13-15, tel, 4q12-13 and 11p12-13, an amplification on 11p12-13 is reported for the first time. By RCP we could demonstrate that amplified domains on 12q13-15 and 4q12-13 in three tumors were maintained in TFS whereas the amplification on 11p12-13 could not be confirmed in TFS. In monolayer cultures, all amplifications which were detected in primary culture were lost until passage 5. The results of this first comparative study of DNA amplification in glioma by analyzing native tumor tissue and tumor fragment spheroids enables us to conclude that TFS seems to be a promising in vitro model for the study of DNA amplification under cell culture conditions.

Adult↗