Biomedical subjects

H Yeger

Publications and source records attributed to H Yeger.

At least 19 recordsLinked to original sources

PubMed1992

Detection of amplified DNA sequences in human tumor cell lines by fluorescence in situ hybridization.

An unambiguous and rapid characterization of amplified DNA sequences in tumor cells is important for the understanding of neoplastic progression. This study was conducted to evaluate the potential of fluorescence in situ hybridization (FISH) to identify such amplified DNA sequences in human tumor cell lines. Applying this technique, we followed the metaphase location and interphase position of amplified DNA sequences corresponding to the SAMK, MYC, and MYCN genes in four cell lines derived from human tumors: two gastric carcinoma lines (KATO III and SNU-16), a neuroblastoma (NUB-7), and a neuroepithelioma (NUB-20) line. In metaphase cells of KATO III, NUB-7, and NUB-20 lines, the amplified regions were clearly visible and easily identified at an intrachromosomal location: in KATO III and NUB-7 at a terminal position and in NUB-20 at an interstitial position. In SNU-16, on the other hand, the amplified SAMK and MYC sequences were identified in extrachromosomal double minute chromosomes (DMs). In this line, the SAMK and MYC sequences were coamplified in the same cells and were colocated on the same DMs. FISH also allowed the identification of amplified DNA sequences in nondividing cells, enabling us to distinguish, at interphase, whether the amplification gave rise to intrachromosomal amplified regions (IARs) or to extrachromosomal DMs. The FISH technique also allowed us to determine at metaphase as well as at interphase the extent of amplification and the size of the IARs.

DNA Probes↗

PubMed1992

Loss of heterozygosity mapping in Wilms tumor indicates the involvement of three distinct regions and a limited role for nondisjunction or mitotic recombination.

Loss of heterozygosity (LOH) for polymorphic markers is a frequently occurring event in some tumors, reflecting the role of allele loss in the development of these tumors. We have determined LOH in 38 cases of Wilms tumor for the 2 known loci on chromosome arm 11p and for a newly detected locus on chromosome arm 16q. Only 7 of the 38 tumors studied showed reduction to homozygosity of 11p13 markers. In 4 of these tumors, reduced expression of WT1 and WIT1, genes located at 11p13 and implicated in Wilms tumorigenesis, was noted. However, this was also found in 2 of 7 tumors showing LOH exclusively of 11p15 markers and in 15 of the remaining 24 tumors in which there was no LOH for 11p markers. This suggests that events not involving mitotic recombination or chromosome nondisjunction are the most common mechanisms for mutations at the 11p Wilms tumor locus. We also noted that mitotic recombination involving 11p15 loci occurred in addition to reduced expression of the 11p13 locus genes in 2 tumors, suggesting a possible interaction between these 2 loci. In addition, LOH for 16q markers was observed in 6 tumors. In one case this was coincident with reduction of WT1 and WIT1 gene expression, and in 3 other cases it occurred in addition to 11p LOH. This indicates that an additional locus on 16q is likely to be involved in Wilms tumorigenesis.

Adolescent↗

PubMed1992

Increased globotriaosylceramide in familial dysautonomia.

Familial Dysautonomia (FD) is an autosomal recessive disease of unknown etiology, occurring primarily in Ashkenazi Jews. Patients are neurologically impaired, with deficits primarily in autonomic and sensory functions. The biochemical and genetic defects have remained elusive, precluding carrier detection and prenatal diagnosis. High-performance liquid chromatography data indicated up to a threefold increase in the neutral glycosphingolipid globotriaosylceramide in Dysautonomic fibroblasts and lymphoblasts. Total ganglioside values, measured by colorimetric, fluorometric or specific sodium borohydride incorporation, were decreased. Affected fibroblasts exhibited a range of pleomorphic phenotypes, such that the usual swirl-like confluent growth pattern of normal fibroblasts was distorted to varying degrees, suggesting abnormalities in the FD plasma membrane, possibly affecting cell-cell contacts. The glycosphingolipid increase could not be accounted for on the basis of markedly decreased alpha-galactosidase activity, as in Fabry's disease, where patients also display decreased autonomic function.

Cell Line↗

PubMed1992

Isolation and culture of neuroendocrine cells from fetal rabbit lung using immunomagnetic techniques.

We describe a novel method for the isolation and subsequent culture of pulmonary neuroendocrine cells (PNEC) from normal fetal rabbit lung using immunomagnetic techniques with a monoclonal antibody, MOC-1. This surface antigen has originally been identified on small cell carcinoma of the lung. Our immunohistochemical studies have shown that MOC-1 cross-reacts with PNEC of human and rabbit fetal lungs on frozen sections, and in fixed cultures of rabbit fetal lung. Using a combination of mechanical and enzymatic disaggregation, a single-cell suspension of fetal rabbit lung was obtained. These cells were incubated with MOC-1 conjugated to magnetic beads. PNEC were selectively removed from the heterogeneous mixture using a magnet, giving up to 2-fold enrichment compared with our previously reported method. These cells were maintained in culture in a functional state for up to 7 days. The ability to prepare PNEC from rabbit fetal lung offers an opportunity to develop in vitro models to investigate the physiologic and biochemical properties of these cells, and ultimately it may lead to a better understanding of their function in health and disease.

Animals↗

PubMed1992

Coordinate expression of Wilms' tumor genes correlates with Wilms' tumor phenotypes.

The cloning and molecular characterization of two putative tumor genes, WT1 and WIT1, from the chromosome 11p13 region has provided a means of evaluating their role in the generation of Wilms' tumor heterogeneity. A series of 29 tumors were analyzed for WT1 and WIT1 expression by Northern blot or RNase protection analyses, and results were compared with tumor histopathology. Tumors were scored for the percentage of mesenchymal and epithelial derived tissue components. Homotypic tumors comprised blastema, tubular epithelium, and a fibroblast-like mesenchyme. In addition to these tissue components, the group of tumors designated as heterotypic also contained ectopic cell phenotypes such as muscle and squamous epithelium. The analyses suggest that heterotypic differentiation patterns occur when WT1 and WIT1 expression is low relative to normal fetal kidney. In situ hybridization using antisense RNA probes showed that WT1 and WIT1 were concordantly expressed in normal fetal kidney and in the blastema of tumors. The ratio of WT1:WIT1 expression remained relatively constant in homotypic tumors but deviated significantly in heterotypic tumors. These results suggest that expression patterns of the WT1 and WIT1 genes can be closely correlated to Wilms' tumor histopathology.

Blotting, Northern↗

PubMed1991

P-glycoprotein expression as a predictor of the outcome of therapy for neuroblastoma.

BACKGROUND AND METHODS: Multidrug resistance in chemotherapy for cancer is characterized by increased genetic expression of P-glycoprotein, which acts as an ATP-dependent drug-efflux pump. To determine whether P-glycoprotein levels are of prognostic value in such cases, we measured these levels immunohistochemically in a retrospective study of sequential tumor samples from 67 children with neuroblastoma. RESULTS: P-glycoprotein was not detected in pretreatment samples from either of the 2 patients with Stage I disease, any of the 21 with Stage II disease, or any of the 8 with Stage IVS disease, but it was detected in the samples from 1 of the 17 patients with Stage III disease (6 percent) and 12 of the 19 with Stage IV disease (63 percent). Of the 44 patients with nonlocalized neuroblastoma (Stage III, IVS, or IV), 26 of the 31 who were negative for P-glycoprotein had a complete response to primary treatment, as compared with 6 of the 13 who were positive for P-glycoprotein (84 percent vs. 46 percent, P = 0.0232 by Fisher's exact test). Log-rank analysis of outcome, with simultaneous stratification according to tumor stage and age, showed that the group that was negative for P-glycoprotein had significantly longer relapse-free survival (P = 0.0011) and overall survival (P = 0.0373) than the group that was positive. CONCLUSIONS: Expression of P-glycoprotein before treatment may predict the success or failure of therapy for nonlocalized neuroblastoma. Neuroblastoma may be a promising tumor to treat with anticancer drug therapy combined with a chemosensitizing agent capable of reversing P-glycoprotein-mediated multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1991

Enhancement by retinoic acid and dibutyryl cyclic adenosine 3':5'-monophosphate of the differentiation and gene expression of human neuroblastoma cells induced by interferon.

Human neuroblastoma cell lines are induced to differentiate and display neuronal phenotypes when treated with interferon (IFN)-alpha 2, retinoic acid (RA), or dibutyryl cyclic AMP (dbcAMP). We investigated the effects of combinations of these agents in induction-differentiation in the neuroblastoma cell line, NUB-6. The inductive effect of IFN-alpha 2 was markedly enhanced when used in combination with RA or dbcAMP. In parallel, RA or dbcAMP also enhanced the level of 2'-5'-oligoadenylate (2-5A) synthetase, and enzyme induced by IFNs and implicated in their biological action. The levels of another IFN-inducible enzyme, p68 kinase, were not enhanced by the combination treatments. The enhancement effects appeared to be exerted largely at the posttranscriptional level as both RA and dbcAMP stabilized IFN-induced 2-5A synthetase mRNA, resulting in increased enzyme activity. Thus, the 2-5A synthetase system is likely involved in mediating the IFN-alpha 2-induced differentiation of neuroblastoma cells and may also mediate the enhancement effects of RA and dbcAMP on IFN activity in these cells. These results also provide a rational basis for establishing a combination therapeutic approach for the treatment of neuroblastoma.

2',5'-Oligoadenylate Synthetase↗

PubMed1990

Tissue, developmental, and tumor-specific expression of divergent transcripts in Wilms tumor.

The Wilms tumor locus on chromosome 11p13 has been mapped to a region defined by overlapping, tumor-specific deletions. Complementary DNA clones representing transcripts of 2.5 (WIT-1) and 3.5 kb (WIT-2) mapping to this region were isolated from a kidney complementary DNA library. Expression of WIT-1 and WIT-2 was restricted to kidney and spleen. RNase protection revealed divergent transcription of WIT-1 and WIT-2, originating from a DNA region of less than 600 bp. Both transcripts were present at high concentrations in fetal kidney and at much reduced amounts in 5-year-old and adult kidneys. Eleven of 12 Wilms tumors classified as histopathologically heterogeneous exhibited absent or reduced expression of WIT-2, whereas only 4 of 14 histopathologically homogeneous tumors showed reduced expression. These data demonstrate a molecular basis for the pathogenetic heterogeneity in Wilms tumorigenesis.

Base Sequence↗

PubMed1990

Wilms tumor locus on 11p13 defined by multiple CpG island-associated transcripts.

Wilms tumor is an embryonal kidney tumor involving complex pathology and genetics. The Wilms tumor locus on chromosome 11p13 is defined by the region of overlap of constitutional and tumor-associated deletions. Chromosome walking and yeast artificial chromosome (YAC) cloning were used to clone and map 850 kilobases of DNA. Nine CpG islands, constituting a "CpG island archipelago," were identified, including three islands that were not apparent by conventional pulsed-field mapping, and thus were at least partially methylated. Three distinct transcriptional units were found closely associated with a CpG island within the boundaries of a homozygous DNA deletion in a Wilms tumor.

Chromosome Mapping↗

PubMed1990

Importance of phenotypic and molecular characterization for identification of a neuroepithelioma tumor cell line, NUB-20.

A neuroblastic-like cell line (NUB-20) was derived from a case of histopathologically diagnosed metastatic neuroblastoma. The metastatic tumor and nude mouse heterotransplant resembled neuroblastoma by histological criteria, in contrast to the primary tumor, which was differentially classified as Ewing's sarcoma. However, the cell line demonstrated a unique phenotype in culture with respect to morphology, immunohistochemical markers, and sensitivity to a battery of differentiation modulators. These characteristics, together with the presence of a chromosomal translocation (11;22),(q24;q12) and amplification with enhanced expression of the c-myc protooncogene rather than N-myc, established this tumor as neuroepithelioma. Neuroepithelioma is a tumor type distinct from, but related to, neuroblastoma in its development from the neural crest lineage. These results emphasize the growing importance of cytogenetic and molecular markers in the classification and characterization of human tumors.

Bucladesine↗

PubMed1990

Complete physical map of the WAGR region of 11p13 localizes a candidate Wilms' tumor gene.

A complete physical map of the 11p13 region containing the Wilms' tumor locus has been developed and used to localize a candidate Wilms' tumor gene. Our strategy to construct the map combined the use of pulsed-field gel electrophoresis and irradiation-reduced somatic cell hybrids. These hybrids, which contain limited segments of human chromosome 11 segregated from the remainder of the human genome, permit direct visualization of restriction fragments located in 11p13 using human interspersed repeated DNA sequences as hybridization probes. The physical map has provided a framework to identify the sites of genes responsible for the complex of disorders associated with hemizygous 11p13 deletion: Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation. The Wilms' tumor locus has been limited to a region of less than 345 kb, and a transcript with many of the characteristics expected for the Wilms' tumor gene has been localized to this region.

Cell Line↗

PubMed1990

Isolation and characterization of a zinc finger polypeptide gene at the human chromosome 11 Wilms' tumor locus.

We have isolated a series of genomic and cDNA clones mapping within the boundaries of constitutional and tumor deletions that define the Wilms' tumor locus on human chromosome 11 (band p13). The transcription unit corresponding to these clones spans approximately 50 kb and encodes an mRNA approximately 3 kb long. This mRNA is expressed in a limited range of cell types, predominantly in the kidney and a subset of hematopoietic cells. The polypeptide encoded by this locus has a number of features suggesting a potential role in transcriptional regulation. These include the presence of four zinc finger domains and a region rich in proline and glutamine. The amino acid sequence of the predicted polypeptide shows significant homology to two growth regulated mammalian polypeptides, EGR1 and EGR2. The genetic localization of this gene, its tissue-specific expression, and the function predicted from its sequence lead us to suggest that it represents the 11p13 Wilms' tumor gene.

Amino Acid Sequence↗

PubMed1990

Definition of the limits of the Wilms tumor locus on human chromosome 11p13.

In a previous report, we described a contiguous restriction map of chromosome band 11p13 that localized the Wilms tumor locus to a small group of NotI fragments. In an effort to identify and isolate the 11p13-associated sporadic Wilms tumor locus, we developed a panel of NotI fragment-specific DNA probes. These probes were selected from genomic libraries constructed using the Chinese hamster ovary-human somatic cell hybrid carrying only human 11p. The libraries were prepared from NotI-digested DNA after size selection by pulsed-field gel electrophoresis. The selected NotI fragments had been previously targeted on the basis of deletion mapping as having a high probability of containing the Wilms tumor locus. We used these newly identified 11p13-specific probes to improve the resolution of the restriction map spanning the Wilms tumor locus. The locus has been defined by a homozygous deletion in a sporadic Wilms tumor. Using these probes, the region of homozygous deletion in this tumor and presumably all or part of the Wilms tumor gene have been confined to two small SfiI fragments spanning less than 350 kb.

Animals↗

PubMed1990

Transformed sweat gland and nasal epithelial cell lines from control and cystic fibrosis individuals.

We undertook to extend the in vitro lifespan of epithelial cell cultures useful for the study of the cellular defect underlying cystic fibrosis (CF). Primary cultures from sweat glands of four CF and four non-CF and from nasal polyps of one non-CF and two CF individuals were transformed using a chimaeric virus, Ad5/SV40 1613 ori-. The extended lifespans ranged from 20 to more than 250 population doublings beyond that of the primary cultures. Despite some degree of aneuploidy (as assayed by total cellular DNA content) all samples tested retained at least one copy of the region of chromosome 7 containing the CF gene (as assayed by probing with flanking DNA markers). Epithelial characteristics, including an epithelioid morphology, tight junctions and desmosomes, apical microvilli, keratin networks, and dome formation were positive in the majority of cells examined, although variably expressed. All cells tested demonstrated outwardly rectifying chloride channels by patch clamp, with some from non-CF cells responsive to the catalytic subunit of cyclic AMP-dependent protein kinase. The cells were used for DNA transfection assays with selectable marker genes in appropriate vectors, in order to develop methodology for assaying the function of the CF gene product and the effects of mutations.

Cell Division↗

PubMed1990

Nickel binding by isolated human trophoblast cells.

Isolated human trophoblast cells were used for the first time to mimic the placental metabolism of nickel. The retention of nickel by the trophoblast cells was assessed in presence of different nickel ligands. Nickel chloride and nickel-albumin showed an initial rapid binding to human trophoblast cells reaching a quasi-steady state after 30 min. Albumin, however, reduced the binding of nickel to the cells. Nickel-histidine showed slow initial binding, although no saturation was achieved. The results for nickel-histidine complexes are similar to those observed in in vivo experiments.

Absorption↗

PubMed1989

Monoclonal antibody to a human pancreatic carcinoma cell line recognizes gastrointestinal neoplasms.

A monoclonal antibody designated RWP1.1 was produced against a human pancreatic carcinoma cell line RWP1. This antibody was shown to recognize an epitope of carcinoembryonic antigen. The reactivity of the antibody was evaluated on formalin-fixed normal tissues, 86 malignant neoplasms, 10 colonic polyps, and 6 cases of chronic pancreatitis using the peroxidase anti-peroxidase technique. RWP1.1 did not react with normal tissues apart from weak staining of fetal pancreatic ducts. This antibody preferentially reacted with primary and metastatic adenocarcinomas of the colon, stomach, pancreas, and colonic polyps but not with most adenocarcinomas from other sites nor with other types of tumors or cases of chronic pancreatitis. It also reacted with colon adenocarcinomas on frozen sections. The restricted specificity of this antibody could be used in differentiating gastrointestinal adenocarcinomas from other types of tumors including adenocarcinomas from other sites and most pancreatic adenocarcinomas from chronic pancreatitis.

Adenocarcinoma↗

PubMed1989

Amplification of N-myc and ornithine decarboxylase genes in human neuroblastoma and hydroxyurea-resistant hamster cell lines.

The genes for the M2 subunit of ribonucleotide reductase (RRM2), ornithine decarboxylase (ODC1), and 55,000-Daltons protein (P5), are amplified in hydroxyurea-resistant hamster and human cell lines. These genomic sequences have been mapped to hamster chromosome 7 and to human chromosome 2p24-25 near the cytogenetic location of the N-myc gene. We now report that genomic sequences homologous to N-myc are amplified in hydroxyurea-resistant hamster lung cell line, 600H, and the N-myc gene segregates with hamster chromosome 7 in mouse-hamster somatic cell hybrids. The conserved linkage group consisting of the RRM2, ODC1, P5, and N-myc in the hamster and human genomes prompted our investigation of human neuroblastomas. We report here that genomic DNA from 1 of 6 primary neuroblastoma tumors containing amplified N-myc also contains amplified sequences homologous to a hamster ODC cDNA.

Adolescent↗

PubMed1988

Homozygous deletion of a DNA marker from chromosome 11p13 in sporadic Wilms tumor.

A random DNA fragment, probe p2.3 (locus D11S87), was cloned from the 11p13 region between a translocation breakpoint associated with familial aniridia and another translocation breakpoint associated with childhood T-cell leukemia. The D11S87 locus maps between the catalase (CAT) locus and the beta subunit of follicle stimulating hormone (FSHB). The D11S87 locus is deleted in a Wilms tumor patient with a constitutional deletion of 11p and in a case of sporadic Wilms tumor (WiT-13) apparently with normal karyotype. In the WiT-13 tumor both maternal and paternal chromosomes 11 are retained; D11S87 is deleted homozygously and FSHB hemizygously. These results suggest two mutational events resulting in homozygous deletion in this patient. The D11S87 homozygous deletion was also demonstrated in WiT-13 nude mouse heterotransplants and in fibroblast-like cell line derived from the primary tumor. The minimum size of the deletion was estimated to be 30 kb as determined by cosmid screening and hybridization. As homozygous deletions in the 11p13 region have not been previously reported for sporadic Wilms tumors, these findings place the D11S87 locus within or approximate to the Wilms tumor gene.

Blotting, Southern↗