Search PubMed⌕ Search

Biomedical subjects

B Vogelstein

Publications and source records attributed to B Vogelstein.

At least 271 records · Page 15Linked to original sources
PubMed1987

Rapid purification of DNA from agarose gels by centrifugation through a disposable plastic column.

A simple and rapid method for purifying DNA from agarose gels is described. Agarose slices containing DNA are placed in a disposable plastic column and the DNA is separated from the agarose by centrifugation in a microfuge. Recoveries averaging 25% are obtained for DNA of 14 kb or less. The recovered DNA can be labeled to high specific activity, cleaved with restriction endonucleases, and ligated efficiently using standard cloning vectors.

Centrifugation↗
PubMed1987

Relationship between gene amplification and chromosomal deviations in malignant human gliomas.

Biopsies of 33 malignant human gliomas were karyotyped and evaluated for amplification (more than eight gene copies per cell) of the epidermal growth factor receptor (EGFR), N-myc, c-myc, and gli genes by Southern blot analysis. Fifteen of 33 tumors showed amplification of EGFR, none had amplified c-myc, one tumor had amplified N-myc, and one had amplification of gli. Thirteen of the 16 (81%) evaluable tumors with gene amplification contained double minutes (DM), and only four of 16 (25%) tumors without demonstrable amplification contained these structures. Polysomy for chromosome #7, in contrast, occurred in 58% of tumors with EGFR amplification and 53% of tumors without amplification of the gene. Structural abnormalities of 7p occurred in two tumors with EGFR amplification and in one tumor without amplification of this gene. These studies suggest that DM are the usual locus for amplified genes (usually EGFR) in human glioma biopsies, but that structural abnormalities of 7p may be associated with EGFR amplification in a small proportion of these tumors. The presence of polysomy 7, however, probably is unrelated to amplification of the EGFR gene.

Chromosome Aberrations↗
PubMed1987

Increased expression of the epidermal growth factor receptor gene in malignant gliomas is invariably associated with gene amplification.

Primary malignant gliomas from 63 patients were analyzed to determine the relationship between amplification of the gene encoding the epidermal growth factor receptor (EGFR) and expression of the corresponding mRNA. Twenty-four tumors were found to have amplified the EGFR gene and amplification of other genes occurred in three additional tumors. Hybridization with synthetic RNA probes was used to quantitate mRNA levels in situ. All 24 tumors with amplification of the EGFR gene had high levels of expression of this gene, while none of the 39 tumors without amplification had increased levels. This shows that, in human gliomas, large increases in the expression of the EGFR gene are invariably associated with alterations in gene structure.

ErbB Receptors↗
PubMed1987

Hypermethylation of the 5' region of the calcitonin gene is a property of human lymphoid and acute myeloid malignancies.

An abnormal increase in numbers of CCGG sites methylated in the 5' region of the human calcitonin (CT) gene occurred in tumor cell DNA samples from 90% (17 of 19) of patients with non-Hodgkin's T and B cell lymphoid neoplasms and in 95% (21 of 22) of tumor cell DNA samples from patients with acute nonlymphocytic leukemia (ANLL). The changes were not seen in patients with chronic myelogenous leukemia (0 of 9). The abnormal methylation patterns appear to be a property only of transformed or malignant cells since they were not found in DNA from nonneoplastic adult tissues including sperm, early myeloid progenitor cells, benign lymphoid hyperplasia, peripheral lymphocytes stimulated to divide, or early myeloid progenitor cells (obtained by immunoaffinity using anti-My-10 antibody), but they did appear after Epstein-Barr virus transformation of lymphocytes. Moreover, during the course of therapy in patients with ANLL, the hypermethylation pattern reflects the presence of the leukemic clone even in normal-appearing granulocytes derived from this clone. The increased methylation of the CT gene may then provide an important molecular marker for biologic events in human cell transformation or tumor progression and may prove clinically useful in monitoring patients with lymphoid and acute myelogenous neoplasms.

Acute Disease↗
PubMed1986

Gene amplification of c-myc and N-myc in small cell carcinoma of the lung.

The relationship of the copy numbers of the c-myc and N-myc oncogenes to tumor formation and progression was studied in small cell carcinoma of the lung. When 96 neoplastic lesions from 45 patients were examined, these lesions could be grouped into three categories: high copy (tumors with greater than 3 copies of the N-myc or c-myc gene per haploid genome), middle copy (1.5 to 3 copies per genome), and normal copy. Fourteen of the patients had middle copy tumors, but this was almost always a result of chromosome duplication rather than the amplification of a small genetic locus. In contrast, five patients had high copy tumors, with the increased copy number in each case due to gene amplification. The amplification did not occur in a heterogeneous fashion within individual patients, since all metastatic lesions from patients with high copy lung tumors were also high copy, while none of 41 metastatic lesions from the other patients were high copy. These data suggest that gene amplification is an important step in neoplastic growth in a subset of patients with small cell carcinoma of the lung and that this genetic event occurs relatively early (before metastasis) in this subset.

Carcinoma, Small Cell↗
PubMed1986

Differentiation of leukemia cells to polymorphonuclear leukocytes in patients with acute nonlymphocytic leukemia.

We used recombinant-DNA techniques to determine the origin of polymorphonuclear leukocytes in patients with acute nonlymphocytic leukemia, at presentation, in remission, and in relapse. Studies using X chromosome-linked DNA polymorphisms strongly suggested that leukemic blast cells in this form of leukemia can differentiate in vivo to form mature granulocytes. Common chromosomal changes seen in blast cells, such as the addition of a chromosome 8 or the loss of a chromosome 7, were found to be present in the mature granulocytes of patients with leukemia. In addition, rearrangement of the immunoglobulin heavy-chain gene was detected in the polymorphonuclear leukocytes of one patient. Finally, we have observed the persistence of a single dominant hemopoietic clone in the granulocytes of 3 of 13 patients in complete remission. These findings demonstrate that recombinant-DNA probes can detect clonal populations of granulocytes in patients with acute nonlymphocytic leukemia, and provide further evidence that in some patients leukemic blast cells differentiate to form mature cells.

Acute Disease↗
PubMed1986

Gene amplification in cancer: a molecular cytogenetic approach.

Many human tumors have been shown to exhibit cytologic evidence of gene amplification. In several instances the amplified genes have proven to be cellular oncogenes. We present an approach for combining cytogenetic and molecular biological techniques to systematically investigate this phenomenon.

Base Sequence↗
PubMed1986

Evidence for rearrangement, amplification, and expression of c-myc in a human glioblastoma.

Amplified cellular genes are frequently manifested in one of two cytologically recognizable forms, double minutes or homogeneously staining regions. Additionally, evidence is accumulating that aberrant expression of cellular genes (including oncogenes) may be mediated by gene amplification. We now describe the amplification and expression of the cellular oncogene c-myc in double-minute-containing cells from a patient with glioblastoma multiforme, and we have shown that the amplification is associated with rearrangement of the c-myc gene. This finding further supports the common association of the myc gene family in neurogenic tumors and provides evidence of myc gene amplification in human brain cancer.

Cell Line↗
PubMed1986

Amplification units containing human N-myc and c-myc genes.

The amplification units in human tumors containing amplified myc genes were examined. The amplification unit in all cases consisted of a large genomic region coamplified with the coding region of the myc genes themselves. In eight independent neuroblastomas containing N-myc amplifications, the amplification unit was estimated to be 290 to 430 kilobases. This amplification unit was highly conserved among the different neuroblastomas, with some neuroblastomas containing almost identical units. In contrast, five tumor cell lines containing c-myc amplifications exhibited amplification units that were more variable in size (90 to 300 kilobases) and sequence content; at least three different patterns of c-myc amplification units could be discerned.

Brain Neoplasms↗
PubMed1986

Immunoglobulin gene rearrangements in remission bone marrow specimens from patients with acute lymphoblastic leukemia.

Recombinant DNA probes for the joining (JH) segment of the immunoglobulin heavy chain gene were used to detect molecular rearrangements of this gene in the DNA of bone marrow cells obtained during remission of acute lymphoblastic leukemia (ALL). This molecular approach was optimized and found to exceed the sensitivity of conventional morphologic screening for detecting residual leukemia cells; one leukemic cell in 500 normal nucleated bone marrow cells was easily detected using this approach. In the present study, bone marrow from three of seven patients in complete clinical remission (defined morphologically) contained leukemic cells in these proportions. This analysis may be of use in evaluating the status of clinical remission in selected ALL patients.

Adolescent↗
PubMed1985

The anatomy of supercoiled loops in the Drosophila 7F locus.

The genome in eucaryotes is organized into a series of supercoiled loops, topologically anchored at their bases by components of the nuclear matrix. Previous studies have shown that active genes are associated with the nuclear matrix. We wished to know whether loops in general were solely organized by active genes. We therefore examined a locus of the Drosophila X-chromosome comprising 163,000 bp of continuous DNA sequences and devoid of known active genes. Of the 52 EcoRI restriction fragments comprising this region, we found 5 anchored fragments which non-randomly organized this region into 4 DNA loops. Each of the 5 anchored fragments contained a transcribed sequence. These results strongly suggest that supercoiled loops are organized in a specific fashion with respect to DNA sequence, with the anchorage points exclusively demarcated by transcriptionally active genes.

Animals↗
PubMed1985

Purification of DNA from formaldehyde fixed and paraffin embedded human tissue.

The ability to isolate DNA from preserved human tissues would provide numerous experimental opportunities. In this report it is shown that DNA can be extracted from tissues prepared for routine histopathological examination (i.e., fixed with formaldehyde and embedded in paraffin). Although the extracted DNA is not intact, it is double stranded, cleavable with restriction endonucleases, and suitable for a variety of standard techniques used in molecular biology.

Carcinoma↗
PubMed1985

Hypomethylation of DNA from benign and malignant human colon neoplasms.

The methylation state of DNA from human colon tissue displaying neoplastic growth was determined by means of restriction endonuclease analysis. When compared to DNA from adjacent normal tissue, DNA from both benign colon polyps and malignant carcinomas was substantially hypomethylated. With the use of probes for growth hormone, gamma-globin, alpha-chorionic gonadotropin, and gamma-crystallin, methylation changes were detected in all 23 neoplastic growths examined. Benign polyps were hypomethylated to a degree similar to that in malignant tissue. These results indicate that hypomethylation is a consistent biochemical characteristic of human colonic tumors and is an alteration in the DNA that precedes malignancy.

Chorionic Gonadotropin↗
PubMed1985

The association of transcribed genes with the nuclear matrix of Drosophila cells during heat shock.

Using the transcriptional modulation afforded by heat shock, we found that the association of active genes with the nuclear matrix was not dependent on their level of transcription. Heat shock genes were matrix associated both before heat shock (when transcription was relatively low), and during heat shock (when transcription was greatly increased). Conversely, the cytoplasmic actin gene was matrix associated during normal growth conditions (when transcription was high) and during heat shock (when transcription was greatly decreased). Removal of greater than 99.7% of nascent RNA during preparation of the matrices did not affect these findings. Detailed examination of the cytoplasmic actin gene revealed that its matrix association was apparently mediated by multiple interactions near the 5' end of the gene.

Actins↗
PubMed1985

Use of restriction fragment length polymorphisms to determine the clonal origin of human tumors.

A novel strategy to determine the clonal origin of human tumors has been devised. The strategy involves the use of a cloned polymorphic X-chromosomal gene and two restriction endonucleases. The first endonuclease distinguishes the paternal and maternal copies of the gene through a DNA polymorphism of restriction fragment length. The second endonuclease distinguishes active from inactive copies of this gene through changes in DNA methylation. As illustrations of this strategy, three human cancers were each shown to be monoclonal. The analysis described should have a wide variety of clinical and experimental applications.

Autoradiography↗
PubMed1983

The association of transcriptionally active genes with the nuclear matrix of the chicken oviduct.

Eucaryotic DNA is organized into a series of supercoiled loops that are anchored to the nuclear matrix. When these DNA loops are cleaved by endonucleases, the DNA sequences which remain associated with the nuclear matrix can be recovered and analyzed for their content of specific genes. Using restriction endonucleases to cleave the loops, we demonstrate that ovalbumin and conalbumin gene sequences are preferentially associated with the nuclear matrix of hen oviduct cells but not with the nuclear matrix of hen brain cells. Furthermore, we determined that several regions of the ovalbumin gene were independently attached to the nuclear matrix of hen oviduct cells. This included sequences located 3.8 kb downstream from the 3' end of the ovalbumin gene transcription unit. To determine whether the nuclear matrix association of the ovalbumin gene was regulated by hormones, we examined the oviduct cells of chicks that underwent primary estrogen stimulation, estrogen withdrawal and secondary estrogen stimulation. Ovalbumin gene sequences selectively dissociated from the chick oviduct nuclear matrix during estrogen withdrawal and reassociated with the nuclear matrix following restimulation.

Animals↗