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Biomedical subjects

R Hancock

Publications and source records attributed to R Hancock.

At least 55 records · Page 3Linked to original sources
PubMed1991

Chromosome recombination and defective genome segregation induced in Chinese hamster cells by the topoisomerase II inhibitor VM-26.

We found that 4'-demethylepipodophyllotoxinthenylidene-beta-D-glucoside (VM-26; Teniposide), which specifically inhibits the enzyme DNA topoisomerase II, induces the formation of quadriradial chromosomes in Chinese hamster ovary cells. VM-26 traps topoisomerase II molecules when they are covalently integrated into DNA during their reaction. Quadriradial chromosomes are formed by reciprocal exchange of double-stranded DNA between single chromatids of two different chromosomes. Using synchronised cells, we found that they were formed after a single replication cycle in the presence of VM-26 at a low concentration (0.008 micro M), which does not affect DNA replication, and occurred in 50% of the mitotic cells at a concentration of 0.16 micro M. They were also formed when VM-26 was present for only 1.5 h before mitosis, after the completion of S-phase DNA replication. Chromatids bearing a translocated segment of another chromatid, which were derived from recombined chromosomes, were observed in late metaphase cells. Segregation of the daughter genomes was defective in many mitotic cells, probably because chromatids with two or no centromeres and kinetochores, formed from chromosomes recombined between their centromeres, could not be segregated. In the light of evidence that topoisomerase II molecules covalently integrated in DNA are trapped and therefore more abundant in the presence of VM-26, and that this enzyme can effect recombination of double-stranded DNA in vitro, we interpret these observations as evidence that topoisomerase II can mediate chromosome recombination in vivo.

Animals↗
PubMed1990

DNA topoisomerase II is required for formation of mitotic chromosomes in Chinese hamster ovary cells: studies using the inhibitor 4'-demethylepipodophyllotoxin 9-(4,6-O-thenylidene-beta-D-glucopyranoside).

To study the biochemical processes which DNA topoisomerase II carries out in mammalian cells, which have not been identified, we have examined the effects on chromosome replication in Chinese hamster ovary cells of an agent which traps molecules of topoisomerase II when they are covalently integrated into DNA during their reaction. This agent, 4'-demethylepipodophyllotoxin 9-(4,6-O-thenylidene-beta-D-glucopyranoside) (VM-26), targets this enzyme specifically according to a compelling body of evidence. Using synchronously growing cells, we found that VM-26 at a cytotoxic concentration (0.08 microM) did not affect DNA replication during the S phase. The formation of mitotic chromosomes was delayed by 4 h, and its rate was reduced thereafter, causing a delay in mitosis of greater than 14 h in 65% of the cells; in some cells, the chromatin was aberrantly condensed, forming diffuse chromosomes or particles. Chromosome formation was completely inhibited at 0.32 microM VM-26. DNA fragments derived from topoisomerase II molecules covalently integrated in DNA and trapped by VM-26 were detected by FIGE analysis in the G2 period, but not during the S phase. The delay of chromosome formation appeared to be caused by two factors: first, a delay in the completion of DNA replication, because progress of some cells to mitosis after removal of VM-26 was prevented by aphidicolin, an inhibitor of DNA polymerases alpha and delta; and second, a delay of chromosome formation in cells which had apparently completed DNA replication. The observations reported here show that topoisomerase II carries out reactions which are essential for formation of mitotic chromosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

An antigen located in the kinetochore region in metaphase and on polar microtubule ends in the midbody region in anaphase, characterised using a monoclonal antibody.

We describe a new component of the kinetochore region of Chinese hamster ovary cells, which was characterised using a monoclonal antibody (mAb). This antigen was localised on the kinetochore regions of purified metaphase chromosomes, but in anaphase it was instead located on the polar microtubules in the midbody region, where they terminate in the stembody. It was not detectable in prophase or interphase cells by immunofluorescence, but was present in the interphase nucleus as shown by immunoblotting after SDS-polyacrylamide gel electrophoresis. The mAb recognised two polypeptides of Mr 140,000 and 155,000. The localisation of this antigen in metaphase on the kinetochore region, where the plus ends of the kinetochore microtubules are temporarily stabilised when they attach, and later in the stembody and midbody where the plus ends of the polar microtubules are stabilised in anaphase and telophase, suggests that it could play a role in stabilising the plus ends of microtubules and thus in the control of microtubule dynamics during mitosis.

Anaphase↗
PubMed1990

Electroporation-mediated uptake of proteins into mammalian cells.

Proteins of up to 230 kilodaltons are taken up by chinese hamster ovary fibroblasts exposed to electroporation under conditions generally similar to those used to mediate DNA transfection. The uptake of catalase, ovalbumin, and histone H1 labelled with fluorescein was visualized by fluorescence microscopy. Under the same conditions, the uptake of colloidal gold particles (20 nm diameter) was visualized by electron microscopy. In optimum conditions, about 25% of the cells remained viable and grew normally and about 25% of these retained labelled proteins during two cycles of further growth. About 6 x 10(4) molecules of catalase were retained per cell. Proteins were taken up when presented to the cells up to 4 h after electroporation, suggesting that mechanisms other than classical electropore formation may operate in these conditions. The proteins were localized in the cytoplasm in a predominantly vesicular pattern and histone H1 entered the nucleus in some cells.

Animals↗
PubMed1986

Isolation and characterization of nuclear lamina from Ehrlich ascites tumor cells.

We have developed a simple and rapid method for isolation of purified nuclear lamina from Ehrlich ascites tumor cells. The procedure employs chromatin structures prepared from whole cells at low ionic strength and is carried out under conditions that minimize the formation of artifactual protein-DNA complexes. When the isolation is performed in the presence of EDTA, nuclear lamina without distinct pore complexes is obtained. In the absence of EDTA, intact pore complexes and a large amount of vimentin 100 A filaments are seen associated with nuclear lamina. The main nuclear lamina proteins are characterized using gel electrophoresis, immunoblotting, and two-dimensional peptide mapping. An extensive structural homology is found between lamin A and lamin C, whose peptide maps differ by only one major spot, whereas lamin B has apparently unrelated pattern.

Animals↗
PubMed1986

Association of DNA with the nuclear lamina in Ehrlich ascites tumor cells.

We have studied in vitro binding of DNA to nuclear lamina structures isolated from Ehrlich ascites tumor cells. At low ionic strength in the presence of Mg++, they bind considerable amounts of mouse and bacterial DNA, forming complexes stable in 2 M NaCl. Single-stranded DNA and pulse-labeled DNA show higher binding efficiencies than native uniformly labeled DNA. When mixing occurs in 2 M NaCl, complex formation is inhibited. When nuclei are digested with DNAse I under conditions that favor chromatin condensation, DNA associated with matrices subsequently prepared from such nuclei is markedly enriched in satellite DNA. If digestion is carried out with DNAse II while nuclei are decondensed in EDTA, no enrichment in satellite DNA is observed. Preparations of purified, high-molecular weight, double-stranded DNA contain variable amounts of fast-sedimenting aggregates, which are insoluble in 2 M NaCl but are dispersed by DNA fragmentation or denaturation. These results point at some artifacts inherent in studies of DNA bound to residual nuclear structures in vivo and suggest conditions expected to avoid these artifacts. Further, using controlled digestion with DNAse II, we have studied the in vivo association of DNA with nuclear lamina isolated from Ehrlich ascites tumor cells. In the course of DNA fragmentation from above 50 kbp to about 20 kbp average size, the following events were observed. The DNA of high molecular weight (much longer than 50 kbp) behaved as if tightly bound to the nuclear lamina, as judged by sedimentation in sucrose and metrizamide density gradients, electron microscopy, and retention on glass fiber filters. As the size of DNA decreased, it was progressively detached from the nuclear lamina, and at about 20 kbp average length practically all DNA was released. The last 1-4% of DNA, although cosedimenting with the nuclear lamina in sucrose gradients, behaved as free DNA, banding at 1.14 g/cm3 in metrizamide density gradients and showing less than 4% retention on filters. At no stage of digestion did the DNA cosedimenting with nuclear lamina show changes in satellite DNA content relative to that of total DNA or enrichment in newly replicated DNA. It was shown, however, that digestion of nuclear lamina-DNA complex with EcoRI or Hae III led to the formation of DNA-protein aggregates, which banded at 1.35 g/cm3 in high salt containing metrizamide density gradients and which were strongly enriched in satellite DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Simultaneous mainstream-sidestream smoke exposure systems I. Equipment and procedures.

Equipment and procedures for the simultaneous exposure of cell cultures and rodents to mainstream and sidestream smoke are described. Whole smoke or the gas phase of smoke can be generated and manipulated under a wide range of conditions. Identical smoke can be used for the exposure of cell cultures, perfused lungs, and for the exposure of experimental animals. The equipment is relatively inexpensive, and can be used by a small laboratory for efficient, controlled smoke exposure studies. Total particulate matter data are used to illustrate the flexibility of smoke generation, manipulation, and dose variables which can be obtained with the exposure systems.

Animals↗
PubMed1985

Chromosomol DNA fragments from mouse cells exposed to an intercalating agent contain a 175-kdalton terminal polypeptide.

A 175 kdalton (kDa) polypeptide is bound covalently to the chromosomal DNA fragments from mouse cells exposed to the intercalating agent 4'-[(9-acridinyl)-amino]methansulphon-m-anisidide. Electron microscopy shows a terminal protein on the DNA fragments, whose 5'-termini are blocked. Since the relative molecular mass of topoisomerase II polypeptide chains is also about 175 kDa and topoisomerase II inhibitors prevent intercalator-induced DNA fragmentation, we propose that the polypeptide bound covalently to the 5'-terminus of the DNA fragments is a polypeptide derived from frequently integrated topoisomerase II operating to normalize torsional stress resulting from intercalation.

Aminoacridines↗
PubMed1984

An improved drug infusion pump for injecting nanoliter volumes subcortically in awake rats.

A new drug infusion pump capable of injecting nanoliter volumes of drug solution into the brains of awake rats has been constructed which incorporates a new "turntable" commutator and a compact tubing compressor mechanism requiring no gears. Injector cannulas inserted into guide cannulas permanently mounted in the rat's skull are connected to the drug pump by spring-protected, PE 10 tubing. Drug solutions are delivered when the pump rollers compress the drug-filled PE 10 tubing. An additional animal-activated switch and motorized mechanism rotates the drug pump in response to the animal's movements so that the PE 10 drug reservoir is not twisted. Testing the drug pump's performance in vivo with injections of 14C-nicotine into the caudate nucleus shows that drug delivery is both reliable and reproducible.

Animals↗
PubMed1984

Correlation of morphologic and biochemical observations in human lipomas.

Our studies of 107 patients with 133 lipomata showed that the male to female ratio for single lipoma was 1.2:1, and for multiple lipoma it was 3.5:1. More than 40% of the men and 30% of women with multiple lipoma had a family history of lipoma. Specimens from 23 patients were analyzed for lipid and protein content. Adipose tissue from lipoma had somewhat more lipid per gram of wet weight than adjacent normal tissue (75.0 versus 72.8%) or 16 additional control adipose tissue samples, from patients undergoing elective abdominal surgery (71.5%), but the differences were not statistically significant (p = 0.085). There were no differences in protein content. In 13 men the lipoprotein lipase activity of lipoma was markedly higher compared with the adjacent normal adipose tissue (40.4 +/- 15.5 versus 14.0 +/- 11.7 nmoles/gm/minute at 37 degrees C, p = 0.001) or to control adipose tissue (9.6 +/- 7.2 nmoles of free fatty acid/gm/minute at 37 degrees C, p = 0.001). It is likely that the high lipoprotein lipase activity of lipoma contributes to the growth of the tumor. Morphologically, there were no clear distinguishing features between the adipocytes of lipoma, adjacent normal adipose tissue, or control adipose tissue. However, the numbers of more immature type or preadipocytes (mesenchymal cells) appeared to be more numerous in lipomata compared with the control tissues. No differences in immunologic reactivity could be detected using antisera to fat cell membranes from lipoma or control adipose tissue.

Adipose Tissue↗
PubMed1984

Histone complements of human tissues, carcinomas, and carcinoma-derived cell lines.

The pattern of subtypes of the nucleosomal histones and of histone H1 was investigated in human cells from adult and fetal lung and liver, from carcinoma tissues and from carcinoma-derived cell lines, with the object of comparing these patterns, and their relationship to cell growth rate, with those in cells of other species. The subtype pattern of the nucleosomal histones H2A and H3 shows a correlation with replication rate. In adult tissues, subtype H3-3 predominates over H3-2 and H3-1, and the subtype H2A-1 and H2A-2 are approximately equally abundant. In fetal tissues, lung carcinoma and cultured carcinoma-derived cell lines, the subtype H3-1 is predominant and H2A-1 is more abundant than H2A-2. The subtype pattern of H1 also differs between normal and carcinoma cells, among different tissues, and in different cell lines derived from the same type of carcinoma. In particular, the relative level of H1 degrees differs in several cell lines showing relatively high rates of replication, and in some cases represents more than 25% of the total H1, similar to the level in slowly replicating normal adult liver and lung tissues. The relative level of H1 degrees does not therefore appear to be correlated in a simple manner with cell growth rate in these human cells.

Animals↗
PubMed1983

Blocked 5'-termini in the fragments of chromosomal DNA produced in cells exposed to the antitumor drug 4'-[(9-acridinyl)-amino]methanesulphon-m-anisidide (mAMSA).

Comparison of the sensitivity of DNA isolated from untreated and mAMSA-treated PY815 mouse mastocytoma cells to hydrolysis by E.coli 3'-exonuclease III and phage lambda or phage T7 5'-exonucleases show that the fragments of chromosomal DNA produced by mAMSA treatment have free 3'-OH termini and blocked 5'-termini.

Aminoacridines↗
PubMed1982

Assembly of new histones into nucleosomes and their distribution in replicating chromatin.

We studied the assembly of new histones into nucleosomes and their distribution in replicating chromatin in growing P815 mouse cells. New histones and new DNA were density-labeled with 13C, 15N, 2H-substituted amino acids together with [3H]arginine or with 5-iododeoxyuridine and [3H]thymidine, respectively, for 1 hr (approximately 20% of S phase). Mono- di-, tri-, tetra- and larger oligonucleosomes were isolated by sucrose gradient centrifugation of micrococcal nuclease-digested chromatin, and their density distribution was analyzed, without fixation, in metrizamide/triethanolamine density gradients [Russev, G. and Tsanev, R. (1976) Nucleic Acids Res. 3, 697-707] in which mono- and oligonucleosomes containing dense amino acids or 5-iododeoxyuridine separate from the corresponding normal nucleosomes. Under these conditions, approximately 74% of the new histones are found in nucleosomes on newly replicated DNA, and the remainder are on unreplicated DNA. The majority of new histones form entirely new nucleosomes; a minor fraction may form hybrid nucleosomes that also contain preexisting histones. New nucleosomes are distributed to both new daughter DNA molecules with approximately equal probability, and our evidence suggests, but does not prove, that they are distributed in a random manner along new DNA.

Animals↗