Search PubMed⌕ Search

Biomedical subjects

I D Hickson

Publications and source records attributed to I D Hickson.

At least 127 records · Page 7Linked to original sources
PubMed1989

Overproduction of topoisomerase II in an ataxia telangiectasia fibroblast cell line: comparison with a topoisomerase II-overproducing hamster cell mutant.

Ataxia telangiectasia (AT) cell lines are characterised by their hypersensitivity to ionizing radiation and bleomycin, and their failure to inhibit DNA synthesis after DNA damage. A recent report [Singh et al. (1988) Nucl. Acids Res. 16, 3919-3929] indicated that a reduction in topoisomerase II (topo II) activity was a feature of AT lymphoblast cell lines. We have studied the possible role of DNA topoisomerases in determining the phenotype of an AT fibroblast cell line. AT5BIVA cells are sensitive to the topo II inhibitors etoposide (VP16) and amsacrine (m-AMSA), compared to normal human fibroblasts (MRC5-V1 and VA13). AT5BIVA cells express a 3-fold higher level of topo II protein than MRC5-V1 cells, and 6-fold higher than VA13. This is reflected in elevated topo II activity in AT5BIVA cells. Untransformed AT5BI cells also show elevated topo II activity compared to untransformed normal cells. The extent of overproduction of topo II in AT5BIVA cells is comparable with that seen in a mutant Chinese hamster cell line, ADR-1, which is similarly hypersensitive to both bleomycin and topo II inhibitors. However, ADR-1 cells show neither hypersensitivity to ionizing radiation nor abnormal inhibition of DNA synthesis following DNA damage. Topo II overproduction per se does not appear sufficient to generate an "AT-like" phenotype. AT5BIVA cells express a reduced level of topoisomerase I (topo I) and are hypersensitive to the topo I inhibitor, camptothecin. ADR-1 cells express a normal level of topo I, indicating that a reduction in the level of topo I is not the inevitable consequence of an elevation in topo II.

Amsacrine↗
PubMed1989

Radiosensitizing and cytotoxic effects of nitroimidazoles in CHO cells expressing elevated levels of glutathione-S-transferase.

A cell line, CHO-Chlr, which has elevated levels of glutathione-S-transferase (GST), is resistant to damage caused by bi-functional nitrogen mustards. This cell line has a similar radiation sensitivity to and value of oxygen enhancement ratio as the wild type cells, CHO-K1, from which the mutant line was derived. Hypoxic cell radiosensitizing efficiencies for misonidazole, etanidazole (SR2508), and pimonidazole (R0 03-8799) are similar in both cell lines with values for C1.6 of 0.5, 0.75, and 0.08 mmol dm-3 for each drug respectively. In contrast, for RSU 1069 the sensitizing efficiency is lower in the cell line showing elevated levels of GST. The hypoxic toxicity of RSU 1069 and misonidazole is similar in each cell line, whereas in air, toxicity is 2 fold less in the CHO-Chlr cells. These results suggest that reaction of the alkylating aziridine part of RSU 1069 contributes to the high sensitizing efficiency of this drug. This is likely to be a consequence of either binding of the sensitizing moiety at the target site (ie, increased local concentration or induction of sub-lethal damage that is only expressed on subsequent irradiation.

Animals↗
PubMed1989

Defective repair of DNA single- and double-strand breaks in the bleomycin- and X-ray-sensitive Chinese hamster ovary cell mutant, BLM-2.

Using filter elution techniques, we have measured the level of induced single- and double-strand DNA breaks and the rate of strand break rejoining following exposure of two Chinese hamster ovary (CHO) cell mutants to bleomycin or neocarzinostatin. These mutants, designated BLM-1 and BLM-2, were isolated on the basis of hypersensitivity to bleomycin and are cross-sensitive to a range of other free radical-generating agents, but exhibit enhanced resistance to neocarzinostatin. A 1-h exposure to equimolar doses of bleomycin induces a similar level of DNA strand breaks in parental CHO-K1 and mutant BLM-1 cells, but a consistently higher level is accumulated by BLM-2 cells. The rate of rejoining of bleomycin-induced single- and double-strand DNA breaks is slower in BLM-2 cells than in CHO-K1 cells. BLM-1 cells show normal strand break repair kinetics. The level of single- and double-strand breaks induced by neocarzinostatin is lower in both BLM-1 and BLM-2 cells than in CHO-K1 cells. The rate of repair of neocarzinostatin-induced strand breaks is normal in BLM-1 cells but retarded somewhat in BLM-2 cells. Thus, there is a correlation between the level of drug-induced DNA damage in BLM-2 cells and the bleomycin-sensitive, neocarzinostatin resistant phenotype of this mutant. Strand breaks induced by both of these agents are also repaired with reduced efficiency by BLM-2 cells. The neocarzinostatin resistance of BLM-1 cells appears to be a consequence of a reduced accumulation of DNA damage. However, the bleomycin-sensitive phenotype of BLM-1 cells does not apparently correlate with any alteration in DNA strand break induction or repair, as analysed by filter elution techniques, suggesting an alternative mechanism of cell killing.

Animals↗
PubMed1988

Nuclear topoisomerase II levels correlate with the sensitivity of mammalian cells to intercalating agents and epipodophyllotoxins.

We have investigated the biochemical basis for the hypersensitivity to intercalating agents and epipodophyllotoxins of a Chinese hamster cell mutant, ADR-1. More topoisomerase II-induced DNA strand breaks are accumulated by ADR-1 than by parental CHO-K1 cells following exposure to the intercalating agent amsacrine. Levels of induced DNA strand breaks correlate with cell killing. Topoisomerase II activity is elevated in ADR-1 cells as a consequence of an increased cellular level of topoisomerase II protein. We have studied the phenotype of cell hybrids generated by fusing parental and mutant cells. The hybrid ADR-1/CHO-K1 exhibits normal levels of resistance to amsacrine and expresses the lower, parental level of topoisomerase II. These results provide additional evidence that topoisomerase II mediates the cytotoxic action of intercalating agents and epipodophyllotoxins and suggest that the intracellular level of topoisomerase II is an important determinant of cellular sensitivity to these drugs. This has implications for antitumor therapy. ADR-1 cells provide a model system for studying the effects of topoisomerase II overproduction on cell proliferation and chromosome organization.

Amsacrine↗
PubMed1988

Bleomycin and X-ray-hypersensitive Chinese hamster ovary cell mutants: genetic analysis and cross-resistance to neocarzinostatin.

We have previously reported the isolation of 3 mutants of Chinese hamster ovary cells which exhibit hypersensitivity to bleomycin. 2 mutants were isolated on the basis of bleomycin-sensitivity [designated BLM-1 and BLM-2, Robson et al., Cancer Res., 45 (1985) 5304-5309] and 1 as adriamycin-sensitive [ADR-1, Robson et al., Cancer Res., 47 (1987) 1560-1565]. Because bleomycin generates DNA-strand breaks via a free-radical mechanism, we have studied the survival response of these mutants to a range of drugs which also generate free radicals and consequently DNA-strand breaks. The mutants are all hypersensitive to phleomycin, which differs from bleomycin in being unable to intercalate due to a modified bithiazole moiety. However, BLM-2 cells alone are hypersensitive to pepleomycin, a semi-synthetic bleomycin analogue. In contrast, BLM-1 cells are more sensitive than BLM-2 to streptonigrin (which operates via a hydroquinone intermediate). ADR-1 cells show wild-type resistance to streptonigrin. The results obtained with neocarzinostatin, an antibiotic requiring thiol activation, are unusual in that both BLM-1 and BLM-2 are approximately 3-fold more resistant than parental cells. However, the steady-state intracellular level of the major non-protein thiol, glutathione, is not altered in BLM-1 or BLM-2 cells. ADR-1 cells show essentially wild-type resistance to neocarzinostatin. Analysis of cell hybrids shows that BLM-1 and BLM-2 cells are phenotypically recessive in combination with parental CHO-K1 cells and represent different genetic complementation groups not only from one another, but also from the bleomycin-sensitive mutant xrs-6, isolated on the basis of X-ray sensitivity by Jeggo and Kemp [Mutation Res., 112 (1983) 313-319]. These results indicate that at least 3 gene products are involved in cellular protection against bleomycin toxicity in mammalian cells.

Animals↗
PubMed1988

Amplification and increased expression of alpha class glutathione S-transferase-encoding genes associated with resistance to nitrogen mustards.

Glutathione-dependent enzymes play a central role in the protection of cells from cytotoxic chemicals and have been implicated in the intrinsic and acquired resistance of tumors to cytotoxic drugs. We have generated a Chinese hamster ovary line resistant to bifunctional nitrogen mustards and in this report have characterized and isolated the protein that represents the major observable phenotypic difference between the drug-sensitive and drug-resistant cell lines. This purified protein is shown to be an alpha class glutathione S-transferase comprising YcYc subunits and possessing a pI value of approximately 8.0. The intracellular level of the Yc subunit is elevated greater than 40-fold in the drug-resistant cell line, which could account for the increase in glutathione S-transferase (RX:glutathione R-transferase; EC 2.5.1.18) activity toward both 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide. Other glutathione S-transferase subunits within this gene family are also elevated. These changes are accompanied by a significant elevation in alpha class mRNA levels. Southern analysis indicates that the genes coding for these proteins are amplified 4- to 8-fold in the drug-resistant cell line. In addition, gamma-glutamyl transpeptidase [(5-glutamyl)-peptide:amino acid 5-glutamyltransferase; EC 2.3.2.2] activity is increased 3.6-fold in the drug-resistant Chinese hamster ovary cell line, which may explain the increase in cellular glutathione level. In this case no gene amplification was seen. These data indicate that gene amplification may be important in drug resistance toward alkylating agents and also that other enzymes in glutathione homeostasis are involved.

Animals↗
PubMed1987

Reduced levels of drug-induced DNA cross-linking in nitrogen mustard-resistant Chinese hamster ovary cells expressing elevated glutathione S-transferase activity.

We have reported previously (C. N. Robson et al., Cancer Res., 46: 6290-6294, 1986) the isolation of a Chinese hamster ovary cell line, designated CHO-Chlr, that exhibits resistance to bifunctional nitrogen mustards while maintaining the normal parental level of sensitivity to several other alkylating agents. We have compared the rate of formation and repair of DNA cross-links induced by mechlorethamine in CHO-Chlr and parental CHO-K1 cells, both in intact cells and in isolated nuclei. Equimolar doses of mechlorethamine induce significantly fewer DNA interstrand cross-links in CHO-Chlr cells than in CHO-K1 cells, but levels of DNA-protein adducts are approximately equivalent in the two lines. There is a correlation between the relative resistance of CHO-Chlr cells to mechlorethamine (34-fold) and the amount of drug required to induce approximately equal numbers of DNA interstrand cross-links in the two cell lines. This strongly implicates DNA-DNA adducts in the cytotoxic action of mechlorethamine. DNA cross-linking studies on isolated nuclei reveal only minor differences between the two lines even with identical drug treatments. The rate of cross-link repair is comparable in the two cell lines. These results, taken together with our earlier observation that the rate of drug accumulation is identical in these two lines, suggest that enhanced cytoplasmic drug detoxification is the underlying resistance mechanism in CHO-Chlr cells. We have measured cellular glutathione S-transferase activity, using both the general substrate 1-chloro-2,4-dinitrobenzene, and substrates with some specificity for the different classes of transferase isoenzymes. Total enzyme activity (as measured with 1-chloro-2,4-dinitrobenzene) is elevated 3-fold in the resistant cells. A 2- and 5-fold increase, respectively, in activity against ethacrynic acid and cumene hydroperoxide is detectable in CHO-Chlr cells. This elevation in catalytic activity in the resistant cells is reflected in higher levels of both the Yf- and Ya-type transferase subunits.

Animals↗
PubMed1987

Cross-sensitivity to topoisomerase II inhibitors in cytotoxic drug-hypersensitive Chinese hamster ovary cell lines.

We have isolated a Chinese hamster ovary cell line, designated ADR-1, which exhibits hypersensitivity to a range of drugs which are thought to inhibit the action of the enzyme topoisomerase II. These include anthracyclines, other classes of intercalating agents, and the epipodophyllotoxin, etoposide. No significant sensitivity to radiation, or to mono- and bifunctional alkylating agents was seen, although mild cross-sensitivity to the radiomimetic agent bleomycin was observed. We have monitored the level of DNA strand breaks induced by topoisomerase II inhibitors in ADR-1 cells using alkaline elution. At equimolar Adriamycin (doxorubicin) doses, more protein-associated DNA strand breaks are induced in ADR-1 cells than in wild-type cells. This enhanced level of drug-induced strand breaks does not appear to be a function of increased drug uptake as both lines accumulate similar levels of radiolabeled daunomycin. Both the rate of repair of strand breaks and the final percentage of strand breaks rejoined was equivalent in the 2 cell lines. These results are consistent with an enhancement in the level of topoisomerase II-dependent DNA breakage in ADR-1 cells following exposure to topoisomerase II inhibitors. We have previously reported the isolation of 2 bleomycin-sensitive Chinese hamster ovary cell lines, BLM-1 and BLM-2 (C. N. Robson et al., Cancer Res. 45:5304-5309, 1985). While BLM-1 exhibited cross-sensitivity only to Adriamycin, BLM-2 was shown to be hypersensitive not only to Adriamycin out also to certain alkylating agents and to ionizing radiation. In this paper, we show that both BLM-1 and BLM-2 also exhibit mild cross-sensitivity to a range of topoisomerase II inhibitors. These results indicate that intercalating agents and epipodophyllotoxins exert their cytotoxicity via common mechanisms and suggest that the maintenance of normal levels of cellular resistance to these agents requires the products of several different genes.

Animals↗
PubMed1987

Isolation of alkylating agent-sensitive Chinese hamster ovary cell lines.

Six derivatives of a Chinese hamster ovary cell line have been isolated by selection for hypersensitivity to the cytotoxic effects of the monofunctional alkylating agent methyl methane sulphonate (MMS). These cells are up to 6-fold more sensitive to MMS than the parental line, as estimated from D37 values, and are cross-sensitive to methyl nitrosourea, as well as to the ethyl derivatives of these drugs. Comparisons of their sensitivities to the bifunctional alkylating agents cis-platinum (II) diammine dichloride, mitomycin C and melphalan reveals marked phenotypic diversity, with only one mutant, designated MMS-2, exhibiting appreciable hypersensitivity to all of these agents. No striking hypersensitivity to radiation or to the purine analogue caffeine is apparent in any of the mutant lines. Based on their profiles of sensitivity to DNA damaging agents, it would appear that these mutants are phenotypically unlike any previously described mammalian cell mutants and probably represent a number of different genetic complementation groups. These mutants may facilitate an investigation into the mechanisms of repair of alkylation damage in mammalian cells and could prove to be suitable hosts for the cloning of human DNA repair genes.

Alkylating Agents↗
PubMed1986

Complete nucleotide sequence of the Escherichia coli recB gene.

The complete nucleotide sequence of the Escherichia coli recB gene which encodes a subunit of the ATP-dependent DNase, Exonuclease V, has been determined. The proposed coding region for the RecB protein is 3543 nucleotides long and would encode a polypeptide of 1180 amino acids with a calculated molecular weight of 133,973. The start of the recB coding sequence overlaps the 3' end of the upstream ptr gene, and the recB termination codon overlaps the initiation codon of the downstream recD gene, suggesting that these genes may form an operon. No sequences which reasonably fit the consensus for an E. coli promoter could be identified upstream of the proposed recB translational start. The predicted RecB amino acid sequence contains regions of homology with ATPases, DNA binding proteins and DNA repair enzymes.

Amino Acid Sequence↗
PubMed1986

Complete nucleotide sequence of recD, the structural gene for the alpha subunit of Exonuclease V of Escherichia coli.

Intracellular amplification of the Escherichia coli RecB and RecC proteins does not result in an increase in Exonuclease V activity unless the level of a third protein, encoded between the recB and argA genes, is also amplified. Nucleotide sequence analysis of this region reveals a 1,824 nucleotide open reading frame which would encode a protein of 608 amino acids with a calculated molecular weight of 66,973. This is assumed to be the structural gene for the alpha subunit of Exonuclease V, recently designated recD. The proposed initiation codon of the recD gene overlaps the termination codon of the upstream recB gene by one nucleotide, suggesting that these genes may form an operon. The deduced amino acid sequence of the RecD protein contains a region which is homologous to highly conserved sequences in adenine nucleotide binding proteins.

Amino Acid Sequence↗
PubMed1986

Complete nucleotide sequence of the Escherichia coli ptr gene encoding protease III.

The nucleotide sequence of a 3120 bp region of the E. coli chromosome that includes the entire ptr gene has been determined. The proposed coding region for Protease III is 2889 nucleotides long, which would encode a protein consisting of 962 amino acids with a calculated molecular mass of 107,719 daltons. The predicted primary structure of the protein includes a 23-residue signal sequence, cleavage of which would give rise to a mature protein of molecular mass 105,124 daltons. At its 3' end, the ptr gene overlaps the start of the recB coding sequence by 8 bases, suggesting that these genes may form part of an operon.

Amino Acid Sequence↗
PubMed1986

Complete nucleotide sequence of the Escherichia coli recC gene and of the thyA-recC intergenic region.

The nucleotide sequence of a 6,000 bp region of the E. coli chromosome that includes the 3' end of the coding region for the thyA gene and the entire recC gene has been determined. The proposed coding region for the RecC protein is 3369 nucleotides long, which would encode a polypeptide consisting of 1122 amino acids with a calculated molecular mass of 129 kDa. Mung bean nuclease mapping of a recC specific transcript produced in vivo indicates that transcription of recC is initiated 80 bp upstream of the translational start point. A weak promoter sequence situated 5' to the transcription initiation point has been identified. In the 1953 bp thyA-recC intergenic region there are three open reading frames that would code for polypeptides of molecular mass 30 kDa, 13.5 kDa and 12 kDa, respectively. Although the first and third of these open reading frames are preceded by possible ribosome binding sites, no obvious promoter sequences could be identified. Moreover, transcripts for these reading frames could not be detected.

Amino Acid Sequence↗
PubMed1986

Genetic analysis of mitomycin-C-sensitive mutants of a Chinese hamster ovary cell line.

5 mutants of a Chinese hamster ovary (CHO) cell line, which exhibit similar levels of sensitivity to killing by mitomycin C, have been analysed genetically to determine whether they represent one or more genetic complementation groups. Hybrids were constructed by fusing cells carrying either the neo or the Ecogpt marker and selecting in medium containing G418 and mycophenolic acid. Selectable markers were introduced into the cells by DNA transfection using pSV5-neo or pSV5-gpt, which represents a quick and convenient method for generating resistant derivatives. Hybrids generated by crosses between any one mutant and the parental cell line exhibited near wild-type resistance to mitomycin C, indicating that the mutants are phenotypically recessive. Self-cross hybrids for all 5 mutants had D37 values for killing by mitomycin C of between 20 and 30 ng/ml. The values obtained for crosses between different mutants were 60-105 ng/ml, with the exception of 1 pairing which gave a value of 33 ng/ml. These results indicate that that the mutants represent at least 4 different genetic complementation groups, suggesting that cellular resistance to mitomycin C is mediated via a number of different mechanisms.

Animals↗
PubMed1986

Isolation and characterization of a Chinese hamster ovary cell line resistant to bifunctional nitrogen mustards.

A drug-resistant derivative of a Chinese hamster ovary cell line has been generated by chronic exposure to progressively higher concentrations of chlorambucil. The cells exhibit greater than 20-fold resistance to the cytotoxic effects of chlorambucil and comparable levels of cross-resistance to mechlorethamine and melphalan. These drugs all belong to a class of bifunctional alkylating agents which generate DNA cross-links by reaction at the N-7 position of guanine. However, no resistance is observed to several other drugs which possess a similar mechanism of action, to cis-platinum diammine dichloride or to bischloroethylnitrosourea and mitomycin C, which cross-link DNA via the O6 position of guanine. Lack of resistance to vincristine, colchicine, or Adriamycin coupled with the failure of the calcium channel blocker verapamil to reverse the phenotype, indicates that the mechanism of resistance is distinct from that characterized by the multidrug-resistant phenotype. Support for this view comes from the finding that no significant alteration in melphalan uptake could be demonstrated. The phenotype is very stable and has been maintained during 12 months of continuous culture without further selection. A slightly elevated basal level of glutathione is present in the resistant cells, but resistance is not overcome by depletion of intracellular glutathione with buthionine sulfoximine. A cytosolic protein with a molecular weight of approximately 25,000 is constitutively overexpressed in the resistant cells. Although these cells have an abnormal karyotype, no conclusive evidence for any cytogenetic indicator of gene amplification could be detected.

Animals↗
PubMed1985

Reconstitution of RecBC DNase activity from purified Escherichia coli RecB and RecC proteins.

The Escherichia coli RecB protein, normally synthesized in low amounts, has been amplified by linkage of the recB gene to the phage lambda leftward promoter in an expression plasmid. From strains harboring this plasmid, RecB protein has been purified to homogeneity by a simple procedure which includes affinity chromatography on a column of RecC protein bound to agarose. The purified RecB protein has DNA-dependent ATPase activity but no exonuclease activity. RecC protein alone has neither ATPase nor exonuclease activity. However, when combined together, the RecB and RecC proteins show the ATP-dependent double-stranded exonuclease properties characteristic of the RecBC DNase.

Adenosine Triphosphatases↗