PubMed Health⌕ Search

Biomedical subjects

C S Downes

Publications and source records attributed to C S Downes.

At least 19 recordsLinked to original sources
PubMed2003

Detection of replicative integrity in small colonic biopsies using the BrdUrd comet assay.

The alkaline single-cell gel electrophoresis or comet assay is a relatively simple method of measuring DNA single-strand breaks and alkali-labile sites in individual cells. Previously, we have used a combination of this with bromodeoxyuridine labelling of DNA and immunolocalisation of the BrdUrd to show that DNA replicative integrity can be assessed in single cultured cells. This study demonstrates the application of the technique to single cells derived from small human colonic biopsies isolated at routine endoscopy. A high level of reproducibility within replicate comet slides and between comet slides prepared from various colonic sites within a single patient is shown. Preliminary results demonstrate that defects in replication can be detected in tumour and premalignant colonic tissue adjacent to the tumour, suggesting that alterations in replicative integrity are an early event in neoplasia, appearing in premalignant mucosal cells. This development deems the BrdUrd comet assay suitable as an ex vivo molecular end point that can be measured easily in tissue collected by biopsy at routine colonic endoscopy. Thus, the BrdUrd comet assay has the potential to facilitate trial investigations of diet- or environment-related factors that may affect replicative integrity in the colon and provides a novel biomarker for colon carcinogenesis.

Aged↗
PubMed2001

The human decatenation checkpoint.

Chromatid catenation is actively monitored in human cells, with progression from G(2) to mitosis being inhibited when chromatids are insufficiently decatenated. Mitotic delay was quantified in normal and checkpoint-deficient human cells during treatment with ICRF-193, a topoisomerase II catalytic inhibitor that prevents chromatid decatenation without producing topoisomerase-associated DNA strand breaks. Ataxia telangiectasia (A-T) cells, defective in DNA damage checkpoints, showed normal mitotic delay when treated with ICRF-193. The mitotic delay in response to ICRF-193 was ablated in human fibroblasts expressing an ataxia telangiectasia mutated- and rad3-related (ATR) kinase-inactive ATR allele (ATR(ki)). BRCA1-mutant HCC1937 cells also displayed a defect in ICRF-193-induced mitotic delay, which was corrected by expression of wild-type BRCA1. Phosphorylations of hCds1 or Chk1 and inhibition of Cdk1 kinase activity, which are elements of checkpoints associated with DNA damage or replication, did not occur during ICRF-193-induced mitotic delay. Over-expression of cyclin B1 containing a dominant nuclear localization signal, and inhibition of Crm1-mediated nuclear export, reversed ICRF-193-induced mitotic delay. In combination, these results imply that ATR and BRCA1 enforce the decatenation G(2) checkpoint, which may act to exclude cyclin B1/Cdk1 complexes from the nucleus. Moreover, induction of ATR(ki) produced a 10-fold increase in chromosomal aberrations, further emphasizing the vital role for ATR in genetic stability.

Ataxia Telangiectasia↗
PubMed2000

Premitotic chromosome individualization in mammalian cells depends on topoisomerase II activity.

When DNA topoisomerase II (topo II) activity is inhibited with a non-DNA-damaging topo II inhibitor (ICRF-193), mammalian cells become checkpoint arrested in G2-phase. In this study, we analyzed chromosome structure in cells that bypassed this checkpoint. We observed a novel type of chromosome aberration, which we call omega-figures. These are entangled chromosome regions that indicate the persistence of catenations between nonhomologous sequences. The number of omega-figures per cell increased sharply as cells evaded the transient block imposed by the topo II-dependent checkpoint, and the presence of caffeine (a checkpoint-evading agent) potentiated this increase. Thus, the removal of nonreplicative catenations, a process that promotes chromosome individualization in G2, may be monitored by the topo II-dependent checkpoint in mammals.

Animals↗
PubMed2000

Mammalian S-phase checkpoint integrity is dependent on transformation status and purine deoxyribonucleosides.

In eukaryotic cells arrested in S-phase, checkpoint controls normally restrain mitosis until after replication. We have identified an array of previously unsuspected factors that modulate this restraint, using transformed hamster cells in which cycle controls are known to be altered in S-phase arrest. Arrested cells accumulate cyclin B, the regulatory partner of the mitotic p34(cdc2) kinase, which is normally not abundant until late G(2) phase; treatment of arrested cells with caffeine produces rapid S-phase condensation. We show here that such S-phase checkpoint slippage, as visualised through caffeine-dependent S-phase condensation, correlates with rodent origin and transformed status, is opposed by reverse transformation, and is favoured by c-src and opposed by wnt1 overexpression. Slippage is also dependent on a prolonged replicative arrest, and is favoured by arrest with hydroxyurea, which inhibits ribonucleotide reductase. This last is a key enzyme in deoxyribonucleotide synthesis, recently identified as a determinant of malignancy. Addition of deoxyribonucleosides shows that rapid S-phase condensation is suppressed by a novel checkpoint mechanism: purine (but not pyrimidine) deoxyribonucleosides, like reverse transformation, suppress cyclin B/p34(cdc2) activation by caffeine, but not cyclin B accumulation. Thus, ribonucleotide reductase has an unexpectedly complex role in mammalian cell cycle regulation: not only is it regulated in response to cycle progression, but its products can also reciprocally influence cell cycle control kinase activation.

Animals↗
PubMed1999

The bromodeoxyuridine comet assay: detection of maturation of recently replicated DNA in individual cells.

The single-cell gel electrophoresis (Comet) assay is a relatively simple method of measuring DNA single strand breaks and alkali-labile sites in individual cells. We have combined this with bromodeoxyuridine (BrdUrd) labeling of DNA and immunolocalization of the BrdUrd to assess DNA replicative integrity on a single-cell basis. We show that the existence of strand discontinuities in recently replicated domains of DNA, caused during semiconservative replication or exacerbated by the arrest of replicative polymerases at UV irradiation- or chemical-induced lesions, can be detected in individual cells. Data obtained from BrdUrd-Comets are consistent with biochemical data derived with a range of techniques showing that DNA replication involves the creation of strand breaks or gaps adjacent to recently replicated material, and that DNA damage prolongs the duration of such discontinuities where DNA polymerases are stalled opposite lesions (R. T. Johnson et al, The Legacy of Cell Fusion, pp. 50-67, Oxford: Science Publications, 1994; R. B. Painter, J. Mol. Biol., 143: 289-301, 1980.). Compared with standard biochemical techniques, the BrdUrd-Comet assay is simple and suitable for the accurate and automatable assessment of replicative integrity in very small numbers of mammalian cells, such as may be obtained by biopsy.

Animals↗
PubMed1999

Radioadaptation in Indian muntjac fibroblast cells induced by low intensity laser irradiation.

Earlier reports have indicated that an adaptive, protective response to ionizing radiation is inducible by pre-treatment with low intensity laser irradiation (LILI). We have investigated the potential of LILI to induce an adaptive response against the damaging effects of ionizing radiation in Indian muntjac fibroblasts. LILI at 660, but not 820 nm, at 11.5 and 23.0 J/cm2, induced an apparent adaptive response in the form of a reduction in the frequency of radiation-induced chromosome aberrations, but not in cell survival. There was also a trend towards a reduction in the level of single-stranded and double-stranded DNA breaks induced by ionizing radiation when cells were preconditioned with LILI. However, this did not contribute to the reduced chromosome aberration frequency. Further analysis revealed that the reduced aberration frequency was caused by a laser-induced extension of G2 delay. The adaptive response was therefore the result of cell cycle modulation by LILI, at a wavelength where there is no known DNA damaging effect to induce the checkpoint mechanisms that are normally responsible for altering cell cycle progression.

Animals↗
PubMed1999

Chemical reverse transformation of CHO-K1 cells induces changes in expression of a candidate tumour suppressor and of a gene not previously characterised as transformation related.

Chemical reverse transformation of CHO-K1 and other cells is a well-established phenomenon, in which oncogenically transformed cells re-acquire fibroblastoid morphology, contact inhibition and anchorage-dependent growth, in response to cyclic AMP and other agents. A limited number of changes in gene transcription and enzyme activity have been demonstrated to coincide with these morphological and physiological changes. We have used a partial differential display to identify four genes that are transcriptionally modulated in reverse transformation. One of these, encoding ribosomal protein S18, is transcriptionally suppressed, probably as a result of the detransforming process. Three others are transcriptionally activated. One has homology to NADH-ubiquinone oxidoreductase chain 4 protein, and is also probably changed as a result of the detransforming process. Another is homologous to a human sequence which encodes a 27 kDa protein, p27(BBP/eIF6), that is involved in the biogenesis of 60S ribosomal subunit, and in cell lines of epithelial origin binds to beta integrin. This has not previously been described as transformation-related, and could have a causative role in reverse transformation. The third has homology, with transcriptional or processing variations, to a human genomic sequence, a positional candidate for a tumour suppressor gene, encoding the Krit1 protein which interacts with the Ras-family GTPase Krev-1.

Animals↗
PubMed1999

Competence for assembly of sister chromatid cores is progressively acquired during S phase in mammalian cells.

Condensed sister chromatids possess a protein scaffold or axial core to which loops of chromatin are attached. The sister cores are believed to be dynamic frameworks that function in the organization and condensation of chromatids. Chromosome structural proteins are implicated in the establishment of sister chromatid cohesion and in the maintenance of epigenetic phenomena. Both processes of templating are tightly linked to DNA replication itself. It is a question whether the structural basis of sister chromatid cores is templated during S phase. As cells proceed through the cell cycle, chromatid cores undergo changes in their protein composition. Cytologically, cores are first visualized at the start of prometaphase. Still, core assembly can be induced in G1 and G2 when interphase cells are fused with mitotic cells. In this study, we asked if chromatid cores are similarly able to assemble in S-phase cells. We find that the ability to assemble cores is transiently lost during local replication, then regained in chromosome regions shortly after they have been replicated. We propose that core templating occurs coincident with DNA replication and that the competence for the assembly of the sister chromatid cores is acquired shortly after passage of replication forks.

Animals↗
PubMed1998

Creation of monosomic derivatives of human cultured cell lines.

Monosomic mammalian cell lines would be ideal for studying gene dosage effects, including gene imprinting, and for systematic isolation of recessive somatic mutants parallel to the invaluable mutants derived from haploid yeast. But autosomal monosomies are lethal in early development; although monosomies appear in tumors, deriving cell lines from these tumors is difficult and cannot provide several syngenic lines. We have developed a strategy for generating stable monosomic human cells, based on random autosomal integration of the gpt plasmid, partial inhibition of DNA topoisomerase II during mitosis to promote chromatid nondisjunction, and selection against retention of gpt. These are likely to be valuable as a source of otherwise inaccessible mutants. The strategy can also be used to generate partial mammalian monosomies, which are desirable as a source of information on recessive genes and gene imprinting.

Anaphase↗
PubMed1998

Emerging applications of the single cell gel electrophoresis (Comet) assay. I. Management of invasive transitional cell human bladder carcinoma. II. Fluorescent in situ hybridization Comets for the identification of damaged and repaired DNA sequences in individual cells.

ABSTRACT I: Management of invasive transitional cell human bladder carcinoma. The two main treatment options for invasive transitional cell bladder carcinoma are radiotherapy or primary cystectomy with urinary diversion or bladder substitution. Approximately 50% of patients fail to respond to radiotherapy and such patients so treated are disadvantaged by the absence of predictive information regarding their radiosensitivity, since the tumour gains additional time for metastatic spread before cystectomy is performed. The SF2 clonogenic assay, which measures the surviving fraction of tumour cells after 2 Gy X-ray irradiation, is regarded as a good measure of radiosensitivity. However, the assay is time consuming and provides results for only approximately 70% of human tumours. In this paper three bladder transitional cell carcinoma cell lines (HT1376, UMUC-3 and RT112) were exposed to X-irradiation (0-10 Gy). We have compared the responses obtained using a clonogenic assay and a more clinically feasible alkaline single cell gel electrophoresis (Comet) assay. A very good inverse correlation was obtained between cell survival (clonogenic assay) and mean tail moment (Comet assay) for the three cell lines, indicating that the Comet assay can be used to predict the radio-responsiveness of individual cell lines. The clinical usefulness of the assay for predicting response to radiotherapy in bladder cancer patients is currently being investigated. ABSTRACT II: Fluorescent in situ hybridization (FISH) Comets for the identification of damaged and repaired DNA sequences in individual cells. In mammalian cells the extent of DNA damage is partly and the rate of DNA repair very considerably dependent on DNA position and transcription. This has been established by biochemical techniques which are labour intensive and require large numbers of cells. The Comet assay for overall DNA damage and repair is relatively simple and allows individual cells to be examined. Here we present a protocol for combination of the Comet assay with fluorescent in situ hybridization (FISH) using a p53 gene probe which allows specific observation of p53 sequences within DNA comets. Chromosome-specific probes can also be used. Optimization of the FISH/Comet protocol to include automation of the analysis is currently underway to facilitate future application of the technique to study selective DNA damage and repair in defined sequences in single mammalian cells.

Carcinoma, Transitional Cell↗
PubMed1996

A protein kinase-dependent block to reinitiation of DNA replication in G2 phase in mammalian cells.

Eukaryotic cells normally replicate their DNA only once between mitoses. Unlike G1 nuclei, intact G2 nuclei do not replicate during incubation in Xenopus egg extract. However, artificial permeabilization of the nuclear membrane of G2 nuclei allows induction of new initiations by Xenopus egg extract. This is consistent with the action of a replication licensing factor which is believed to enter the nucleus when the nuclear membrane breaks down at mitosis. Here, we show that G2 nuclei will initiate a new round of replication in the absence of nuclear membrane permeabilization, if they are preexposed to protein kinase inhibitors in vivo. Competence to rereplicate is generated within 30 min of drug treatment, well before the scheduled onset of mitosis. This demonstrates that a protein kinase-dependent mechanism is continually active in G2 phase to actively prevent regeneration of replication capacity in mammalian cells. Kinase inhibition in G2 cells causes nuclear accumulation of replication protein A. Rereplication of kinase-inhibited G2 nuclei also depends on factors supplied by Xenopus egg extract, which are distinct from those required for replication licensing.

Adenine↗
PubMed1995

A postprophase topoisomerase II-dependent chromatid core separation step in the formation of metaphase chromosomes.

Metaphase chromatids are believed to consist of loops of chromatin anchored to a central scaffold, of which a major component is the decatenatory enzyme DNA topoisomerase II. Silver impregnation selectively stains an axial element of metaphase and anaphase chromatids; but we find that in earlier stages of mitosis, silver staining reveals an initially single, folded midline structure, which separates at prometaphase to form two chromatid axes. Inhibition of topoisomerase II prevents this separation, and also prevents the contraction of chromatids that occurs when metaphase is arrested. Immunolocalization of topoisomerase II alpha reveals chromatid cores analogous to those seen with silver staining. We conclude that the chromatid cores in early mitosis form a single structure, constrained by DNA catenations, which must separate before metaphase chromatids can be resolved.

Animals↗
PubMed1995

Cell cycle regulation of RNA polymerase III transcription.

Inactivation of the TATA-binding protein-containing complex TFIIIB contributes to the mitotic repression of RNA polymerase III transcription, both in frogs and in humans (J. M. Gottesfeld, V. J. Wolf, T. Dang, D. J. Forbes, and P. Hartl, Science 263:81-84, 1994; R. J. White, T. M. Gottlieb, C. S. Downes, and S. P. Jackson, Mol. Cell. Biol. 15:1983-1992, 1995). Using extracts of synchronized proliferating HeLa cells, we show that TFIIIB activity remains low during the early part of G1 phase and increases only gradually as cells approach S phase. As a result, the transcription of all class III genes tested is significantly less active in early G1 than it is in S or G2 phase, both in vitro and in vivo. The increased activity of TFIIIB as cells progress through interphase appears to be due to changes in the TATA-binding protein-associated components of this complex. The data suggest that TFIIIB is an important target for the cell cycle regulation of RNA polymerase III transcription during both mitosis and interphase of actively proliferating HeLa cells.

Cell Cycle↗
PubMed1995

Mitotic regulation of a TATA-binding-protein-containing complex.

The mitotic state is associated with a generalized repression of transcription. We show that mitotic repression of RNA polymerase III transcription can be reproduced by using extracts of synchronized HeLa cells. We have used this system to investigate the molecular basis of transcriptional repression during mitosis. We find a specific decrease in the activity of the TATA-binding-protein (TBP)-containing complex TFIIIB. TBP itself is hyperphosphorylated at mitosis, but this does not appear to account for the loss of TFIIIB activity. Instead, one or more TBP-associated components appear to be regulated. The data suggest that changes in the activity of TBP-associated components contribute to the coordinate repression of gene expression that occurs at mitosis.

Base Sequence↗
PubMed1994

A topoisomerase II-dependent G2 cycle checkpoint in mammalian cells/.

The enzyme DNA topoisomerase II, which removes the catenations formed between the DNA molecules of sister chromatids during replication and is a structural component of chromosome cores, is needed for chromosome condensation in yeast and in Xenopus extracts. Inhibitors of topoisomerase II arrest mammalian cells before mitosis in the G2 phase of the cell cycle, but also produce DNA damage, which causes arrest through established checkpoint controls. It is open to question whether cells need topoisomerase II to leave G2, or control late-cycle progression in response to its activity. Bisdioxopiperazines are topoisomerase II inhibitors that act without producing direct DNA damage; the most potent, ICRF-193, blocks mammalian entry into but not exit from mitosis. Here we show that checkpoint-evading agents such as caffeine override this block to produce abortively condensed chromosomes, indicating that topoisomerase II is needed for complete condensation. We find that exit from G2 is regulated by a catenation-sensitive checkpoint mechanism which is distinct from the G2-damage checkpoint.

Animals↗
PubMed1993

Fine tuning of DNA repair in transcribed genes: mechanisms, prevalence and consequences.

Cells fine-tune their DNA repair, selecting some regions of the genome in preference to others. In the paradigm case, excision of UV-induced pyrimidine dimers in mammalian cells, repair is concentrated in transcribed genes, especially in the transcribed strand. This is due both to chromatin structure being looser in transcribing domains, allowing more rapid repair, and to repair enzymes being coupled to RNA polymerases stalled at damage sites; possibly other factors are also involved. Some repair-defective diseases may involve repair-transcription coupling: three candidate genes have been suggested. However, preferential excision of pyrimidine dimers is not uniformly linked to transcription. In mammals it varies with species, and with cell differentiation. In Drosophila embryo cells it is absent, and in yeast, the determining factor is nucleosome stability rather than transcription. Repair of other damage departs further from the paradigm, even in some UV-mimetic lesions. No selectivity is known for repair of the very frequent minor forms of base damage. And the most interesting consequence of selective repair, selective mutagenesis, normally occurs for UV-induced, but not for spontaneous mutations. The temptation to extrapolate from mammalian UV repair should be resisted.

Animals↗
PubMed1993

Reversible effects of nuclear membrane permeabilization on DNA replication: evidence for a positive licensing factor.

We have investigated the mechanism which prevents reinitiation of DNA replication within a single cell cycle by exploiting the observation that intact G2 HeLa nuclei do not replicate in Xenopus egg extract, unless their nuclear membranes are first permeabilized (Leno et al., 1992). We have asked if nuclear membrane permeabilization allows escape of a negative inhibitor from the replicated nucleus or entry of a positive activator as proposed in the licensing factor hypothesis of Blow and Laskey (1988). We have distinguished these possibilities by repairing permeabilized nuclear membranes after allowing soluble factors to escape. Membrane repair of G2 nuclei reverses the effects of permeabilization arguing that escape of diffusible inhibitors is not sufficient to allow replication, but that entry of diffusible activators is required. Membrane repair has no significant effect on G1 nuclei. Pre-incubation of permeable G2 nuclei in the soluble fraction of egg extract before membrane repair allows semiconservative DNA replication of these nuclei when incubated in complete extract. Addition of the same fraction after membrane repair has no effect. Our results provide direct evidence for a positively acting "licensing" activity which is excluded form the interphase nucleus by the nuclear membrane. Nuclear membrane permeabilization and repair can be used as an assay for licensing activity which could lead to its purification and subsequent analysis of its action within the nucleus.

Animals↗