Search PubMed⌕ Search

Biomedical subjects

C S Downes

Publications and source records attributed to C S Downes.

At least 37 records · Page 2Linked to original sources
PubMed1993

Topoisomerase II inhibition prevents anaphase chromatid segregation in mammalian cells independently of the generation of DNA strand breaks.

Yeast temperature-sensitive mutants of DNA topoisomerase II are incapable of chromosome condensation and anaphase chromatid segregation. In mammalian cells, topoisomerase II inhibitors such as etoposide (VP-16-123) have similar effects. Unfortunately, conclusions drawn from work with mammalian cells have been limited by the fact that the standard inhibitors of topoisomerase II also generate DNA strand breaks, which when produced by other agents (e.g. ionizing radiation) are known to affect progression into and through mitosis. Here we show that the anti-tumour agent ICRF-193, recently identified as a topoisomerase II inhibitor operating by a non-standard mechanism, generates neither covalent complexes between topoisomerase II and DNA, nor adjacent DNA strand breaks, in mitotic HeLa. However, the drug does prevent anaphase segregation in HeLa and PtK2 cells, with effects similar to those of etoposide. We therefore conclude that topoisomerase II function is required for anaphase chromosome segregation in mammalian cells, as it is in yeast.

Anaphase↗
PubMed1992

The nuclear membrane prevents replication of human G2 nuclei but not G1 nuclei in Xenopus egg extract.

We have used synchronized HeLa cells to investigate the role of the nuclear membrane in preventing rereplication in a single cell cycle. Nuclei were prepared with intact nuclear membranes using streptolysin-O or digitonin and assayed for replication in Xenopus egg extracts. Intact G1 nuclei replicate semiconservatively, but intact G2 nuclei do not replicate in egg extract. However, permeabilizing the nuclear membranes of G2 nuclei by treatment with NP-40 allows them all to replicate in egg extract under cell cycle control, suggesting that integrity of the nuclear membrane is required to distinguish G2 from G1 human nuclei and to prevent rereplication within a single cell cycle. The results are discussed in terms of the previously proposed licensing factor model.

Animals↗
PubMed1992

Radiomimetic cell cycle delay induced by tetranodecanoyl phorbol acetate is enhanced by caffeine and by the protein kinase inhibitor 2-aminopurine.

The tumour promoter and protein kinase C agonist, 12-O-tetranodecanoyl-phorbol-13-acetate (TPA), has been reported to show a radiomimetic action because it transiently delays the passage of HeLa cells through the G2 phase, as do ionizing radiation and other DNA damaging agents. Caffeine is known to override the G2 delay imposed by DNA damage; it is shown here that caffeine does not override the radiomimetic delay imposed by TPA in HeLa, but instead enhances it, without affecting G2 progression in control cells. Most of the other agents which more specifically affect some of the diverse range of caffeine targets either do not affect G2 progression after TPA, or delay G2 progression in control cells and exert a further delay in the presence of TPA. The exception is 2-aminopurine, a protein kinase inhibitor which has been shown to have an action similar to that of caffeine is allowing progression of the cell cycle to mitosis after the inhibition of DNA synthesis, without affecting normal cycle progression through G2. This agent, like caffeine, also has the contrary action of retarding cycle progression after TPA. It is concluded that the G2 delays induced by ionizing radiation and by TPA operate by different mechanisms, which are modulated in opposite senses by mechanisms involving protein kinase inhibition.

2-Aminopurine↗
PubMed1991

Inhibitors of DNA topoisomerase II prevent chromatid separation in mammalian cells but do not prevent exit from mitosis.

DNA topoisomerase II (EC 5.99.1.3) is necessary for chromosome condensation and disjunction in yeast but not for other functions. In mammalian cells, it has been reported to be necessary for progression toward mitosis but not for transit through mitosis. We have found, on the contrary, that specific inhibition of topoisomerase II (but not of topoisomerase I) interferes with mammalian mitotic progression. Metaphase is prolonged, and anaphase separation of chromatids is completely inhibited, in cells given high concentrations of topoisomerase II inhibitors; nevertheless these cells attempt cleavage, sometimes generating nucleate and anucleate daughters. Lower concentrations of inhibitors interfere with anaphase and produce abnormalities of segregation. DNA topoisomerase II activity is therefore necessary for mammalian chromatid separation, but it is not tightly coupled to the control of other mitotic events.

Anaphase↗
PubMed1991

Abnormal sister-chromatid exchange induction by 3-aminobenzamide in an SV40-transformed Indian muntjac cell line: relationships with DNA maturation and DNA-strand breakage.

In SVM cells, an SV40-transformed line of Indian muntjac fibroblasts, levels of sister-chromatid exchanges are known to be abnormally high after UV-irradiation or alkylation. The SVM line is also known to have a defect in the processing of DNA-strand breaks. Sister-chromatid exchange in other cells is known to be stimulated by the poly(ADP-ribose) transferase inhibitor, 3-aminobenzamide, which also retards DNA-break sealing. Sister-chromatid exchanges in SVM cells are found to be hypersensitive to 3-aminobenzamide, or to nicotinamide deprivation which similarly inhibits poly(ADP-ribosyl)ation; DNA-strand breaks are likewise induced by 3-aminobenzamide. Bromodeoxyuridine, needed to detect sister-chromatid exchanges, is more toxic to SVM cells and itself induces sister-chromatid exchanges to a greater extent than it does in normal muntjac cells. However, in contrast to the situation reported for other cell types prone to sister-chromatid exchange (the Chinese hamster ovary mutant EM9 and human Bloom's Syndrome cells), SVM cells do not show an abnormal delay in DNA maturation when, under the influence of bromodeoxyuridine and 3-aminobenzamide, they show a high level of sister-chromatid exchange. The mechanism by which BrdU exerts its effects can largely be explained in terms of familiar effects on deoxyribonucleotide pools and DNA integrity. 3-Aminobenzamide, however, induces sister-chromatid exchanges in SVM cells by another mechanism.

Animals↗
PubMed1990

Action of caffeine on DNA replication after ultraviolet irradiation in Indian muntjac cells: no connection with action on cell cycle delay.

Indian muntjac fibroblasts of the SV40-transformed line SVM are hypersensitivity to UV, and after UV irradiation have defective post-replication recovery and a high level of sister chromatid exchanges and chromosome aberrations. The lethal and clastogenic effects of UV on SVM have elsewhere been shown to be aggravated by caffeine, which overcomes the block to cycle traverse imposed by DNA damage; however, in DM cells, an Indian muntjac line of normal UV sensitivity, caffeine has no effect on cycle traverse, but nevertheless enhances UV killing and sister chromatid exchanges. In this paper, the effects of caffeine on irradiated DM cells are shown to be due to its inhibition of post-replication recovery, with subsequent formation of DNA double-strand breaks at the strand gaps thus produced. By contrast, in SVM cells the limited capacity for post-replication recovery is relatively insensitive to caffeine after UV fluences which permit significant cell survival; however, caffeine still strongly induces DNA double-strand breaks and chromosome aberrations, apparently by an alternative mechanism. The SVM and DM cell lines therefore exemplify separate actions of caffeine on mammalian cells, deficient in the caffeine effects on post-replication recovery and cell cycle progression, respectively.

Animals↗
PubMed1990

Caffeine overcomes a restriction point associated with DNA replication, but does not accelerate mitosis.

Mitotic chromosome condensation is normally dependent on the previous completion of replication. Caffeine spectacularly deranges cell cycle controls after DNA polymerase inhibition or DNA damage; it induces the condensation, in cells that have not completed replication, of fragmented nuclear structures, analogous to the S-phase prematurely condensed chromosomes seen when replicating cells are fused with mitotic cells. Caffeine has been reported to induce S-phase condensation in cells where replication is arrested, by accelerating cell cycle progression as well as by uncoupling it from replication; for, in BHK or CHO hamster cells arrested in early S-phase and given caffeine, condensed chromosomes appear well before the normal time at which mitosis occurs in cells released from arrest. However, we have found that this apparent acceleration depends on the technique of synchrony and cell line employed. In other cells, and in synchronized hamster cells where the cycle has not been subjected to prolonged continual arrest, condensation in replication-arrested cells given caffeine occurs at the same time as normal mitosis in parallel populations where replication is allowed to proceed. This caffeine-induced condensation is therefore "premature" with respect to the chromatin structure of the S-phase nucleus, but not with respect to the timing of the normal cycle. Caffeine in replication-arrested cells thus overcomes the restriction on the formation of mitotic condensing factors that is normally imposed during DNA replication, but does not accelerate the timing of condensation unless cycle controls have previously been disturbed by synchronization procedures.

Animals↗
PubMed1988

Caffeine induces uncoordinated expression of cell cycle functions after ultraviolet irradiation. Accelerated cycle transit, sister chromatid exchanges and premature chromosome condensation in a transformed Indian muntjac cell line.

Caffeine enhances the lethal effect of DNA-damaging agents. It also affects the timing of events in the cell cycle; the enhanced cytotoxicity may be partly due to caffeine's ability to overcome the protective damage-induced delay in S or G2 phase. When the effects of caffeine are compared in a normal Indian muntjac cell line and a simian virus 40 (SV40)-transformed, ultraviolet light (u.v.)-sensitive line in which u.v. induces many sister chromatid exchanges, different cell cycle sensitivities are seen. In the SV40-transformed line, caffeine over-rides the delay in DNA synthesis imposed by DNA damage; it initiates late cycle events after u.v. irradiation, and in some cases it induces S-phase premature chromosome condensation, apparently by inducing mitotic factors in cells where the chromatin is still replicating. Caffeine has been reported to induce similar premature chromosome condensation in cells arrested with DNA polymerase inhibitors; this is the first recognition that such events occur in response to caffeine treatment after DNA damage. Some apparent paradoxes of caffeine's action on irradiated cells can be thus explained. In the normal line, caffeine neither affects progression through the cycle nor induces premature chromosome condensation after irradiation. In both lines, caffeine increases killing and sister chromatid exchanges after u.v. irradiation; but these effects are more pronounced when exerted through the cell cycle-related mechanism.

Animals↗
PubMed1987

Automatic nitrous oxide synchronization of mitotic human cell cultures.

Large numbers of human cells can be reversibly arrested in mitosis by high-pressure nitrous oxide. The optimum schedule for this arrest requires that the high-pressure block be started around midnight to provide a mitotic population that can be released the next morning to progress in synchrony through the next cell cycle. We describe a simple and safe device which can be set up at the end of the day and automatically exposes the cells to high-pressure nitrous oxide overnight.

Cells, Cultured↗
PubMed1987

Action of etoposide (VP-16-123) on human cells: no evidence for topoisomerase II involvement in excision repair of u.v.-induced DNA damage, nor for mitochondrial hypersensitivity in ataxia telangiectasia.

The excision repair of u.v. damage has been supposed to involve an initial action of DNA topoisomerase II, since some pre-incision step is sensitive to novobiocin, a topoisomerase II inhibitor. But novobiocin also affects mitochondrial structure and ATP metabolism, and this may account for its apparent inhibition of energy-dependent excision repair. We have investigated the effects of etoposide, another inhibitor of topoisomerase II, on u.v.-irradiated human cells: it is a more specific agent with no immediate side-effects on mitochondria. But etoposide is without effect on cellular excision repair, at the pre-incision stage or at the later stages of either DNA resynthesis or strand break ligation; nor does it potentiate cell killing after u.v. irradiation. The chromosome decondensation that accompanies incomplete excision repair in mitotic cells is likewise not greatly affected by etoposide. Therefore, if topoisomerase II is involved in excision repair or its regulation, it acts through a process that in whole cells is insensitive to etoposide. In ataxia telangiectasia cells, which are known to be hypersensitive to etoposide, the mitochondrial activities are not abnormally affected.

Ataxia Telangiectasia↗
PubMed1987

DNA repair under stress.

When the excision repair process of eukaryote cells is arrested by inhibitors of repair synthesis including hydroxyurea (HU), 1-beta-D-arabinofuranosylcytosine (araC) or aphidicolin, major cellular changes follow the accumulation of repair-associated DNA breaks. These changes, each of which reflects more or less severe cellular stress, include cycle delay, chromosome behaviour, fall in NAD level, the development of double-stranded DNA breaks, rapid chromosome fragmentation and cell killing. Disruption of the repair process by agents such as araC after therapeutic DNA damage may, therefore, have some potential value in cancer treatment. The extreme cellular problems associated with the artificial arrest of repair may have their subtler counterparts elsewhere, and we discuss several systems where delays in the completion of excision repair in the absence of repair synthesis inhibitors have marked repercussions on cell viability. We also show that the average completion time of an excision repair patch varies according to the state of cell culture, and that completion time is extended after treatment with insulin or following trypsin detachment. Under certain growth conditions ultraviolet irradiation followed by mitogenic stimulation results in double-stranded DNA breakage and additional cell killing, and we discuss these data in the light of protocols that have been used successfully to transform human or rodent cells in vitro. Finally, we consider whether the rejoining of DNA breaks accumulated by repair synthesis inhibitors is a valid model system for studying ligation, and show that this protocol provides an extremely sensitive assay for most incision events and, thereby, a means for discriminating between normal human cells on the one hand, and Cockayne's Syndrome cells and their heterozygotes on the other.

Aphidicolin↗
PubMed1986

Qualitative differences between replicative and repair synthesis of DNA in normal and transformed mouse cells as measured by precursor discrimination.

Inhibitors of DNA polymerase alpha such as aphidicolin (APC) or 1-beta-D-arabinofuranosyl-cytosine (araC) cause DNA-strand breaks to accumulate after UV-irradiation, at sites where repair resynthesis is inhibited. Transformed cells accumulate fewer such breaks than normal cells do; this may be due to differences in the extent, or the nature, of excision-repair synthesis in transformed and in normal cells. We have looked for differences in the nature of repair synthesis, comparing the labelling of DNA by deoxycytidine (dC) and araC through UV-induced repair in normal and transformed mouse cells. We have made parallel determinations of precursor discrimination in replicative synthesis, and find that normal cells discriminate better against araC in replicative synthesis than do transformed cells. But repair synthesis discriminates against araC less than normal replicative synthesis does, to a similar extent in both cell types. Thus, there are qualitative differences between the DNA polymerases engaged in UV excision repair and replication in normal and transformed mouse cells; but there is no evidence for a predominantly araC-insensitive repair synthesis in transformed cells, such as might account for the difference in break accumulation.

Animals↗
PubMed1986

Defective post-replication recovery and u.v. sensitivity in a simian virus 40-transformed Indian muntjac cell line.

The responses to u.v. of two cell lines derived from the Indian muntjac are described. The u.v. sensitivity of the diploid cell falls within the range of most normal mammalian cells while the other, a heteroploid cell, transformed by SV40, is much more sensitive to killing. This hypersensitivity cannot be explained by defective excision repair: the two cell types are indistinguishable in this activity as judged by inhibitor-associated DNA break accumulation and unscheduled DNA synthesis. Rather, the SV40 transformed cells have a pronounced inability to recover normal DNA replication after u.v. These cells are, therefore, defective in a post-replication recovery mechanism and in this respect resemble the behaviour of the variant form of xeroderma pigmentosum. Their limited ability to recover normal levels of RNA synthesis after u.v. hints at the complexity of the phenotype.

Animals↗
PubMed1985

Novobiocin inhibition of DNA excision repair may occur through effects on mitochondrial structure and ATP metabolism, not on repair topoisomerases.

Novobiocin inhibits DNA topoisomerases. It also inhibits excision repair of DNA photodamage, blocking both repair synthesis and the earlier step of incision at u.v. damage sites (as measured by the accumulation of DNA strand breaks in u.v.-irradiated interphase cells treated with DNA synthesis inhibitors such as hydroxyurea or cytosine arabinoside). It has been supposed, therefore, that novobiocin affects repair by blocking a putative topoisomerase step prior to incision. But we find that novobiocin also has a marked dose- and time-dependent effect on mitochondria: in cells exposed to novobiocin, mitochondria swell and their cristae become disrupted, and the intracellular ATP:ADP ratio is lowered, though the membrane potential is maintained as judged by rhodamine 123 fluorescence. Mitotic cells are more resistant to mitochondrial disruption by novobiocin than are interphase cells. This correlates with a relative resistance of u.v.-irradiated mitotic cells to the inhibition of incision by novobiocin. The chromosomal decondensation that results from the accumulation of DNA breaks due to incision when u.v.-irradiated mitotic cells are treated with hydroxyurea and cytosine arabinoside is largely suppressed by novobiocin. Furthermore, the suppression of induced strand break accumulation is partly due to a suppression by novobiocin of the uptake and phosphorylation of cytosine arabinoside; breaks accumulated in u.v.-irradiated cells in the presence of aphidicolin, an inhibitor of DNA polymerase alpha that does not require phosphorylation, are less novobiocin-sensitive. We conclude that the effects of novobiocin on excision repair are more likely to be due to a non-specific effect on ATP metabolism than to a specific effect on a repair-related topoisomerase.

Adenosine Triphosphate↗