Biomedical subjects

D A Lydall

Publications and source records attributed to D A Lydall.

4 recordsLinked to original sources

PubMed2007

Modelling the checkpoint response to telomere uncapping in budding yeast.

One of the DNA damage-response mechanisms in budding yeast is temporary cell-cycle arrest while DNA repair takes place. The DNA damage response requires the coordinated interaction between DNA repair and checkpoint pathways. Telomeres of budding yeast are capped by the Cdc13 complex. In the temperature-sensitive cdc13-1 strain, telomeres are unprotected over a specific temperature range leading to activation of the DNA damage response and subsequently cell-cycle arrest. Inactivation of cdc13-1 results in the generation of long regions of single-stranded DNA (ssDNA) and is affected by the activity of various checkpoint proteins and nucleases. This paper describes a mathematical model of how uncapped telomeres in budding yeast initiate the checkpoint pathway leading to cell-cycle arrest. The model was encoded in the Systems Biology Markup Language (SBML) and simulated using the stochastic simulation system Biology of Ageing e-Science Integration and Simulation (BASIS). Each simulation follows the time course of one mother cell keeping track of the number of cell divisions, the level of activity of each of the checkpoint proteins, the activity of nucleases and the amount of ssDNA generated. The model can be used to carry out a variety of in silico experiments in which different genes are knocked out and the results of simulation are compared to experimental data. Possible extensions to the model are also discussed.

Cell Cycle↗

PubMed1991

The Walker 256 carcinoma: a cell type inherently sensitive only to those difunctional agents that can form DNA interstrand crosslinks.

The Walker 256 rat tumour has been maintained in vivo for over 60 years and until recently was used as a primary screen for new antitumour agents. This screen was particularly useful in identifying difunctional alkylating agents as potentially useful anticancer agents and it would seem that the Walker tumour is composed of cells sensitive towards this type of agent. A cell line (WS) established from the Walker tumour retained the sensitivity of the tumour towards difunctional agents and we have examined its phenotype in comparison to a derived, resistant, cell line (WR). The response of WR cells to a range of cytotoxic agents was similar to other established cell lines whilst WS cells were much more sensitive only towards difunctional reacting agents. There were no significant differences in the binding of these agents to the DNA of WS or WR cells. All the agents towards which WS cells showed sensitivity were, without exception, capable of reacting with DNA in Walker cells and forming DNA-DNA interstrand crosslinks. WS cells were not sensitive to busulphan, BCNU, CCNU or Me-CCNU but these agents did not produce interstrand crosslinks in the DNA of either WS or WR cells. Thus WS cells are intrinsically sensitive to specific DNA damage and this is probably a DNA interstrand crosslink. Hybrid cells produced by fusion of WS with WR cells lacked the inherent sensitivity of the WS cells towards cisplatin; sensitivity was therefore a recessive characteristic. Transfection of WS cells with human DNA also gave rise to 2 cisplatin-resistant clones, although it could not be ascertained if these clones were true transfectants or revertants. The survival of these resistant clones, after treatment with cisplatin, was about the same as WR cells a finding which would be consistent with complementation by a transferred gene or reversion of a single gene defect in WS cells. In their sensitivity only to difunctional compounds and lack of an apparent DNA excision repair defect the phenotype of Walker cells strongly resembles those cells from human patients suffering from Fanconi's anaemia and also of yeast snm1 mutant cells. The mechanisms giving rise to this failure to tolerate specific DNA damage (which seems to involve the inability to recover from the initial inhibition of DNA synthesis and may involve a single defect of a gene involved in the late steps of crosslink repair), do not involve drug uptake, drug binding to DNA, cell size, cell doubling time or DNA excision repair.

Animals↗

PubMed1987

The effect of monofunctional or difunctional platinum adducts and of various other associated DNA damage on the expression of transfected DNA in mammalian cell lines sensitive or resistant to difunctional agents.

The effects of introducing various DNA damage into pSV2gpt DNA on the subsequent expression of xanthine guanine phosphoribosyltransferase (XGPRT), after its transfection into two Walker 256 cell lines, one which is inherently sensitive only to difunctional agents while the other shows a normal sensitivity, have been examined. Both the sensitive (WS) and the relatively resistant (WR) cell lines were shown to be equally capable of both ligation of DNA double-strand breaks (although the efficiency varied with the actual site of the break) introduced into pSV2gpt and homologous recombination of pSV2gpt fragments (recombination events are thought to be important in the repair of DNA-DNA interstrand crosslinks). Reacting the plasmid with either the difunctional platinum compound, Cisplatin, or the monofunctional reacting Pt(Dien) caused a dose-dependent decrease in the subsequent expression of XGPRT. This decrease was about the same with either agent in either cell line when expressed as a function of dose of drug. However, when the actual binding of platinum to DNA by these compounds was measured, a large difference (due to the higher specific binding of Pt(Dien) to DNA) in the effects of the difunctional, as opposed to the monofunctional agent, was apparent and this was a reflection of the relative cytotoxicities of these compounds towards mammalian cells. Although at doses of Cisplatin equitoxic to WS and WR cells 20-fold less Pt is bound to the DNA of WS cells, no significant difference was seen on the expression of pSV2gpt, reacted with this agent, between WS or WR cells. Based upon a knowledge of the proportions of adducts formed in DNA reacted with Cisplatin, the lesion that inactivates expression of XGPRT was probably the intrastrand crosslink and it was calculated that due to the size of the plasmid, the interstrand crosslink was unlikely to be present at these inactivating doses. It is suggested that the inherent sensitivity of WS cells only to difunctional agents is due to their response to such relatively rare lesions such as a DNA-DNA interstrand crosslink.

Animals↗

PubMed1986

Mechanism of cytotoxicity of anticancer platinum drugs: evidence that cis-diamminedichloroplatinum(II) and cis-diammine-(1,1-cyclobutanedicarboxylato)platinum(II) differ only in the kinetics of their interaction with DNA.

The kinetics of the aquation reactions of cisplatin and carboplatin and their subsequent reactions with DNA, both in vitro and in vivo, have been measured. The results have been extrapolated to indicate the expected cytotoxicity of these compounds in cells obtained from human cancer patients. Rate constants for the aquation at 37 degrees C of cisplatin and carboplatin of 8 X 10(-5) and 7.2 X 10(-7) s-1, respectively, were calculated from the half-life of these compounds in phosphate buffer, pH 7. This difference in their rate of activation was matched by their rates of binding to DNA. By use of a 14C-labeled ligand, carboplatin was shown to bind monofunctionally to DNA, after which there was a time-dependent formation of difunctional interstrand cross-links, formed from some of these initially monofunctional adducts. A similar, although faster, accumulation of cross-links was seen when cisplatin was bound to DNA. The loss of the 14C-CBDCA ligand of carboplatin was calculated to occur with a rate constant of 1.3 X 10(-5) s-1 which was similar to that for the rate of formation of interstrand cross-links and faster than that for the monofunctional reaction with DNA. It was concluded therefore that the CBDCA ligand becomes a more labile leaving group once carboplatin has been monoaquated. In contrast, both chloro-ligands of cisplatin were shown to leave at similar rates. The fact that other difunctional lesions were formed to the same extent, by equal bound doses of cisplatin or carboplatin, was indicated by the unwinding of supercoiled plasmid DNA. The effects of cisplatin and carboplatin on this DNA were the same once bound to the same extent. About a 100-fold larger dose of carboplatin was, as predicted by their rates of aquation, required to produce equivalent binding to plasmid DNA. In vivo, equal binding of the two drugs to DNA of various cell systems resulted in equal cytotoxicity. Again a much larger dose (20- to 40-fold) of carboplatin was required to produce this equal binding. In general a DNA bound platinum level of about 20 nmol/g reduced cell survival by 90%, although certain cell lines were shown to be much more sensitive to DNA bound platinum. Similar binding values, to those above, were obtained in the DNA extracted from cells of human cancer patients treated with cisplatin. It was inferred that the cytotoxic effect of this level of platinum on DNA would be (unless the cells were of a sensitive phenotype) about 90%.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗