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Biomedical subjects

M J Tilby

Publications and source records attributed to M J Tilby.

30 records · Page 2Linked to original sources
PubMed1991

Sensitive detection of DNA modifications induced by cisplatin and carboplatin in vitro and in vivo using a monoclonal antibody.

An assay that is based upon a monoclonal antibody (ICR4) is described that enables the quantitation of cisplatin-induced adducts on DNA down to 3 nmol Pt/g DNA (i.e., 1 Pt adduct/10(6) bases), the level necessary to produce toxic effects in cells in vitro and in vivo, using just a few micrograms of DNA. Detection is possible below this level (although probably not necessary for in vivo studies) but the cross-reactivity of unmodified DNA sequences complicates absolute quantitation of adducts. Therefore, it will be possible to investigate the distribution of clinically useful platinum drugs in patients undergoing chemotherapy. Rats of strain F344 appeared to be the best, among several tested, for the production of antibodies to modified DNA, and they were used for the production of hybridomas. Fifteen hybridomas which secreted antibodies that bound to DNA that was highly modified with cisplatin but not to normal DNA were obtained. One (ICR4) was chosen for further characterization because of its relatively strong binding to DNA modified to a moderate level with cisplatin. The characterization included the development of a sensitive competitive enzyme-linked immunoabsorbent assay and the use of DNA that had been reacted with cisplatin both in vitro and in vivo. The levels of platination of both types of DNA samples were determined by atomic absorbance spectroscopy. For DNA that had been exposed to cisplatin in vitro, 50% inhibition of antibody binding was caused by about 15 fmol of total DNA-bound Pt/assay well. At moderate levels of platination, heating of the DNA solution at 100 degrees C for 5 min increased its immunoreactivity such that 50% inhibition was caused by 2.5 fmol Pt adducts/well. Pt adducts on DNA extracted from cells that had been treated with cisplatin were less immunoreactive than DNA treated with cisplatin in vitro, but after heating the immunoreactivity increased such that 50% inhibition in the assay was caused by 2 fmol Pt adduct/well. This sensitivity was invariant over a wide range of levels of platinum adduct frequency. DNA adducts formed by the second generation anticancer drug carboplatin were recognized similarly to the adducts formed by cisplatin, but those formed by the clinically inactive trans-diamminedichloroplatinum(II) or chloro(diethylenetriamine)-platinum(II)-chloride were not significantly immunoreactive. Control DNA cross-reacted in the competitive assay but the immunoreactivity per mol base was 10(7) times lower than the immunoreactivity of cisplatin adducts.

Animals↗
PubMed1990

Alkylation of DNA by melphalan in relation to immunoassay of melphalan-DNA adducts: characterization of mono-alkylated and cross-linked products from reaction of melphalan with dGMP and GMP.

A product expected to result from cross-linking of guanine bases in DNA by melphalan (4-(2-(di-guanin-7-yl))ethylamino-L-phenylalanine) was obtained from hydrolysis of melphalan-treated sodium deoxyguanylate at pH7 and characterized by U.V. and mass spectra. When tested in a competitive immunoassay using an antibody specific for melphalan-alkylated DNA it showed an affinity intermediate between that of melphalan-alkylated DNA and melphalan. From this and other assays it seemed possible that the cross-linked moiety in DNA was recognised by the antibody, but that its conformation differed from that of the free base tested, sufficiently to account for the discrepancy. It seemed possible that cross-linked guanine nucleotides would provide a better model, and these were therefore isolated, characterised and tested. Products derived from cross-linking of guanylic acid moieties through N-7 and N-7, and through N-7 and phosphate, had higher affinity than the cross-linked base, approximately the same as for alkylated native DNA, but less than for alkylated denatured DNA or RNA.

Alkylation↗
PubMed1987

Immunological detection of DNA damage caused by melphalan using monoclonal antibodies.

Immunological detection of melphalan adducts on DNA should permit new types of clinical and experimental investigations. Five cloned rat hybridoma cell lines were derived, each producing an antibody that bound to DNA alkylated with melphalan (phenylalanine mustard) but not to normal DNA. Further characterization of one melphalan specific antibody (MP5/73) used a competitive fluorogenic enzyme-linked immunoabsorbent assay. Using denatured DNA, 50% inhibition of antibody binding was caused by 30 fmol of total melphalan adducts (determined using radioactive melphalan) per assay well. Denatured control DNA caused 16 to 24% inhibition at 45 micrograms (130 nmol)/well, the maximum concentration tested. Adducts on RNA behaved similarly to those on denatured DNA. Adducts on native DNA caused 50% inhibition at 272 to 1335 fmol/well dependent upon alkylation frequency and sonication treatment. Native control DNA caused no detectable inhibition at 45 micrograms/well. The adducts recognized by the antibody were thermo- and alkali labile. Denaturation of the alkylated DNA by moderate heating in the presence of 75% formamide gave 50% inhibition at 50 fmol/well, indicating that only 5% of the recognized adducts could bind antibody in native DNA.

Animals↗
PubMed1986

Comparative studies of total cross-linking, cell survival and cell cycle perturbations in Chinese hamster cells treated with alkylating agents in vitro.

The toxicities of 4-hydroperoxycyclophosphamide (4-OOH CY), phosphoramide mustard (PM), melphalan (MEL) and busulphan (BU) have been compared in Chinese hamster cells, V-79-753B. The initial total amount of cross-linking was a determining factor for the clonogenic survival of cells treated with MEL or PM. Although 4-OOH CY generated cross-links in this cell line, this damage did not account for the toxicity of the compound. There was no evidence for cross-link formation in cells treated with BU, even at a dose of the drug (1000 micrograms/ml) that was too toxic to measure clonogenic survival. Comparison for the four compounds at equitoxic doses showed that both PM and MEL caused the arrest of the cell cycle at G2 which persisted after drug removal. This was accompanied by a decline in the population growth rate and a decrease in total cell count. In contrast, both BU and 4-OOH CY caused a temporary arrest of the cell cycle G2, 24 hr after drug removal. However, the cell cycle distribution returned the control values within 3-4 days after treatment. Both BU and 4-OOH CY showed little effect on the initial growth rate of the cells. It is concluded that the initial amount of cross-links contributes to the toxicity of PM and MEL. However, it is unlikely that the generation of cross-links is of major importance for the toxicity of either 4-OOH CY or BU.

Alkylating Agents↗
PubMed1984

An effect of elevated postirradiation pH on the yield of double-strand breaks in DNA from irradiated bacterial cells.

Exposure of DNA isolated from irradiated cells of Escherichia coli to a pH of 9.6 caused a marked increase in the yield of double-strand breaks (dsb). The dsb were measured by sedimentation analysis of E. coli chromosomal DNA using neutral sucrose gradients. After incubation for 4 hr at 37 degrees C and pH 9.6 the dsb yields were 95% and 71% higher than when incubation was at pH 7.0 for irradiation under oxic and anoxic conditions, respectively. This effect was not apparent when dsb were induced enzymatically and it was linearly related to radiation dose. After oxic irradiation, the increase in dsb at pH 9.6 was consistent with first-order kinetics over greater than 2 half-lives (t1/2 = 1.6 hr at 37 degrees C). The effect of elevated pH was largely additive to a previously reported increase in dsb yield caused by ethanol. It is proposed that the effects of elevated pH and of ethanol revealed the presence in intracellularly irradiated DNA of previously unidentified sites where both strands of the DNA were damaged as a result of single radiation events. The possible nature of the proposed sites and the relevance of these findings to the "neutral" elution technique are discussed.

DNA, Bacterial↗
PubMed1983

Measurements of DNA double-strand break yields in E. coli after rapid irradiation and cell inactivation: the effects of inactivation technique and anoxic radiosensitizers.

A study has been made of methods for rapidly inactivating cells of E. coli at neutral pH to prevent enzymatic, chemical, or physical modification to DNA damaged by irradiation. The radiation was delivered in a fraction of a second using an electron accelerator. Cell inactivation was with ethanol or a solution (CSE) containing detergent, EDTA, and chloroform. It was found that DNA could be released from irradiated and inactivated cells simply by incubating them with the protease Pronase, and this DNA appeared to be in a form suitable for centrifugational analysis in neutral sucrose gradients. When cells were irradiated in the presence of oxygen, inactivation with ethanol gave a radiation-induced double-strand break (dsb) yield 1.8-fold higher than when inactivation was with CSE. Possible explanations of this are discussed. Using CSE inactivation, an oxygen enhancement ratio for dsb formation of 4.3 was observed and yields of dsb per Gray were in good agreement with results from other laboratories. At concentrations which enhanced cell killing 2.6-fold the "electron-affinic" type anoxic radiosensitizer misonidazole enhanced dsb formation 2.0-fold, whereas the nitroxyl free radical anoxic radiosensitizer norpseudopelletierine-N-oxyl had no significant effect on dsb yield although there was a possible slight enhancement at the higher doses used.

DNA Repair↗
PubMed1978

Detergent-resistant variants of Bacillus subtilis with reduced cell diameter.

Variants of Bacillus subtilis resistant to the detergent Triton X-100 may exhibit: (i) normal cell morphology, (ii) reduced cell diameter, or (iii) helical cell shape. One variant of type ii was studied in some detail. Triton resistance, cell diameter reduction, and poor sporulation all may have resulted from a single mutation. High concentrations of Triton caused rapid lysis of wild-type cells. B. subtilis adapted to low Triton concentrations such that, upon subsequent exposure to higher concentrations, growth continued, although it bacame inhibited at very high concentrations. The variant studied retained its sensitivity to Triton-induced lysis but, after adaptation, grew at very high Triton levels. In this strain, cell diameter and cross-sectional area were reduced to about 73 and 50%, respectively, of those of wild type, yet the cells grew at normal rates, and DNA/protein/RNA ratios were largely unaltered. Peptidoglycan content per unit of cell surface area was higher in the variant than in the wild type under at least certain growth conditions.

Bacillus subtilis↗
PubMed1976

Tryptophan biosynthesis in Coprinus lagopus: a genetic analysis of mutants.

Thirty-one tryptophan-requiring mutants of Coprinus lagopus have been assigned by genetic and complementation analyses to four loci designated trp-I, trp-2, trp-3 and trp-4. The trp-1 and trp-3 loci were located in group III and trp-2 in group G of the linkage map. The trp-4 locus showed no linkage to the other trp loci or to markers in three additional linkage groups tested. From auxanographic tests and a study of accumulated biosynthetic intermediates, the enzymes controlled by each locus have been provisionally assigned. The trp-2 and trp-3 loci both appear necessary for anthranilate synthetase activity since mutants accumulated no intermediates. Only the trp-3 mutant could utilize anthranilic acid, therefore the trp-2 locus must also be involved in a subsequent step in the pathway. The trp-4 mutants utilized indole and accumulated anthranilic acid, and hence this locus is involved in the conversion of anthranilic acid to indoleglycerol phosphate. The trp-1 mutants utilized only tryptophan and accumulated indoleglycerol phosphophate and anthranilic acid. They are therefore blocked in the final steps of the pathway catalysed by tryptophan synthetase.

Agaricales↗
PubMedDate not supplied

The comparative pharmacokinetics and pharmacodynamics of cisplatin and carboplatin in paediatric patients: a review.

Cisplatin and carboplatin are important drugs in the treatment of childhood cancers. Extensive data for carboplatin, and more limited results with cisplatin, indicate that there is significant inter-patient pharmacokinetic variability such that adaptive dosing may be necessary for the optimal use of these drugs. Glomerular filtration rate based dosing formulae have been developed for carboplatin and their use is reviewed. Further studies are needed with cisplatin in order to define the reasons for inter-patient pharmacokinetic variability and its relevance in clinical practice. Only very limited data are available on the clinical or molecular pharmacodynamics of cisplatin and carboplatin in paediatric patients. By analogy with adult studies, pharmacologically-guided platinum complex treatment should offer significant advances over empiric dosing.

Adolescent↗