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S M Cutts

Publications and source records attributed to S M Cutts.

21 records · Page 2Linked to original sources
PubMed1995

Stability of adriamycin-induced DNA adducts and interstrand crosslinks.

The stability of adriamycin-induced DNA adducts and interstrand crosslinks was measured at 37 degrees C by three independent procedures. The loss of [14C]-labelled adducts was described by two first-order decays with half-lives of 7.4 h (60% amplitude) and 39 h (40%). The loss of the drug chromophore also exhibited a biphasic character, with half-lives of 6 h (65%) and approximately 150 h (35%). The decay of transcriptional blockages at an isolated, apparent interstrand GpC crosslinking site was described by two first-order processes, with half-lives of 3 h (65%) and 40 h (35%), whereas the decay of transcriptional blockages at an isolated guanine residue (apparent site of monoadduct) was completely described by a first-order decay with a half-life of 5.3 h. The loss of interstrand crosslinks was measured using a gel electrophoresis assay, and the decay was characterised by a single first-order process with a half-life of 4.7 h. Collectively, these values serve to define a model of the interstrand crosslink with unstable sites of attachment at both ends of the crosslink, with half-lives at either end being approximately 5 and 40 h. The adducts exhibited increasing lability with increasing pH, and were particularly unstable at pH 12, with a half-life of approximately 0.5 h. The adducts were also heat labile, with an overall melting temperature of 67 degrees C (10 min exposure) and this was also the thermal lability measured at three individual adduct sites probed by lambda exonuclease.

Animals↗
PubMed1994

Formation of adriamycin--DNA adducts in vitro.

Adriamycin is known to induce the formation of adducts with DNA when reacted under in vitro transcription conditions. The factors affecting the extent of adduct formation were examined in order to establish the critical components and optimal conditions required for the reaction, and to gain insight into the nature of the DNA-adduct complex. There was a strong dependence on reaction temperature (with a 40-fold increase of adducts at 40-50 degrees C compared to 10 degrees C), pH (maximum adducts at pH 7), but little dependence on the oxygen level. There was an absolute requirement for a reducing agent, with adducts detected with DTT, beta-mercaptoethanol and glutathione, maximal adducts were formed at high levels of DTT (5-10 mM). Adducts were also formed with a xanthine oxidase/NADH reducing system, with increasing amounts of adducts detected with increasing NADH; no adducts were detected in the absence of either the enzyme or NADH. Of fourteen derivatives studied, only four yielded a similar extent of adduct formation as adriamycin; there was no absolute requirement for a carbonyl at C13 or hydroxyl at C14. Adducts were also observed with ssDNA but required a longer reaction time compared to dsDNA. The sequence specificity of adduct formation with ssDNA was examined using a primer-extension assay; almost all adducts were associated with a guanine residue. Overall, the results are consistent with a two-step reaction mechanism involving reductive activation of adriamycin, with the activated species then reacting with the guanine residues of either dsDNA or ssDNA.

DNA↗