Several studies have shown that non-cycling cells are resistant to the cytotoxic effects induced by amsacrine (m-AMSA; 4'-(9-acridinylamino)methanesulphon-m-anisidide). This resistance may limit the activity of m-AMSA and related 9-anilinoacridine antitumour agents against solid tumours. The biochemical mechanism(s) for this resistance have been investigated using spontaneously transformed Chinese hamster fibroblast (AA8 cells) in log- and plateau-phase spinner cultures. In early plateau phase most cells entered a growth-arrested state with a G1-G0 DNA content and showed a marked decrease in sensitivity to cytotoxicity after a 1-h exposure to m-AMSA or its solid tumour-active analogue, CI-921. Studies with radiolabelled m-AMSA demonstrated that changes in sensitivity to m-AMSA-induced cell killing were not due to a difference in uptake or retention of drug by log- and plateau-phase cells, and there was no significant metabolism of drug by either log- or plateau-phase cells. Thus, after a 1-h exposure to [3H]-m-AMSA at 37°C, a small proportion (1%) of cell-associated radioactivity was covalently bound to macromolecules, but most of the cell-associated radioactivity represented unchanged m-AMSA. There was no evidence for any oxidative metabolism to reactive quinoidal species in these tumour cells. However, studies with a fluorescence assay for DNA unwinding indicated that plateau-phase cells were 3 to 4 times less sensitive to m-AMSA-induced DNA breakage than log-phase cells, and changes in sensitivity to m-AMSA-induced DNA breakage correlated with changes in sensitivity to cell killing by m-AMSA as cell progressed from log to plateau phase. Further studies showed that the decrease in sensitivity to m-AMSA-induced DNA stand breakage correlated with a decrease in sensitivity to covalent DNA-protein complex formation in plateau-phase cells after m-AMSA treatment. Combined with evidence that the DNA lesions rapidly disappeared from both log- and plateau-phase cells following the removal of m-AMSA (half-time approx. 4 min), this indicated that the lesions detected by the FADU assay probably arose from the stimulation of DNA-topoisomerase II (topo II) cleavable complex formation by m-AMSA. K+/SDS precipitation assays with [32p] 3’-end-labelled pBR322 DNA indicated that nuclear extracts containing topo II activity from plateau-phase cells were 3- to 4-fold less sensitive to stimulation of DNA-topo II complex formation by m-AMSA than nuclear extracts from log-phase cells. This change in sensitivity to m-AMSA-induced DNA-topo II complex formation was therefore similar to that observed with intact cells. However, P4 unknotting assays indicated that topo II activity in nuclear extracts from plateau-phase cells was only 2-fold lower than that in nuclear extracts from log-phase cells. Resistance to treatment with m-AMSA may therefore reflect a decrease in topo II activity and/or a decrease in sensitivity of topo II enzymes to stimulation of cleavable complex formation by m-AMSA in non-cycling cells.
| Identifer | oai:union.ndltd.org:ADTP/275544 |
| Date | January 1988 |
| Creators | Robbie, Maxine Ann |
| Publisher | ResearchSpace@Auckland |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated., http://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm, Copyright: The author |
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