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The role of NAD(P)H dependent reductase mediated redox cycling in DNA strand breakage and cytotoxicity by doxorubicin (DOX), mitozantrone (MIT), CI941 and a series of alkylaminoanthraquinones (AQ) based on MIT has been investigated in MCF-7 human breast cancer cells. lAQ, 1,8AQ and DOX were found to redox cycle in MCF-7 cells as evidenced by stimulation of NADPH oxidation, superoxide anion formation and hydroxyl radical formation associated with generation of a drug free radical in MCF-7 S9 cell fraction. Furthermore, DOX formed a free radical, stimulated oxygen consumption and produced hydroxyl radicals in intact viable MCF-7 cells. 1,5AQ formed a free radical species and produced hydroxyl radicals but did not stimulate NADPH oxidation or superoxide anion formation in MCF-7 S9 fraction. Reactive oxygen formation correlated with the DNA strand breakage and cytotoxicity produced by IAQ, I,SAQ, 1,8AQ and DOX in MCF-7 cells. DOX also produced plasmid DNA strand breakage in the presence of purified cytochrome P4S0 reductase or xanthine oxidase. lAQ, 1,SAQ and 1,8AQ mediated plasmid DNA strand breakage only in the presence of purified cytochrome P4S0 reductase. MIT, CI941 and 1,4AQ did not redox cycle in MCF-7 cell S9 fraction yet produced equivalent cellular DNA strand breakage to DOX and AQ's at LDSO concentrations. The order of cytotoxicity of these compounds after a 1 hour exposure ~nd at least 6 d~ys further cell growth was;- (LDSO);- CI941(I.SxlO-lUM) >MIT(S.2xlO- YM) >lAQ(6.0xlO- 8M) >1,8AQ(O.5xlO- 6M) >1,4AQ(I.2xIO- 6M) >DOX(3.0xlO- 6M) >1,SAQ(12.3xIO- 6M). Variation in cellular uptake between the agents did not account for differences in cytotoxicity and DNA strand breakage observed. The 1,4 substitution pattern of MIT, CI941 and 1,4AQ appears to prevent metabolic activation. In further studies;- i) MIT and CI941 were more potent inhibitors of 3 MCF-7 topoisomerase I activity than other quinones, ii) MIT and DOX-Fe + complexes mediated plasmid DNA strand breakage in the presence of reduced glutathione iii) Only MIT was oxidatively activated by horseradish peroxidase!H202' producing plasmid DNA damage. The results indicate that future development of quinone anti tumour agents should concentrate on compounds which do not redox cycle but have functional groups that assist iron binding, activation by peroxidases and/or topoisomerase inhibition. In this respect, these agents should possess chromophore hydroxyl groups as these appear to be responsible for the increased cytotoxicity of MIT and CI941 compared to the other AQ's.

The generation of human antibodies to the multiple drug resistance protein, P-glycoprotein (Pgp), has been a challenge in the field of cancer chemotherapy since drug resistance was first described. Pgp was identified as being an intrinsic factor in the resistance to chemotherapeutic drugs, as its levels were found to escalate after primary drug challenges. Further investigation into the structure and function of P-glycoprotein elucidated that its activity was ATP dependent, and that it had broad spectrum specificity. In order to augment the success of chemotherapeutic treatment of patients it was deemed necessary to regulate the activity of this protein. This was possible by physical inhibition of its activity or through regulation at the genetic level. This project addresses the former of the two options, the physical inhibition of Pgp activity. It was considered that by the generation of antibodies to the extracellular regions of Pgp it would be possible to inhibit its activities. For such antibodies to be effective in the human model, the antibodies would have to bear human determinants in order that the human immune system would not attach and destroy these antibodies before they had the desired effect. Therefore it was decided that recombinant phage technology should be used. This is a system which selects single chain antibodies with human determinants from a large and diverse population of antibodies with a variety of specificities. Initially, attempts were made to generate a phage library <I>de novo</I> using a commercial kit designed for this purpose. However, after repeated attempts at this process, this approach was found to be beyond technical abilities, and was rejected in favour of a more rudimentary approach. Two libraries were screened for antibodies which would bind to membrane preparations from two leukaemic cell lines, CCRF CEM and CCRF CEM VLB. The former cell line was a drug-sensitive cell line which responded favourably to drug challenge, and the latter was a drug-resistant cell line which thrived in drug-rich environments. The drug-resistant cell line was derived from the drug-sensitive cell line, and was considered to be identical in all respects except drug sensitivity. Antibodies obtained from a large multicombinatorial library, the Lox library, were found to display selective binding to CCRF CEM VLB cell membranes in favour of CCRF CEM cell membranes. Antibodies were also generated to peptides representing the 6 extracellular loops of Pgp. From these selections antibodies were generated which were found to selectively bind the peptide to which they had been raised.