Abstract
Sensitive radioimmunoassays (RIAs) were developed and validated for deoxyuridine triphosphate (dUTP) and deoxycytidine triphosphate (dCTP), while partial development and validation was achieved for RIAs for deoxythymidine triphosphate (dTTP) and cytosine arabinoside triphosphate (ara. CTP). Because of a lack of complete antibody specificity, pre-RIA separation of these four compounds from cross-reacting nucleotides was required. Liquid and affinity chromatographic and degradative techniques were optimised for this purpose. Antibody specificity was also improved for dUTP and dTTP by the use of affinity chromatography to remove a crossreacting portion of the immunoglobulin. The two validated assays have been given initial applications in measuring intracellular pool sizes of dUTP and dCTP. dUTP, not found in untreated cells and implicated in the toxicity of certain anti-cancer agents, was found to occur in increasing, dose-related, concentrations in a human lung carcinoma cell line treated with a thymidylate synthase inhibitor. dCTP, an endogenous and highly successful competitor of ara. CTP, the active form of the anti-leukaemic drug, cytosine arabinoside (ara. C), has been measured in both ara. C-sensitive and ara. C-resistant human leukaemic cell lines and L5178Y mouse lymphoma cells, in order to see if a correlation existed between such resistance and raised dCTP concentrations. These RIAs represent a novel way of measuring compounds which were previously difficult to quantify in small numbers of cells -(10[6]) - due to their presence in very low concentrations. The dUTP assay in particular achieves a unique combination of specificity and sensitivity, in a validated assay, for the measurement of this compound in the presence of dTTP. The dTTP assay, nearing validation, will provide similar advantages over other published methods, for measuring low concentrations of dTTP in the presence of high levels of dUTP. The dCTP and ara. CTP assays may, it is hoped be of use where cases of resistance to ara. C occur clinically. It is also anticipated that the three assays for the endogenous dNTPs will be used for studies on the mode of action of new anti-cancer agents both in vitro and in vivo.