2-Chloro-2'-deoxyadenosine (CldAdo, cladribine) is a new and effective therapy for indolent lymphoid cancers. CldAdo is unique in its ability to kill both dividing and nondividing cells, however, its toxicity to resting cells is not well understood. The primary side effect of CldAdo in patients is moderate to severe myelosuppression. While the specificity and activity of CldAdo has been linked to a high deoxycytidine kinase to 5'- nucleotidase ratio in lymphoid cells, this relationship has not proven consistent. In vitro studies from this laboratory demonstrated that upon incorporation into DNA, CIdATP inhibits essential biochemical processes including DNA synthesis, RNA transcription, and DNAIprotein interactions. The overall goal of this project is to understand the mechanism responsible for CldAdo toxicity in dividing and resting cells, to elucidate the basis of its myelosuppressive effects, and to extend its use to other tumor types. We hypothesize that CldATP incorporation into cellular DNA is the critical factor for an initial and prolonged cytotoxic response to CldAdo in both cultured and patient cells. Furthermore, we propose that such incorporation l) reduces cellular gene expression, 2) interferes with mitochondrial function causing toxicity in resting cells and myelosuppression in patients, and 3) effectively sabotages cellular repair processes when incorporated during DNA repair synthesis. These predictions will be tested as follows.
In Aim l, we will characterize the extent of CIdATP incorporation into cellular DNA by detecting initial levels and sites of ClAde residues in both dividing and nondividing cultured cells using sensitive polymerase chain reaction (PCR) techniques, by examining the role of cellular repair in modulating levels of ClAde in DNA, and by extending this study to a patient population undergoing CldAdo treatment. Experiments in Aim 2 will determine if CldATP incorporation into DNA alters cellular gene expression by examining expression of a control and ClAde-substituted luciferase reporter vector in transiently transfected human lymphocytes.
Aim 3 will analyze effects of CldAdo on mitochondrial function by examining mitochondrial DNA synthesis in the presence of CldATP using isolated mitochondria, determining mitochondrial DNA content, lactic acid production, and the extent and sites of CIdATP incorporation in mitochondrial DNA in CldAdo-treated dividing and nondividing lymphocytes, and measuring mitochondrial mRNA levels after cellular exposure to CldAdo.
In Aim 4, the effects of CldAdo on cellular DNA repair synthesis will be investigated by quantitating the levels and kinetics of repair of damaged plasmid DNA in the presence of CldATP using human leukemia cell-free extracts, studying the effects of CldAdo on UV- irradiation- and cisplatin-induced toxicity in human leukemia cell lines, and extending these studies to human breast and ovarian tumor cell lines, including those with a drug resistance/enhanced DNA repair phenotype.
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