This study is designed to fill crucial gaps in knowledge concerning cytosine arabinoside (ara-C) mechanism of action and cytotoxicity.
Our specific aims focus on (1.) utilization of pH-step alkaline elution in conjunction with a cell-free human DNA synthetic system to define more precisely how ara-C interferes with the DNA replicative process in intact human cells, and (2.) determining the biological consequences of ara-C incorporated into DNA in terms of its effects on the recognition of a replication origin in DNA by a known DNA synthesis initiator protein, and on the transcription of messenger RNA. To accomplish the first aim, we will utilize a human multi-protein complex that contains the enzymatic components necessary for DNA replication. This complex has a sedimentation coefficient of 21S, and is fully competent to carry out the cell-free replication of the Simian virus 40 (SV40) genome, provided that the SV40-specific initiator protein, large T-antigen (T-Ag) is present. We will strive to develop this 21S multi-protein complex system into a cell-free model that emulates the DNA synthetic apparatus in the intact cell, thereby facilitating the study of the effects of ara-C on the intricate process of human DNA replication. The validity of the 21S replication complex model to mimic cellular events will be monitored by studies of ara-C effects on DNA synthesis in intact cells, using the pH- step alkaline elution technique. For the second aim, we will use recombinant DNA methods to insert ara-C into selected sites in a defined DNA template. We have selected for this template the well characterized SV40 viral genome, or plasmids containing the SV40 origin of replication, ori. These sites for ara-C insertion include a specific gene (T-Ag), and the replication origin, ori. We will then test the effects of the incorporated ara-C on initiator protein (T-Ag) recognition of ori, on RNA transcription of the T-Ag gene, and on the properties and biologic function of any transcript of T-Ag that may be made from an ara-C containing template. Although these studies utilize a viral gene and DNA template, any effects of ara-C incorporation observed should directly reflect the same consequences of ara-C on biological function in human cancer cells. No one has ever studied the effects of ara-C on these processes in such a direct manner. the information obtained from these studies may contribute substantially to improving ara-C therapy, and in devising new and improved methods for the detection of ara-C resistance in acute leukemia patients.
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