Cytosine Arabinoside Substitution Decreases Transcription Factor–DNA Binding Element Complex Formation

Abstract

The pyrimidine nucleoside analog, cytosine arabinoside (Ara-C), is an effective therapeutic agent for acute leukemia. The phosphorylated triphosphate, cytosine arabinoside triphosphate, competes with deoxycytosine triphosphate as a substrate for incorporation into DNA. Once incorporated into DNA, it inhibits DNA polymerase and topoisomerase I and modifies the tertiary structure of DNA. To determine if the substitution of Ara-C for cytosine in double-stranded oligonucleotides that contain 4 specific transcription factor binding sites (TATA, GATA, C/EBP, and AP-2alpha) alters transcription factor binding to their respective DNA binding elements. Transcription factors were obtained from nuclear extracts from human promyelocytic leukemia HL-60 cells. [32P]-end-labeled double-stranded oligonucleotides that contained 1 or 2 specific transcription factor binding sites with or without Ara-C substitution for cytosine were used to assess transcription factor binding by electrophoretic mobility shift assay. The substitution of Ara-C for cytosine within and outside the transcription factor binding element (AP-2alpha, C/EBP), outside the binding element only (GATA, TATA), or within the binding element only (AP-2alpha) all result in a reduction in transcription factor binding to their respective DNA binding element. The reduction of the binding capacity of transcription factors with their respective DNA binding elements may depend on structural changes within oligonucleotides induced by Ara-C incorporation. This altered binding capacity of transcription factors to their DNA binding elements may represent one mechanism for Ara-C cytotoxicity secondary to inhibition of transcription of new messenger RNAs and, subsequently, translation of new proteins.