Ape-1/ref-1 Mediated Redox Regulation of Retinoic Acid Receptor Transcription Function in Myeloid Leukemia Cell Differentiation and Apoptosis.

Ape1/ref-1is a multifunctional base excision DNA repair protein that is involved in the repair of abasic sites in DNA. However, it also has a distinct role in the redox regulation of a variety of cellular proteins, such as Fos, Jun, p53, NFkB, PAX, HIF-1a, HLF, and others. Ape-1/ref-1 maintains these proteins in a reduced state thereby facilitating their DNA binding and transcriptional activation capability. HL-60 cells are known to respond to retinoic acid (RA) with terminal granulocytic differentiation and apoptosis, which is mediated through the RA receptors. Previous experiments suggested that Ape1/ref-1 expression is related to apoptosis. To further define this relationship, we used retroviral gene transduction to over-express HA-tagged Ape1/ref-1 in HL-60 myeloid leukemia cells. We observed that the RA-induced growth inhibition, apoptosis, and differentiation of HL-60 cells over-expressing Ape1/ref-1 was significantly enhanced compared to wild type HL-60 cells. To further understand the mechanism of this effect we performed

1. gel shift experiments in vitro with Ape1/ref-1, retinoic acid receptor alpha (RAR-α), and a retinoic acid response element (RARE) under varying redox conditions and

2. co-transfection experiments in CV-1 cells with Ape1/ref-1 and RAR-α using an RARE linked to a luciferase reporter.

Results:

1. gel shift experiments demonstrate a redox dependent binding of RXR-α and RAR-α to their RARE which is mediated by Ape1/ref-1;

2. western blot analysis of transfected CV-1 cells revealed proper expression of each transfected construct including RAR-α, RXR-α and Ape1/ref-1; and

3. examination of RA-treated CV-1 cells for RARE-linked luciferase expression demonstrated Ape1/ref-1 enhancement of RAR activated transcription of the luciferase reporter.

In conclusion, our data supports the contention that Ape1/ref-1 expression may be important for enhancing RA-induced myeloid differentiation and programmed cell death through a redox based mechanism in transcription of target genes.