Redox Regulation of Retinoic Acid Receptor Function in Myeloid Leukemia Cell Differentiation and Apoptosis.

Ape1/ref-1 is 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 of HL-60 cells over-expressing Ape1/ref-1 was significantly enhanced compared to wild type HL-60 cells. To determine if the growth inhibition was related to enhanced programmed cell death and differentiation, we treated Ape1/ref-1 transduced and vector-only (LXSN) transduced HL-60 cells with RA and evaluated the expression of Ape1/ref-1 and the development of apoptosis and markers of differentiation.

Results: 1) RA induced expression of the retroviral Ape1/ref-1 construct as determined by Western blot resulting in a higher (ie retroviral + endogenous Ape1/ref-1) overall expression of Ape1/ref-1 compared to control cells; 2) analysis of RA-treated cells for apoptosis by propidium iodide, TUNEL, and Annexin V staining as well as morphology, unexpectedly demonstrated enhanced programmed cell death in cells expressing the transduced Ape1/ref-1; 3) Ape-1 over-expression enhanced the retinoid differentiation response by morphology and expression of CD11b. Additional mobility shift experiments demonstrated the redox dependence of retinoic acid receptor binding to retinoid response elements mediated by Ape-1/ref-1. In conclusion, our data supports the contention that Ape1/ref-1 expression may be important for mediating RA-induced myeloid differentiation and programmed cell death.