Abstract

Ape-1/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, NFκB, PAX, HIF-1α, 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 elevated Ape-1/ref-1 expression is related to differentiation and apoptosis. To further define the role of the redox function of Ape-1/ref-1 in this relationship, redox function was blocked using two techniques. First, we used retroviral gene transduction to over-express a redox-inactive C-65 mutant of Ape1/ref-1 in HL-60 myeloid leukemia cells and examined the response to retinoic acid. In a second set of experiments we used an Ape-1/ref-1 specific small molecule inhibitor to pharmacologically block the redox function and again examined the response to retinoic acid. Differentiation was evaluated by morphologic change in differential cell counts and expression of CD11b by flow cytometry. Apoptosis was assayed by annexin-PI staining on flow cytometry and cell cycle analysis was examined with propidium iodide flow cytometry.

Results:

  1. HL-60 cells expressing high levels of C-65 Ape-1/ref-1 responded to retinoic acid with a significantly higher level of differentiation and a moderate increase in apoptosis.

  2. Pharmacologic blockade of Ape-1/ref-1 redox function resulted in a profound increase in differentiation and a moderate increase in apoptosis compared to controls.

  3. dose dependent studies with retinoic acid demonstrated a similar degree of differentiation (CD11b expression) in cells treated with 10 μmolar retinoic acid and those treated with the redox inhibitor + 0.1 μmolar retinoic acid; alllowing HL-60 cells in the presence of the redox inhibitor to give a similar response to a 100 fold lower dose of retinoic acid.

The redox inhibitor alone did not induce differentiation and induced only a minimal amount of apoptosis but did increase the number of cells in S phase significantly. In conclusion, our data supports the contention that redox function of Ape-1/ref-1 may be important for controlling RA-induced myeloid differentiation and programmed cell death. The implication of these findings is that myeloid leukemia cells may be sensitized to retinoids by manipulation of the redox status of Ape-1/ref-1.

Author notes

Disclosure: No relevant conflicts of interest to declare.