Role of DNA Methylation of the G₀/G₁ Switch Gene 2 (G0S2) in the Proliferation of Myeloid Leukemia Cells

Abstract 3520

The G₀/G₁ switch gene 2 (G0S2) was originally identified in human blood mononuclear cells as an early response gene. We recently reported that G0S2 maintains quiescence in hematopoietic stem cells (Yamada et al., Plos One, 2012). In addition to hematopoietic stem cells, G0S2 also inhibits proliferation of lymphocytes and hematopoietic progenitor cells. Methylation of the G0S2 gene has been reported in lung, and head and neck cancer, suggesting that G0S2 has potential tumor suppressor function in solid tumors. However, the role of G0S2 in hematological malignancies has not been studied yet. To evaluate the impact of DNA demetylation of the G0S2 gene in human leukemia, we cultured a panel of myeloid (HEL, K562, HL-60, Kasumi) and lymphoid human leukemia cell lines (Jurkat, DND41, and H9) with 10 μM of 5-Azacytidine (5-Aza). Treatment with 5-Aza led to a significant increase of G0S2 expression in K562 cells (24.1-fold) and HL-60 cells (4.9-fold) and reduced proliferation. We show that retroviral overexpression of G0S2 in K562 cells caused inhibition of proliferation. Conversely, gene silencing of G0S2 in 5-Aza-treated K562 cells increased proliferation. Differentiation of K562 with hemin and HL-60 with all-trans retinoic acid was associated with an increase of G0S2 expression of 6.7-fold and 9.9-fold, respectively. Taken together, gain- and loss-of function studies revealed that G0S2 regulates cell proliferation and differentiation in human myeloid leukemia cells. Since G0S2 does not have a direct known function on the cell cycle, we hypothesized that G0S2 interacts with proteins involved in the control of cell proliferation. Mass spectrometric analysis of proteins pulled down with G0S2 in hematopoietic cells revealed that nucleolin, a molecule known to regulate ribosome biogenesis, physically interacts with G0S2 resulting in a perinuclear retention of nucleolin. This interaction was confirmed in K562 cells overexpressing G0S2 by reciprocal co-immunoprecipitation. This data suggest that silencing of G0S2 in leukemic cells prevents sequestration of nucleolin in the cytosol inhibiting its pro-proliferation functions. Collectively, our studies uncovered an important inhibitory role of G0S2 in the proliferation of leukemic cells, suggesting a possible tumor suppressor function in myeloid malignancies.