Effects of 5-Androstenediol on Survival, Clonogenicity, and Expression of IL-6 and NFKB in Irradiated Human Osteoblast and Hematopoietic CD34+ Cells.

We previously reported that 5-androstenediol (5-AED) administration enhanced survival in mice exposed to whole-body ionizing-radiation (IR). 5-AED also induced hematopoiesis and hematopoietic growth factor expression in blood-forming tissue. Since osteoblasts comprise an important part of the hematopoietic niche, we evaluated the radioprotective effects of 5-AED in a human fetal osteoblast cell line (hFOB) and primary human hematopoietic CD34+ progenitor cells. 5-AED (1000 ng/ml) increased hFOB cell survival from 39% (vehicle-treated) to 85% 3 days after a radiation dose of 2 Gy, and from 56% to 64% after 4 Gy. In clonogenic assays of CD34+ cells starting with 10⁴ cells/dish, BFU-E colonies in 5-AED (1000 ng/ml)-treated cultures increased from 163±17 (vehicle-treated) to 418±20 24 h after 2 Gy, and from 59±5 to 118±11 after 4 Gy. CFU-GM colonies in 5-AED-treated cultures rose from 89±6 to 177±13 after 2 Gy, and from 27±3 to 51±3 cells after 4 Gy. Furthermore, 5-AED administration to irradiated CD34+ cell cultures led to an elevated frequency of CD11b+ cells (granulocytes, monocytes and NK cells) as determined by flow cytometry 22 days after irradiation. To test our hypothesis that 5-AED acts via initiation of a cytokine cascade in hematopoietic cells, endogenous IL-6 protein expression was evaluated using ELISA. Administration of 5-AED before and after IR elicited a 10-fold increase in IL-6 levels in CD34+ cultures 24 h after IR. In contrast, IL-6 levels were not elevated in 5-AED-treated, unirradiated CD34+ cells. Recent studies showed that nuclear factor kappa B (NFkB) may play an important role in IR induced damage. We assessed NFkB activation in human FOB and CD34+ cells and now report for the first time that NFkB subunit NFkB₁ (p50) expression was significantly inhibited by IR in CD34+ cells in a dose-dependent manner. Moreover, 5-AED at least partially blocked the downregulation of NFkB₁ expression in irradiated cells. Taken together, our results demonstrate that 5-AED counters the effects of IR in human hematopoietic CD34+ and osteoblast cells, and that these effects may be mediated by modulation of IL-6 and NFkB.