Figure 2.

MST4 overexpression enables tumor colony formation and promotes proliferation under long-term hypoxia. A, Photomicrograph of soft agar colony growth in vector and MST4 stable transfectants under normoxia (20% O₂) or hypoxia (5% O₂) for 14 days. B, MST4 was overexpressed in LβT2 pituitary cells and protein expression detected by immunoblot (insert). MST4 overexpression increased colony formation under hypoxia. Number of colonies in vector control and MST4 transfectants were counted at 14 days. Colony numbers of transfectants cultured under 5% O₂ were compared with the transfectants cultured under 20% O₂ (n = 3). C, Images of the BrdU staining of LβT2 transfectants grown under normoxic or hypoxic conditions for 14 days. LβT2 MST4 and control transfectants were grown under 20% or 5% O₂, and cell proliferation was determined by BrdU staining of nuclei (white arrows). Bar, 20 μm. D, MST4 increased rates of cell proliferation under chronic hypoxia. The proliferation rate of LβT2 transfectants was analyzed from 1 to 14 days and was expressed as percentage of BrdU-positive cells to total cells (n = 3. E, p38MAPK and AKT are hyperactivated in gonadotrope pituitary tumors and LBT2-MST4 transfectants. Levels of endogenous kinases (total p38MAPK, ERK, and AKT) and their phosphorylated forms (P-p38MAPK, P-ERK, and P-AKT) were analyzed by immunoblot in individual human gonadotrope tumor samples compared with normal pituitary (left panel), and the same analysis was performed in LβT2-MST4 transfectants compared with vector control cells (right panel) (n = 3). F and G, p38MAPK and AKT are activated during MST4-induced proliferation. After exposure to hypoxia (1% O₂ for 3 d), cell proliferation was assessed by BrdU staining of nuclei in the presence or absence of a p38MAPK inhibitor (SB203580, 10 μM), AKT inhibitor (LY294002, 10 μM), or MEK inhibitor (PD98059, 30 μM), respectively. F, Images represent BrdU-positive cells of MST4 transfectants after the inhibitor treatment. Bar, 20 μm. G, The proliferation rate was expressed as BrdU-positive cells to total cells (n = 3). Values represent mean ± SEM of three independent experiments. *, P = .02, **, P < .01, MST4 cells compared with vector control cells; #, P < .01, inhibitor-treated MST4 cells compared with DMSO vehicle treatment.