Stimulation of HIF-1 transcriptional activity and HIF-1α protein stabilization by hypoxia or CoCl₂. (A) PC-3 cells were transfected with a pHRE-Luc reporter plasmid. After 24 h, the cells were cultured for 6 h in either 2 or 0.1% FBS and then pretreated for 30 min with solvent vehicle, 50 μM LY294002, or 100 nM rapamycin. The cells were then exposed for 19 h to normoxic conditions, hypoxia, or 150 μM CoCl₂. Luciferase activities were measured with a luminometer, and are presented as relative light units (RLU). Error bars represent the variance of duplicate samples. (B) Effect of rapamycin on GLUT1 mRNA expression. PC-3 cells were cultured in medium containing 2% serum for hypoxic samples (upper panel) or in medium containing 0.1% serum for CoCl₂ stimulation (lower panel). The cells were exposed for 8 h to hypoxia or 150 μM CoCl₂ before harvest of total cellular RNA for semiquantitative RT-PCR analysis.

(A) PC-3 cells were precultured in medium containing 2 or 0.1% FBS, treated with the indicated drugs, and then exposed to normoxia (Co) or hypoxia as described in the Fig. legend. The cells were lysed, and detergent-soluble proteins were resolved by SDS-PAGE. The membrane was immunoblotted sequentially with α-HIF-1α MAb and α-PLCγ1 antibodies. The immunoblot was subjected to densitometric analysis, and values were normalized to that obtained in the normoxic control sample. (B) PC-3 cells were treated as described above, except that cells were stimulated with 150 μM CoCl₂. (C) Cells were exposed to hypoxia for 2 or 6 h in the absence of presence of rapamycin, and samples were processed as described in panel A. The band intensities were quantitated by densitometry, and numbers below the bottom panel indicate the percentage of the non-drug-treated control for each time point.

Correlative suppressive effects of rapamycin on HIF-1 activity and HIF-1α expression. PC-3 cells were transfected with the pHRE-Luc reporter plasmid and, after 24 h, the cells were placed in 2% serum-containing medium. After 8 h, the indicated samples were treated with 100 nM rapamycin and were cultured for an additional 16 h under either normoxic or hypoxic conditions. (Upper panel) Samples were harvested for determinations of luciferase activities. Bars and error flags represent the means ± the standard deviation from triplicate samples. (Lower panel) Parallel samples were harvested, and detergent-soluble proteins were immunoblotted with α-HIF-1α MAb. The blot was stripped and reprobed with PLCγ1-specific antibodies to control for sample loading.

Effect of rapamycin on HIF-1α turnover. (A) Proteasome inhibition antagonizes the suppressive effect of rapamycin on hypoxia-induced HIF-1α expression. (Left panel) PC-3 cells were incubated for 6 h in 2% FBS before pretreatment for 45 min with solvent vehicle, rapamycin (Rap), or LLnV. Cells were incubated under normoxic conditions (−) or hypoxia (H). The cells were harvested after 16 h, and soluble proteins were resolved by SDS-PAGE. (Right panel) PC-3 cells were precultured for 16 h in 0.1% FBS prior to the indicated drug treatments and were then stimulated for 6 h with 150 μM CoCl₂ (C). The blotted proteins were probed sequentially with α-HIF-1α MAb and α-PLCγ1 antibodies. Expression of HIF-1α protein was quantitated by densitometry and was normalized to the value obtained in the corresponding normoxic control samples. (B) LLnV-induced HIF-1α accumulation. PC-3 cells were treated for the indicated times with 10 μM LLnV under normoxic conditions, in the absence or presence of 100 nM rapamycin. HIF-1α expression was determined by immunoblotting, and the blot was stripped and reprobed with α-PLCγ1 antibody to control for sample loading. The results shown are representative of those obtained in three independent trials. (C) Effect of rapamycin on HIF-1α degradation. Cells were cultured in medium containing 2% serum and exposed to hypoxia overnight. The cells were then treated with CHX to block new protein synthesis, and the indicated samples were concomitantly exposed to 100 nM rapamycin. The cells were maintained under hypoxic conditions for the indicated times, and HIF-1α levels were determined by immunoblotting as described in panel A. PLCγ1 served as a sample loading control as described above.

Activation of HIF-1 in stably transfected PC-3 sublines. PC-3 cells were stably transfected with an expression plasmid encoding either AU1-tagged, wild-type AmTOR (WT), or a rapamycin-resistant (Ser²⁰³⁵→Ile) AmTOR mutant (SI). Clonal sublines were cotransfected with the pHRE-luc reporter, together with a Renilla luciferase reporter (as a control for transfection efficiency). After 24 h, the culture medium was changed to RPMI 1640 supplemented with 0.1% FBS, and the cells were cultured for an additional 6 h. The cells were pretreated for 30 min with solvent vehicle or 100 nM rapamycin and then were stimulated for 16 h with 150 μM CoCl₂ or hypoxia as indicated. Cell lysates were subjected to the dual luciferase assay, and sample values were normalized for variations in transfection efficiency based on the Renilla luciferase activity. Error bars represent the variance of duplicate samples. Aliquots of cell extracts were assayed for expression of the recombinant AmTOR protein by α-AU1 immunoblotting (upper panels). (A) AmTOR-WT-transfected subclones. (B) Comparison of AmTOR-WT- and AmTOR-SI-transfected subclones. (C) PC-3 cells and stably transfected WT or SI sublines wereprecultured for 8 h in medium containing 0.1% serum and then treated with either vehicle or rapamycin. After 30 min, the cells were stimulated for 16 h with hypoxia. Detergent-soluble proteins were immunoblotted sequentially with α-HIF-1α MAb and α-PLCγ1 antibodies. HIF-1α levels were determined by densitometric analysis, and the percent inhibition by rapamycin of hypoxia-induced HIF-1α expression was calculated for each sample set. In the upper panel, separate aliquots of the cell extracts were immunoblotted with α-AU1 MAb to detect the expression of the AmTOR-WT or AmTOR-SI proteins.

Role of the ODD domain in the regulation of HIF-1α expression by mTOR. (A) Schematic diagram of HIF-1α functional domains. The domain structure of HIF-1α includes the basic helix-loop-helix (bHLH) and PER-ARNT-SIM (PAS) motifs, the carboxyl-terminal transactivation domains (TAD N and C), and the ODD domain. The regulatory Pro-402 and Pro-564 residues (see the text) are also highlighted. (B) Effect of rapamycin on CoCl₂-induced stabilization of G4-HIF-1α proteins. PC-3 cells were transiently transfected with empty vector (mock) or with plasmids encoding the indicated G4-HIF-1α fusion proteins. After 24 h, the transfected cells were cultured for 2 h in 2% serum-containing medium, and the indicated samples were exposed to hypoxia for 16 h, in the absence or presence of 100 nM rapamycin. Detergent-soluble proteins were separated by SDS-PAGE and immunoblotted with α-G4 MAb, followed by the addition of PLCγ1-specific antibody as a control.