Schematic illustration of dicistronic reporter gene constructs. The control dicistronic reporter gene, in plasmid pRF, contains cDNAs encoding sea pansy (Renilla reniformis) luciferase and firefly luciferase, separated by a short linker sequence. Expression is driven by the SV40 early promoter. pstemRF is similar to pRF, but with the addition of a stem-loop at the 5′ end of the transcript to suppress cap-dependent translation. Plasmids pRhifF, pRmycF, and pRvegfF contain the murine HIF-1α, c-myc, and VEGF 5′ UTRs, respectively, inserted between the two reporter reading frames. Plasmids pstemRhifF, pstemRmycF, and pstemRvegfF are identical to their corresponding vectors described above, except they also contain the stable stem-loop at the 5′ end of the transcript.

Effect of hypoxia on protein synthesis. (A) Time course of incorporation of ³⁵S-methionine/cysteine into TCA-precipitable protein. Cells were preincubated for 24 h under normoxia or hypoxia (1% O₂) or incubated for 23 h under hypoxia, followed by 1 h of normoxia. Also shown is the incorporation into normoxic cells in the presence of 10 μg/ml cycloheximide. Each measurement was normalized with respect to total protein as determined by Bradford assay. (B) Effect of hypoxia on the translation of HIF-1α, β-actin, and GAPDH mRNAs determined by polysome profile analysis. NIH3T3 cells were incubated for 24 h in normoxia or hypoxia (1% O₂), and cytoplasmic extracts were fractionated by 15–50% sucrose gradient centrifugation. Examples of polysome profiles showing the location of 80S ribosomes and free ribosome subunits are shown. The arrow indicates where the sensitivity of the UV monitor was increased fivefold. Transcripts that were being translated at the time of extract preparation are polysome-associated and sediment in fractions 7–16, with the most actively translated transcripts toward the bottom of the gradient. Cytoplasmic extracts from cells grown under normoxic or hypoxic conditions were fractionated, RNA was prepared from each fraction, and the HIF-1α, β-actin, and GAPDH mRNAs were detected by RNase protection assay. Each mRNA from the RNase protection assay was quantitated by phosphorimager and calculated as a percentage of the total for that mRNA. Data for fractions 1–6 and 7–16 were pooled and represented as translated or untranslated regions of the gradient, respectively. Black bars show data for normoxic cells, and white bars show data for hypoxic cells. The data represent the mean (±SEM) from three independent experiments.

Relative enhancement of expression of the downstream reporter enzyme by the HIF-1α 5′UTR in various cell types. NIH3T3, HEK293, and HeLa cells were transiently transfected with pRF or pRhifF (Figure 1) as indicated. Renilla and firefly luciferase activities were determined 24 h posttransfection, and IRES activities represented as ratios of firefly to Renilla luciferase. The ratios for each cell line are graphed relative to pRF, which was given a value of 1. Data presented are the mean (±SEM) of triplicate samples from three independent experiments.

The HIF-1α 5′UTR contains an IRES. (A) Effect of a 5′ stem-loop on relative expression of the downstream reporter enzyme. NIH3T3 and HeLa cells were transfected with pRF, pRhifF, or pstemRhifF (described in Figure 1) as indicated. IRES activities are represented as ratios of firefly luciferase to Renilla luciferase. All luciferase ratios are graphed relative to pRF, which was given a value of 1. Data are presented as mean (±SEM) of triplicate samples from three independent experiments. (B) Ribonuclease protection mapping of the intercistronic region of the dicistronic mRNA. A schematic showing the dicistronic mRNA containing the HIF-1α 5′ UTR hybridized to the antisense RNA probe used for RNase protection analysis is shown. The lengths of probe regions protected by the Renilla luciferase coding region, HIF1-α 5′UTR, and firefly luciferase coding region are indicated. The RNase protection gel is shown below, with sizes relative to molecular weight markers indicated. Lane 1, undigested RNA probe. Lane 2, 2 μg yeast tRNA. Lane 3, Poly(A)⁺ mRNA from NIH3T3 cells transfected with pRhifF. Lane 4, Poly(A)⁺ mRNA from mock transfected NIH3T3 cells. Lane 5, Poly(A)⁺ mRNA from mock transfected HeLa cells. Lane 6, Poly(A)⁺ mRNA from HeLa cells transfected with pRhifF. (C) Northern analysis of pRhifF mRNA in NIH3T3 cells. Poly(A)⁺ RNA was isolated from NIH3T3 cells either transiently transfected with pRhifF, or mock transfected, as indicated. Northern analysis was performed using ³²P-labeled DNA probes for the Renilla (RL) or firefly (FF) luciferase regions as indicated. The migration of RNA size standards is indicated.

Comparison of the activities of the HIF-1α, c-myc, and VEGF IRESs. NIH3T3 and HeLa cells were transiently transfected with the various dicistronic reporter constructs described in Figure 1. Renilla and firefly luciferase activities were determined 24 h posttransfection, and IRES activities were represented as ratios of firefly to Renilla luciferase, relative to the ratio obtained with pRF, which was given a value of 1. Data are presented as the mean (±SEM) of triplicate samples from three independent experiments.

IRES activity is maintained during hypoxia. NIH3T3 cells were transiently transfected with pRhifF (A), pRvegfF (B), or pRmycF (C). Four hours posttransfection, cells were subjected to either hypoxia, serum starvation, or serum starvation and hypoxia together or were left under control conditions for a further 24 h before harvesting and assay for Renilla and firefly luciferase. Each luciferase activity is shown relative to the activity under normoxia, which is expressed as 100%. The histograms at left show Renilla luciferase activity, the middle histograms show firefly luciferase, and the histograms at right show the ratio of firefly to Renilla. The ratios are graphed relative to the ratio obtained with pRF under normoxia, which was given a value of 1. The data shown are means (±SEM) of triplicate samples from each of five independent experiments. Statistical significance of the difference between normoxic and treatment was tested using the two-tailed Student's t test (*p < 0.01; **p < 0.001; ***p < 0.0001). The significance level for the effect of hypoxia on serum-starved cells (H+SS vs. SS) is shown in brackets.