Tissue hypoxia is common in solid tumors. Hypoxia activates a transcription factor - hypoxia-inducible factor 1 (HIF-1) - through posttranslational modifications of HIF-1alpha subunit. During the process, HIF-1alpha accumulation is an important molecular event that contributes to HIF-1 activity in regulation of HIF-1-targeted genes including vascular endothelial growth factor (VEGF), a critical factor involved in angiogenesis. Like tissue hypoxia, an elevated interstitial fluid pressure is often found in solid tumors, and it is associated with angiogenesis, poor prognosis and so on. Elevated interstitial fluid pressure has been documented in numerous solid tumors with the value as high as 60 mmHg in some of them. In this study, an in vitro model was established to mimic a setting with elevated interstitial fluid pressure and to test whether the environmental pressure stress adds to hypoxic effects on HIF-1alpha level and HIF-1 activity in cultured cell lines. Cells were cultured in three systems: control, hypoxia and hypoxia plus pressure. Control cells were placed in a common cell culture incubator containing about 20% O2. Hypoxic cells were cultured in an inflatable and plastic chamber filled with hypoxic gas (1% O2, 5% CO2 and balanced N2) in 80% volume, lacking any potential to create interior pressure throughout experiments. Cells in the conditions of hypoxia plus pressure were placed in a modular incubator chamber made of solid materials in a fixed size, and the chamber was fully filled with the hypoxic gas so as to create an intermediate (40 mmHg) or a high (70 mmHg) interior pressure as measured by a water-mercury manometer. Cells were cultured at 37 oC for 6 to 24 hours. We found that an intermediate pressure (40 mmHg) enhanced hypoxic induction of HIF-1alpha level, HIF-1 transcriptional activity and VEGF production in a cerebellar medulloblastoma Daoy cell line. However, a higher pressure (70 mmHg) did not enhance hypoxic induction of HIF-1alpha level, but resulted in growth inhibition and morphological changes. Interestingly, hypoxia plus intermediate pressure markedly increased the levels of phosphor-AKT, phosphor-p70S6K and phosphor-p44/42 as compared to control group or hypoxia alone group whereas the levels of PHDs were not changed. Above dynamic changes in HIF-1alpha and protein phosphorylations were not shown in other cell lines tested, such as prostate cancer cell line LNCaP, another brain tumor cell line SK-N-SH and HUVEC cell line. These results indicate that intermediate interstitial pressure within solid tumors can augment hypoxia induced HIF-1alpha level and lead to a further increase in HIF-1 activity likely in a cell type dependent manner. Underlying mechanisms are being investigated. (Meredith Holley, a Furman University biology senior, was supported by the WCI summer student program)

[Proc Amer Assoc Cancer Res, Volume 46, 2005]