SECRETION RATES AND LEVELS OF VASCULAR ENDOTHELIAL GROWTH-FACTOR IN CLONE-A OR HCT-8 HUMAN COLON-TUMOR CELLS AS A FUNCTION OF OXYGEN CONCENTRATION

Molecular and in situ hybridization studies have shown, in a number of cell types, that under hypoxic conditions, vascular endothelial growt h factor (VEGF) mRNA expression is up-regulated and VEGF protein is co ncomitantly increased. To establish a quantitative relationship betwee n VEGF protein levels and oxygenation, we exposed exponentially growin g clone A or HCT-8 human colon tumour cells in vitro (22h at 37 degree s C) to oxygen concentrations from 21% (air mixture) to 0.01%. Protein levels in cells and medium were then assayed using an enzyme-linked i mmunoabsorbent assay (ELISA). Intracellular levels of VEGF in clone A or HCT-8 cells exposed to either air (21% O-2) or the 0.01% O-2 mixtur e respectively increased from about 73 to 1270, and 1.5 to 1180 pg/10( 6) cells (about 17- and 80-fold increases), The shapes of the response curves (log of the intracellular VEGF concentrations v. log oxygen co ncentration) for both cell types were sigmoidal. However, intracellula r VEGF levels in HCT-8 cells were always less than that of clone A cel ls until levels of about 0.3 to 0.1% O-2 were reached. Levels of VEGF in the supernatant were also increased after the 22h hypoxic exposures . Because cell proliferation and clonogenicity were also measured, it was possible to estimate the secretion rates of VEGF for both cell lin es as a function of oxygen percentage. For clone A cells, the secretio n rate (pg/10(6) cells/h) in 21% O-2 was 62.5. This rate increased to 428.8 pg/10(6) cells/h at 0.01% O-2, a 7-fold increase. For HCT-8 cell s, levels in the medium at 21% O-2 were too low to be measured by ELIS A. However, between 10% and 0.01% O-2, secretion rates increased from 5.0 to 376.0 pg/10(6) cells/h, a 75-fold increase. Therefore, at very low O-2 levels, VEGF secretion rates were similar in the two cell line s. We propose that the different VEGF responses of clone A and HCT-8 c olon tumour cells to hypoxic stress in vitro are related to the in viv o observation that the respective hypoxic percentages of solid neoplas ms originating from these cell lines are markedly different (i.e. abou t 3 versus 80%) at equivalent volumes of 750 mm(3).