Leith J T, Michelson S
Department of Radiation Medicine, Brown University School of Medicine, Providence, Rhode Island 02912, USA.
Cell Prolif. 1995 Aug;28(8):415-30. doi: 10.1111/j.1365-2184.1995.tb00082.x.
Molecular and in situ hybridization studies have shown, in a number of cell types, that under hypoxic conditions, vascular endothelial growth factor (VEGF) mRNA expression is up-regulated and VEGF protein is concomitantly increased. To establish a quantitative relationship between VEGF protein levels and oxygenation, we exposed exponentially growing clone A or HCT-8 human colon tumour cells in vitro (22 h at 37 degrees C) to oxygen concentrations from 21% (air mixture) to 0.01%. Protein levels in cells and medium were then assayed using an enzyme-linked immunoabsorbent assay (ELISA). Intracellular levels of VEGF in clone A or HCT-8 cells exposed to either air (21% O2) or the 0.01% O2 mixture 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 concentration) for both cell types were sigmoidal. However, intracellular VEGF levels in HCT-8 cells were always less than that of clone A cells until levels of about 0.3 to 0.1% O2 were reached. Levels of VEGF in the supernatant were also increased after the 22 h hypoxic exposures. Because cell proliferation and clonogenicity were also measured, it was possible to estimate the secretion rates of VEGF for both cell lines as a function of oxygen percentage. For clone A cells, the secretion rate (pg/10(6) cells/h) in 21% O2 was 62.5. This rate increased to 428.8 pg/10(6) cells/h at 0.01% O2, a 7-fold increase. For HCT-8 cells, levels in the medium at 21% O2 were too low to be measured by ELISA. However, between 10% and 0.01% O2, secretion rates increased from 5.0 to 376.0 pg/10(6) cells/h, a 75-fold increase. Therefore, at very low O2 levels, VEGF secretion rates were similar in the two cell lines. We propose that the different VEGF responses of clone A and HCT-8 colon tumour cells to hypoxic stress in vitro are related to the in vivo observation that the respective hypoxic percentages of solid neoplasms originating from these cell lines are markedly different (i.e. about 3 versus 80%) at equivalent volumes of 750 mm3.
分子和原位杂交研究表明，在多种细胞类型中，在缺氧条件下，血管内皮生长因子（VEGF）的mRNA表达上调，且VEGF蛋白随之增加。为了建立VEGF蛋白水平与氧合之间的定量关系，我们将呈指数生长的克隆A或HCT-8人结肠肿瘤细胞在体外（37℃，22小时）暴露于21%（空气混合物）至0.01%的氧浓度下。然后使用酶联免疫吸附测定（ELISA）检测细胞和培养基中的蛋白水平。分别暴露于空气（21% O₂）或0.01% O₂混合物中的克隆A或HCT-8细胞内VEGF水平分别从约73增加到1270，以及从1.5增加到1180 pg/10⁶细胞（增加了约17倍和80倍）。两种细胞类型的反应曲线形状（细胞内VEGF浓度的对数对氧浓度的对数）均为S形。然而，直到氧浓度达到约0.3%至0.1%之前，HCT-8细胞内的VEGF水平始终低于克隆A细胞。缺氧暴露22小时后，上清液中的VEGF水平也升高了。由于还测量了细胞增殖和克隆形成能力，因此可以估算两种细胞系中VEGF的分泌率与氧百分比的函数关系。对于克隆A细胞，在21% O₂中的分泌率（pg/10⁶细胞/小时）为62.5。在0.01% O₂时，该分泌率增加到428.8 pg/10⁶细胞/小时，增加了7倍。对于HCT-8细胞，在21% O₂时培养基中的水平过低，无法通过ELISA测量。然而，在10%至0.01% O₂之间，分泌率从5.0增加到376.0 pg/10⁶细胞/小时，增加了75倍。因此，在极低的O₂水平下，两种细胞系中的VEGF分泌率相似。我们提出，克隆A和HCT-8结肠肿瘤细胞在体外对缺氧应激的不同VEGF反应与体内观察结果有关，即在相同体积750 mm³时，源自这些细胞系的实体瘤各自的缺氧百分比明显不同（即约3%对80%）。
