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
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
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DOI：
10.1111/j.1365-2184.1995.tb00082.x
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发表时间：
1995-08-01
影响因子：
8.5
通讯作者：
MICHELSON, S
中科院分区：
文献类型：
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作者：
LEITH, JT;MICHELSON, S
分子和原位杂交研究表明，在许多细胞类型中，在缺氧条件下，血管内皮生长因子（VEGF） mRNA表达上调，VEGF蛋白随之升高。为了建立VEGF蛋白水平与氧合之间的定量关系，我们将指数生长的克隆a或HCT-8人结肠癌细胞在体外（37℃下22小时）暴露于21%（空气混合物）至0.01%的氧气浓度。然后用酶联免疫吸附试验（ELISA）检测细胞和培养基中的蛋白质水平。在暴露于空气（21% O-2）或0.01% O-2混合物中的克隆A或HCT-8细胞中，细胞内VEGF水平分别从73至1270和1.5至1180 pg/10(6)个细胞（分别增加了17倍和80倍），两种细胞类型的响应曲线（细胞内VEGF浓度的对数与氧浓度的对数）的形状均为s型。然而，HCT-8细胞内的VEGF水平一直低于克隆A细胞，直到约0.3 ~ 0.1%的O-2水平。缺氧22小时后，上清液中VEGF水平也升高。由于还测量了细胞增殖和克隆原性，因此可以估计两种细胞系的VEGF分泌率与氧气百分比的关系。对于无性系A细胞，21% O-2的分泌率（pg/10(6)个细胞/h）为62.5。在0.01% O-2时，该速率增加到428.8 pg/10(6)个细胞/h，增加了7倍。对于HCT-8细胞，21% O-2的培养基水平太低，无法通过ELISA检测。然而，在10%和0.01% O-2之间，分泌率从5.0增加到376.0 pg/10(6)个细胞/h，增加了75倍。因此，在非常低的O-2水平下，两种细胞系的VEGF分泌率相似。我们提出克隆A和HCT-8结肠肿瘤细胞对体外缺氧应激的不同VEGF反应与体内观察有关，即在同等体积为750 mm时，源自这些细胞系的实体肿瘤各自的缺氧百分比显着不同（即约3对80%）(3)。
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 (22h 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% O-2) or the 0.01% O-2 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% 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 lines as a function of oxygen percentage. For clone A cells, the secretion 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 cells, levels in the medium at 21% O-2 were too low to be measured by ELISA. 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 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 mm(3).