Yamasaki H, Drevon C, Martel N
Carcinogenesis. 1982;3(8):905-10. doi: 10.1093/carcin/3.8.905.
Specific and saturable binding sites for [20-3H]phorbol 12,13-dibutyrate ([3H]PDBu) were demonstrated in intact Friend erythroleukemia cells (FELC), in which inducible erythroid differentiation is reversibly inhibited by phorbol esters. The binding of [3H]PDBu to intact cells was maximal within only 15 min of incubation at 37 degrees C, after which there was a gradual decrease; binding at 4 degrees C however, was a slow process, requiring greater than 180 min for maximal binding. A Scatchard analysis showed that the dissociation constant for binding of [3H]PDBu is 8.3 nM; at saturation, approximately 1.75 x 10(5) molecules of [3H]PDBu are bound per cell. The binding of [3H]PDBu is blocked by 12-O-tetradecanoyl phorbol-13-acetate, phorbol 12,13-didecanoate, mezerein, 4-O-methyl-12-O-tetradecanoyl phorbol-13-acetate and resiniferatoxin, but not by phorbol or 4 alpha-phorbol 12,13-didecanoate. There was, in general, a good correlation between the potency of these agents in inhibiting [3H]PDBu binding and their activity in promoting tumors on mouse skin. Inducers of differentiation, such as hexamethylene bisacetamide, dimethyl sulfoxide and butyric acid, as well as inhibitors of cell differentiation, dexamethasone and local anesthetics, did not significantly block the binding of [3H]PDBu to intact FELC. When FELC were induced to differentiate with 4 mM hexamethylene bisacetamide (approximately 80% of cells were benzidine-positive), a slight decrease (10-20%) in the number of binding sites at saturation was seen, but the dissociation constant was not changed. When the cells were precultured with non-radioactive phorbol esters, a significant decrease in [3H]PDBu binding was observed, suggesting a homologous down regulation of phorbol ester receptors. Scatchard analysis indicated that the decrease in [3H]PDBu binding was due to a decrease in the number of binding sites and not to a change in affinity. Such specific phorbol ester binding sites might mediate a number of biochemical and biological effects of phorbol esters on FELC.
在完整的Friend红白血病细胞（FELC）中证实了[20 - 3H]佛波醇12,13 - 二丁酸酯（[3H]PDBu）的特异性和可饱和结合位点，在该细胞中佛波醇酯可逆转抑制诱导性红系分化。[3H]PDBu与完整细胞的结合在37℃孵育仅15分钟内达到最大值，之后逐渐下降；然而在4℃时结合是一个缓慢的过程，最大结合需要超过180分钟。Scatchard分析表明[3H]PDBu结合的解离常数为8.3 nM；饱和时，每个细胞约结合1.75×10(5)个[3H]PDBu分子。[3H]PDBu的结合被12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯、佛波醇12,13 - 二癸酸酯、大戟二萜醇、4 - O - 甲基 - 12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯和树脂毒素阻断，但不被佛波醇或4α - 佛波醇12,13 - 二癸酸酯阻断。一般来说，这些试剂抑制[3H]PDBu结合的效力与其促进小鼠皮肤肿瘤的活性之间有良好的相关性。分化诱导剂，如六亚甲基双乙酰胺、二甲基亚砜和丁酸，以及细胞分化抑制剂地塞米松和局部麻醉剂，均未显著阻断[3H]PDBu与完整FELC的结合。当用4 mM六亚甲基双乙酰胺诱导FELC分化时（约80%的细胞对联苯胺呈阳性），饱和时结合位点数量略有减少（10 - 20%），但解离常数未改变。当细胞用非放射性佛波醇酯预培养时，观察到[3H]PDBu结合显著减少，提示佛波醇酯受体的同源性下调。Scatchard分析表明[3H]PDBu结合的减少是由于结合位点数量的减少而非亲和力的改变。这种特异性佛波醇酯结合位点可能介导佛波醇酯对FELC的许多生化和生物学效应。
