Hoffman R, Dennis I F, Donaldson J
Clinical Oncology and Radiotherapeutics Unit, MRC Centre, Cambridge, UK.
Cancer Chemother Pharmacol. 1995;36(4):316-24. doi: 10.1007/BF00689049.
Inhibition of growth factor-stimulated DNA synthesis carried out in defined medium is often compared with inhibition of serum-stimulated DNA synthesis so as to assess the selectivity of growth-factor-receptor tyrosine kinase inhibitors such as tyrphostins. We investigated whether protein binding may influence the interpretation of these experiments. Protein binding of tyrphostins was determined by ultrafiltration, equilibrium dialysis or spectrophotometer, and was quantitated by high-performance liquid chromatography (HPLC). For growth factor-stimulated DNA synthesis, we used the non-small-cell lung cancer cell line L23/P stimulated by transforming growth factor alpha (TGF alpha). The epidermal growth factor (EGF)-receptor kinase was assayed by phosphorylation of a peptide substrate or by receptor autophosphorylation. Protein binding of a number of tyrphostins ranged from 64% to 98%. There was a positive correlation (r = 0.995) between the degree of protein binding and the hydrophobicity. Inhibition of the EGF-receptor tyrosine kinase activity by the highly protein-bound tyrphostin B56 [N-(4-phenylbutyl)-3,4-dihydroxybenzylidene cyanoacet-amide] was reduced by bovine serum albumin (BSA), but BSA had less of an effect on inhibition of the EGF-receptor kinase by the weakly protein-bound tyrphostin A47 (RG 50864: 3,4-dihydroxybenzylidene cyanothioacetamide). Tyrphostins B46 [N-(3-phenylpropyl)-3,4-dihydroxybenzylidene cyanoacetamide] and B56 (both highly protein-bound) inhibited DNA synthesis of L23/P cells with approximately 3-fold greater potency in 0.5% serum than in 10% serum, but the inhibition of DNA synthesis in 0.5% serum was reduced by the addition of BSA. Tyrphostins B46 and B56 inhibited DNA synthesis stimulated by TGF alpha in defined medium to a greater extent than DNA synthesis stimulated by serum. However, this apparent selectivity for inhibition of TGF alpha-stimulated DNA synthesis was lost when the protein concentration in the defined medium was made equivalent to that in the serum-containing medium. By contrast, BSA enhanced the selective inhibition of TGF alpha-stimulated DNA synthesis by tyrphostin A47. These results demonstrate that protein binding accounts for the apparent selectivity of some highly protein-bound tyrphostins for TGF alpha-stimulated DNA synthesis of L23/P cells. Therefore, protein binding should be taken into consideration in assessments of the selectivity of tyrphostins.
在限定培养基中进行的生长因子刺激的DNA合成抑制实验，常与血清刺激的DNA合成抑制实验相比较，以评估诸如 tyrphostins 等生长因子受体酪氨酸激酶抑制剂的选择性。我们研究了蛋白质结合是否会影响这些实验的解读。通过超滤、平衡透析或分光光度计测定 tyrphostins 的蛋白质结合情况，并通过高效液相色谱（HPLC）进行定量分析。对于生长因子刺激的DNA合成，我们使用了由转化生长因子α（TGFα）刺激的非小细胞肺癌细胞系L23/P。通过肽底物的磷酸化或受体自身磷酸化来检测表皮生长因子（EGF）受体激酶活性。多种 tyrphostins 的蛋白质结合率在64%至98%之间。蛋白质结合程度与疏水性之间存在正相关（r = 0.995）。高度蛋白质结合的 tyrphostin B56 [N-(4-苯基丁基)-3,4-二羟基亚苄基氰基乙酰胺] 对EGF受体酪氨酸激酶活性的抑制作用会被牛血清白蛋白（BSA）减弱，但BSA对弱蛋白质结合的 tyrphostin A47（RG 50864：3,4-二羟基亚苄基氰硫代乙酰胺）抑制EGF受体激酶的影响较小。Tyrphostins B46 [N-(3-苯基丙基)-3,4-二羟基亚苄基氰基乙酰胺] 和B56（两者均为高度蛋白质结合）在0.5%血清中抑制L23/P细胞DNA合成的效力比在10%血清中高约3倍，但在0.5%血清中添加BSA后，DNA合成的抑制作用会减弱。Tyrphostins B46和B56在限定培养基中对TGFα刺激的DNA合成的抑制程度大于对血清刺激的DNA合成的抑制程度。然而，当限定培养基中的蛋白质浓度与含血清培养基中的蛋白质浓度相当时，这种对TGFα刺激的DNA合成抑制的明显选择性就消失了。相比之下，BSA增强了 tyrphostin A47对TGFα刺激的DNA合成的选择性抑制作用。这些结果表明，蛋白质结合是一些高度蛋白质结合的tyrphostins对L23/P细胞TGFα刺激的DNA合成具有明显选择性的原因。因此，在评估tyrphostins的选择性时应考虑蛋白质结合因素。
