The effects of Thymidine phsphorylase on tumor angiogenesis and on the sensitivity to anti-tumor drugs

人胸苷磷酸化酶（dThdPase）被认为与一种血管生成因子，血小板来源的内皮细胞生长因子（PD-ECGF）相同。然而，dThdPase的整个氨基酸序列仍然是未知的。dThdPase分离的人胎盘n端氨基酸序列为Ac-AALMTPGTGAPPAPG。与PD-ECGFcDNA预测的序列比较，发现dThdPase的2-16残基与PD-ECGF的相同。dThdPase的主要翻译产物将从启动翻译的蛋氨酸残基中提取一个氨基酸进行加工，并使Ala-2乙酰化。dThdPase具有血管生成活性。为了研究PD-ECGF/dThdpase的dThdpase活性是否与其血管生成活性有关，我们采用定点诱变法制备了3个无dThdpase活性的PD-ECGF/dThdpase突变体。转染野生型PD-ECGF/dThdpase cDNA的COS细胞裂解物具有血管生成活性，而转染突变型PD-ECGF/dThdpase的COS细胞裂解物无血管生成活性。dThdpase的抑制剂6-氨基-5-氯尿嘧啶抑制纯化dThdpase的血管生成活性。这些发现表明dThdpase活性是PD-ECGF/dThdpase血管生成活性不可或缺的。转染PD-ECGF cDNA的人KB表皮癌细胞表达55-kDa蛋白，经抗dThdPase抗体检测，细胞裂解液具有dThdPase活性。转染后的细胞对5′-脱氧-5-氟吡啶的敏感性高于未转染的KB细胞。这些结果表明dThdPase参与了这些抗癌药物的激活。

Human thymidine phsphorylase (dThdPase) is thought to be indentical to an angiogenic factor, Platelet-derived endothelial cell growth factor (PD-ECGF) .However, the whole amino acid sequence of dThdPase is still unknown. N-termina amino acid sequencing of dThdPase isolated human placenta gave the sequence Ac-AALMTPGTGAPPAPG.Comparison with the sequence predicted from the PD-ECGFcDNA reveals that residues 2-16 of dThdPase are indentical to that of PD-ECGF.The primary translational product of dThdPase would be processed one amino acid from the translation-initiating methionine residue and Ala-2 acetylated.dThdPase has angiogenic activity. To investigate whether the dThdpase activity of PD-ECGF/dThdpase is indispensable to its angiogenic activity, three PD-ECGF/dThdpase mutant which have no dThdpase activity were made by site-derected mutagenesis. The lysates of COS cells transfected with the wild-type PD-ECGF/dThdpase cDNA had angiogenic activity, but those transfected with the mutant PD-ECGF/dThdpase did not. An inhibitor of dThdpase, 6-amino-5-chlorouracil, inhibited the angiogenic activity of purified dThdPase. These findings indicate that dThdpase activity is indispensable to angiogenic activity of PD-ECGF/dThdpase.Human KB epidermal carcinoma cells transfected with PD-ECGF cDNA expressed a 55-kDa protein that was detected with anti-dThdPase antibody and the cell lysate had dThdPase activity. The sensitivity of the transfected cells to 5'-deoxy-5-fluorouridine was higher than that of untransfected KB cells. These results demonstrate that dThdPase is involved in the activation of these anticancer agents.