Keppler-Hafkemeyer A, Brinkmann U, Pastan I
Laboratory of Molecular Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Building 37, Room 4E16, 37 Convent Drive MSC 4255, Bethesda, Maryland 20892, USA.
Biochemistry. 1998 Dec 1;37(48):16934-42. doi: 10.1021/bi980995m.
Immunotoxins composed of antibodies linked to plant or bacterial toxins are being evaluated in the treatment of cancer. It is known that the toxin moieties of immunotoxins, including Pseudomonasexotoxin A (PE), diphtheria toxin, and ricin, are capable of inducing apoptosis. Since the efficiency of induction of apoptosis and the apoptosis pathway may have direct effects on the therapeutic usefulness of immunotoxins, we have studied how B3(Fv)-PE38, a genetically engineered immunotoxin in which the Fv fragment of an antibody is fused to a mutated form of PE, induces apoptosis of the MCF-7 breast cancer cell line. We show for the first time that a PE-containing immunotoxin activates ICE/ced-3 proteases, now termed caspases, and causes characteristic cleavage of the "death substrate" poly(ADP)-ribose polymerase (PARP) to an 89 kDa fragment with a time course of cleavage comparable to that induced by TNFalpha. Also the fluorescent substrate, DEVD-AFC, is cleaved 2-4-fold more rapidly by lysates from B3(Fv)-PE38 treated MCF-7 cells than untreated control cells, suggesting that a CPP32-like caspase is involved in B3(Fv)-PE38-mediated apoptosis. B3(Fv)-PE38-induced PARP cleavage is inhibited by several protease inhibitors known to inhibit caspases (zVAD-fmk, zDEVD-fmk, zIETD-fmk) as well as by overexpression of Bcl-2 providing additional evidence for caspase involvement. zVAD-fmk, a broad spectrum inhibitor of most mammalian caspases, prevents the early morphological changes and loss of cell membrane integrity produced by B3(Fv)-PE38, but not its ability to inhibit protein synthesis, arrest cell growth, and subsequently kill cells. Despite inhibition of apoptosis, the immunotoxin is still capable of selective cell killing, which indicates that B3(Fv)-PE38 kills cells by two mechanisms: one requires caspase activation, and the other is due to the arrest of protein synthesis caused by inactivation of elongation factor 2. The fact that an immunotoxin can specifically kill tumor cells without the need of inducing apoptosis makes such agents especially valuable for the treatment of cancers that are protected against apoptosis, e.g., by overexpression of Bcl-2.
由与植物或细菌毒素相连的抗体组成的免疫毒素正在癌症治疗中接受评估。已知免疫毒素的毒素部分，包括绿脓杆菌外毒素A（PE）、白喉毒素和蓖麻毒素，能够诱导细胞凋亡。由于诱导细胞凋亡的效率和凋亡途径可能对免疫毒素的治疗效用有直接影响，我们研究了B3（Fv）-PE38，一种基因工程免疫毒素，其中抗体的Fv片段与PE的突变形式融合，如何诱导MCF-7乳腺癌细胞系的凋亡。我们首次表明，一种含PE的免疫毒素激活ICE/ced-3蛋白酶，现在称为半胱天冬酶，并导致“死亡底物”聚（ADP）-核糖聚合酶（PARP）特征性地裂解为89 kDa片段，裂解的时间进程与TNFα诱导的相当。同样，荧光底物DEVD-AFC被B3（Fv）-PE38处理的MCF-7细胞的裂解物比未处理的对照细胞更快地裂解2-4倍，表明一种类似CPP32的半胱天冬酶参与了B3（Fv）-PE38介导的细胞凋亡。B3（Fv）-PE38诱导的PARP裂解被几种已知抑制半胱天冬酶的蛋白酶抑制剂（zVAD-fmk、zDEVD-fmk、zIETD-fmk）以及Bcl-2的过表达所抑制，为半胱天冬酶的参与提供了额外证据。zVAD-fmk，一种大多数哺乳动物半胱天冬酶的广谱抑制剂，可防止B3（Fv）-PE38产生的早期形态变化和细胞膜完整性丧失，但不能阻止其抑制蛋白质合成、阻止细胞生长并随后杀死细胞的能力。尽管细胞凋亡受到抑制，免疫毒素仍能够选择性地杀死细胞，这表明B3（Fv）-PE38通过两种机制杀死细胞：一种需要半胱天冬酶激活，另一种是由于延伸因子2失活导致蛋白质合成停滞。免疫毒素能够特异性地杀死肿瘤细胞而无需诱导细胞凋亡这一事实使得此类药物对于治疗例如通过Bcl-2过表达而免受细胞凋亡影响的癌症特别有价值。
