Li G, Segu V B, Rabaglia M E, Luo R H, Kowluru A, Metz S A
Medical Service, Middleton Veterans Administration Hospital, Madison, Wisconsin 53705, USA.
Endocrinology. 1998 Sep;139(9):3752-62. doi: 10.1210/endo.139.9.6207.
Inhibitors of IMP dehydrogenase, such as mycophenolic acid (MPA) and mizoribine, which deplete cellular GTP, are used clinically as immunosuppressive drugs. The prolonged effect of such agents on insulin-secreting beta-cells (HIT-T15 and INS-1) was investigated. Both MPA and mizoribine inhibited mitogenesis, as reflected by [3H]thymidine incorporation. Cell number, DNA and protein contents, and cell (metabolic) viability were decreased by about 30%, 60%, and 80% after treatment of HIT cells with clinically relevant concentrations (e.g. 1 microg/ml) of MPA for 1, 2, and 4 days, respectively. Mizoribine (48 h) similarly induced the death of HIT cells. INS-1 cells also were damaged by prolonged MPA treatment. MPA-treated HIT cells displayed a strong and localized staining with a DNA-binding dye (propidium iodide), suggesting condensation and fragmentation of DNA, which were confirmed by detection of DNA laddering in multiples of about 180 bp. DNA fragmentation was observed after 24-h MPA treatment and was dose dependent (29%, 49%, and 70% of cells were affected after 48-h exposure to 1, 3, and 10 microg/ml MPA, respectively). Examination of MPA-treated cells by electron microscopy revealed typical signs of apoptosis: condensed and marginated chromatin, apoptotic bodies, cytosolic vacuolization, and loss of microvilli. MPA-induced cell death was almost totally prevented by supplementation with guanosine, but not with adenosine or deoxyguanosine, indicating a specific effect of GTP depletion. An inhibitor of protein isoprenylation (lovastatin, 10-100 microM for 2-3 days) induced cell death and DNA degradation similar to those induced by sustained GTP depletion, suggesting a mediatory role of posttranslationally modified GTP-binding proteins. Indeed, impeding the function of G proteins of the Rho family (via glucosylation using Clostridium difficile toxin B), although not itself inducing apoptosis, potentiated cell death induced by MPA or lovastatin. These findings indicate that prolonged depletion of GTP induces beta-cell death compatible with apoptosis; this probably involves a direct impairment of GTP-dependent RNA-primed DNA synthesis, but also appears to be modulated by small GTP-binding proteins. Treatment of intact adult rat islets (the beta-cells of which replicate slowly) induced a modest, but definite, death by apoptosis over 1- to 3-day periods. Thus, more prolonged use of the new generation of immunosuppressive agents exemplified by MPA might have deleterious effects on the survival of islet or pancreas grafts.
肌苷-5'-单磷酸脱氢酶（IMP dehydrogenase）抑制剂，如霉酚酸（MPA）和咪唑立宾，可消耗细胞内的鸟苷三磷酸（GTP），在临床上用作免疫抑制药物。本研究调查了此类药物对胰岛素分泌β细胞（HIT-T15和INS-1）的长期影响。MPA和咪唑立宾均抑制有丝分裂，这可通过[3H]胸苷掺入来反映。用临床相关浓度（如1微克/毫升）的MPA分别处理HIT细胞1天、2天和4天后，细胞数量、DNA和蛋白质含量以及细胞（代谢）活力分别下降了约30%、60%和80%。咪唑立宾（处理48小时）同样诱导了HIT细胞死亡。INS-1细胞也因MPA的长期处理而受损。经MPA处理的HIT细胞用一种DNA结合染料（碘化丙啶）染色后显示出强烈且局限的染色，表明DNA发生了浓缩和片段化，通过检测约180碱基对倍数的DNA梯带来证实这一点。MPA处理24小时后观察到DNA片段化，且呈剂量依赖性（分别用1、3和10微克/毫升MPA处理48小时后，受影响的细胞分别为29%、49%和70%）。通过电子显微镜检查经MPA处理的细胞，发现了凋亡的典型特征：染色质浓缩并边缘化、凋亡小体、胞质空泡化以及微绒毛丧失。补充鸟苷可几乎完全阻止MPA诱导的细胞死亡，但腺苷或脱氧鸟苷则不能，这表明GTP耗竭具有特异性作用。蛋白质异戊烯化抑制剂（洛伐他汀，10 - 100微摩尔，处理2 - 3天）诱导的细胞死亡和DNA降解与持续GTP耗竭诱导的相似，提示翻译后修饰的GTP结合蛋白起介导作用。实际上，阻碍Rho家族G蛋白的功能（通过艰难梭菌毒素B进行糖基化），虽然其本身不诱导凋亡，但可增强MPA或洛伐他汀诱导的细胞死亡。这些发现表明，GTP的长期耗竭诱导与凋亡相符的β细胞死亡；这可能直接损害了GTP依赖的RNA引发的DNA合成，但似乎也受到小GTP结合蛋白的调节。对完整成年大鼠胰岛（其β细胞复制缓慢）进行处理，在1至3天的时间内诱导了适度但明确的凋亡性死亡。因此，以MPA为代表的新一代免疫抑制剂使用时间过长可能对胰岛或胰腺移植的存活产生有害影响。
