Gregoli P A, Bondurant M C
Department of Veterans Affairs Medical Center, Nashville, Tennessee 37212, USA.
J Cell Physiol. 1999 Feb;178(2):133-43. doi: 10.1002/(SICI)1097-4652(199902)178:2<133::AID-JCP2>3.0.CO;2-5.
Erythropoietin (EP) is required by late stage erythroid progenitor cells to prevent apoptosis. In a previous study (Gregoli and Bondurant, 1997, Blood 90:630-640), it was shown that rapid proteolytic conversion of procaspase 3 to the fully activated enzyme occurred when erythroblasts were deprived of EP for as little as 2 h. In the present study, protein and mRNA analyses of erythroblasts indicated the presence of the proenzyme precursors of caspases 1, 2, 3, 5, 6, 7, 8, and 9. The effects of various caspase inhibitors on caspase 3 processing and on apoptosis were examined. These inhibitors were benzyloxycarbonyl (z-) and fluoromethyl-ketone (FMK) derivatives of peptides that serve as substrates for selected caspases. z-VAD-FMK, t-butoxycarbonyl-aspartate-FMK (Boc-D-FMK), and z-IETD-FMK blocked the initial cleavage of procaspase 3, while z-DEVD-FMK, z-VEID-FMK, and z-VDVAD-FMK did not block the initial cleavage but had some effect on blocking apoptosis. The peptide inhibitor z-FA-FMK, which inhibits cathepsins B and L but is not known to inhibit caspases, altered caspase 3 processing to a final 19 kDa large subunit that appeared to retain enzymatic activity. The action of z-FA-FMK in preventing the usual conversion to a 1 7 kDa subunit suggests the possibility that a noncaspase protease may be involved in caspase 3 processing. Studies with the peptide inhibitors and EP were done to determine the short- and long-term effectiveness of the caspase inhibitors in protecting EP-deprived cells from apoptosis. Although several of the inhibitors were effective, z-IETD-FMK was studied most extensively because of its specificity for enzymes which cleave procaspase 3 at aspartate 175 (IETD175). Large percentages of EP-deprived erythroblasts treated with z-IETD-FMK appeared morphologically normal and negative by a DNA strand breakage (TUNEL) assay at 24 h (75%) compared to EP-deprived controls (10%) which were not treated with inhibitor. However, inhibitor-treated erythroid progenitors deprived of EP for 24 h and then resupplied with EP showed only a modest improvement in long-term survival compared to cells which did not receive the caspase inhibitor during the 24 h EP deprivation. Thus, while the manifestations of apoptosis were delayed in most cells by inhibiting caspase activity, the processes initiating the loss of cell viability due to EP deprivation were irreparablein the majority of the cells and eventually led to their deaths.
晚期红系祖细胞需要促红细胞生成素（EP）来防止细胞凋亡。在先前的一项研究中（Gregoli和Bondurant，1997年，《血液》90:630 - 640），研究表明，当成红细胞被剥夺EP仅2小时时，procaspase 3会迅速发生蛋白水解转化为完全活化的酶。在本研究中，对成红细胞进行的蛋白质和mRNA分析表明存在caspases 1、2、3、5、6、7、8和9的酶原前体。研究了各种caspase抑制剂对caspase 3加工过程及细胞凋亡的影响。这些抑制剂是作为选定caspases底物的肽的苄氧羰基（z -）和氟甲基酮（FMK）衍生物。z - VAD - FMK、叔丁氧羰基 - 天冬氨酸 - FMK（Boc - D - FMK）和z - IETD - FMK可阻断procaspase 3的初始切割，而z - DEVD - FMK、z - VEID - FMK和z - VDVAD - FMK虽不能阻断初始切割，但对阻断细胞凋亡有一定作用。肽抑制剂z - FA - FMK可抑制组织蛋白酶B和L，但未知其是否抑制caspases，它将caspase 3加工为最终的19 kDa大亚基，该大亚基似乎保留了酶活性。z - FA - FMK阻止通常转化为17 kDa亚基的作用表明，可能有非caspase蛋白酶参与caspase 3的加工过程。使用肽抑制剂和EP进行研究，以确定caspase抑制剂在保护被剥夺EP的细胞免受凋亡方面的短期和长期有效性。尽管几种抑制剂都有效，但由于z - IETD - FMK对在天冬氨酸175（IETD175）处切割procaspase 3的酶具有特异性，因此对其进行了最广泛的研究。与未用抑制剂处理的被剥夺EP的对照（10%）相比，在24小时时，用z - IETD - FMK处理的被剥夺EP的成红细胞中，很大比例（75%）在形态上正常且通过DNA链断裂（TUNEL）检测呈阴性。然而，与在24小时EP剥夺期间未接受caspase抑制剂的细胞相比，用抑制剂处理的被剥夺EP 24小时然后再补充EP的红系祖细胞在长期存活方面仅略有改善。因此，虽然通过抑制caspase活性在大多数细胞中凋亡表现被延迟，但由于EP剥夺导致细胞活力丧失的起始过程在大多数细胞中是无法修复的，最终导致细胞死亡。
