Study of the molecular mechanisms of cell death induced by the dNTP pool imbalance.

我们研究了5-氟-2'-脱氧尿苷（FUdR）诱导细胞死亡的分子机制。在小鼠乳腺肿瘤FM3A细胞中，FUdR引起细胞死亡，诱发dNTP池失衡和DNA双链断裂。我们观察到蛋白酶抑制剂（如TLCK、TPCK、PMSF、p-APMSF、Pefabloc SC和Z-Asp-CH_2-DCB）阻断细胞内酸化、DNA断裂和fudr诱导的细胞死亡。我们还发现，fudr诱导的FM3A细胞死亡与细胞c-jun和c-fos基因的表达增加有关。这些基因表达的增加是通过蛋白激酶c依赖途径介导的。用反义寡脱氧核苷酸阻断c-jun的表达可延缓细胞死亡。这些发现表明，编码参与细胞增殖的转录因子的c-jun和c-fos基因的激活在fudr诱导的细胞死亡中起作用。我们还发现，在FM3A野生型F28-7克隆细胞培养6个月后，FUdR诱导的细胞死亡模式发生了变化。在原始储存的F28-7克隆中，fudr诱导的细胞死亡伴随着坏死样细胞肿胀和DNA断裂至100-200 kbp，没有证据表明存在相关的寡核体DNA断裂。在培养6个月的F28-7细胞亚克隆F28-7- a中，观察到凋亡小体和核小体dna阶梯片段。此外，我们还发现，在FUdR处理24小时后，F28-7细胞的caspase-3样活性增加了24倍，F28-7- a细胞的caspase-3样活性增加了5.3倍。caspase和丝氨酸蛋白酶抑制剂抑制fudr诱导的细胞死亡和两个克隆中caspase-3样活性的增加。研究这些克隆之间细胞死亡方式的差异对于阐明在决定细胞凋亡或坏死的后续命运中起关键作用的分子机制具有重要意义。

We have investigated the molecular mechanism of cell death induced by 5-fluoro-2'-deoxyuridine (FUdR). FUdR caused cell death to induce dNTP pool imbalance and following DNA double strand breaks in mouse mammary tumor FM3A cells. We observed that protease inhibitors (such as TLCK,TPCK,PMSF,p-APMSF,Pefabloc SC,and Z-Asp-CH_2-DCB) blocked intracellular acidification, DNA fragmentation, and FUdR-induced cell death. We also revealed that FUdR-induced death of FM3A cells was found to be associated with an increased expression of cellular c-jun and c-fos genes. The increases in these gene expressions was mediated through the protein kinase C-dependent pathway. Blockage of the expression with the use of antisense oligodeoxynucleotide for c-jun delayd the cell death. These findings suggest that the activation of c-jun and c-fos genes, which encode transcription factors participating in cell proliferation, plays a role in FUdR-induced cell death. We have also found that mode of cell death induced by FUdR changed in wild-type F28-7 clone of FM3A cells after six-month culture. In the original stocked F28-7 clone, FUdR-induced cell death was accompanied by necrosis-like cell swelling and DNA fragmentation to 100-200 kbp with no evidence of associated oligonucleosomal DNA fragmentation. In subclone F28-7-A isolated from F28-7 cells which were cultured for six months, apoptotic bodies and nucleosomal DNA-ladder fragments were observed. Furthemore, we revealed additional findings that at 24 h after treatment with FUdR,caspase-3-like activity increased 24-fold in F28-7 cells and 5.3-fold in F28-7-A cells. Inhibitors of caspases and serine proteases suppressed FUdR-induced cell death and the increase in caspase-3-like activity in both clones. Investigation of differences in mode of cell death between these clones would be important to elucidate the molecular mechanism of pivotal role in deciding subsequent fates of cells toward either apoptosis or necrosis.