Senescent human fibroblasts resist programmed cell death, and failure to suppress bcl2 is involved.
Senescent human fibroblasts resist programmed cell death, and failure to suppress bcl2 is involved.
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作者：
E. Wang
程序性细胞死亡（细胞凋亡）是一种主动过程，细胞通过该过程启动其自身的自我毁灭。越来越多的证据表明，这一事件是由独特的基因表达的激活控制的;一些作为生存基因，如bcl 2，其他作为杀手基因，如ced 3或白细胞介素转化酶。同样，外部因素，如细胞微环境中刺激的存在或不存在，在引导细胞走向生存或自杀命运方面起着关键作用。以前，我和其他人已经报道，从培养基中提取血清可以诱导接触抑制的静止小鼠3 T3成纤维细胞经历快速程序性细胞死亡，如24小时内大量DNA片段的存在所证明的。我现在报告，虽然相同的血清提取过程能够诱导静止的年轻人成纤维细胞凋亡，但该过程需要长达2周。反复尝试在相同的血清撤出与文化的衰老的人成纤维细胞显示，细胞凋亡的表型迹象，如DNA片段化和细胞活力的丧失，没有观察到长达4周，我认为，在体外老化的人成纤维细胞耐经历程序性细胞死亡。我已经研究了bcl 2的存在水平作为一个可能的保护衰老的人成纤维细胞凋亡死亡;生化表征表明，在小鼠以及人成纤维细胞，bcl 2是作为一个容易提取的（0.1%曲拉通）细胞质蛋白。Bcl 2水平与年轻和衰老的人成纤维细胞之间诱导凋亡性死亡的容易程度成反比关系。免疫荧光染色显示，在衰老的人成纤维细胞中，bcl 2不仅存在于小鼠和年轻人成纤维细胞的细胞质点状斑点中，而且存在于细胞核以及细胞核周围的大颗粒中。在血清剥夺后，bcl 2水平在小鼠3 T3成纤维细胞中在24小时内以及在年轻和中等年龄的人成纤维细胞中在2周内降低至检测不到;然而，在血清剥夺2周后，其在衰老的人成纤维细胞中保持不变。这些发现使我得出结论，衰老的成纤维细胞对血清剥夺诱导的细胞凋亡有抵抗力。此外，我认为，在体外老化过程中反复连续传代无意中建立了一种分子机制，使bcl 2水平不能被血清剥夺后抑制，这可能随后使衰老的成纤维细胞寿命长，并保护自我毁灭。
Programmed cell death (apoptosis) is an active process by which cells initiate their own self-destruction. Growing evidence shows that this event is controlled by the activation of unique gene expression; some function as survival genes, such as bcl2, and others as killer genes, such as ced3 or interleukin converting enzyme. Likewise, external factors, such as the presence or absence of stimuli in the microenvironment of a cell, play a key role in ushering it towards survival or suicidal fate. Previously, I and others have reported that withdrawal of serum from culture medium can induce contact-inhibited quiescent mouse 3T3 fibroblasts to undergo rapid programmed cell death, as evidenced by the presence of massive DNA fragmentation within 24 h. I now report that, although the same process of serum withdrawal is capable of inducing apoptotic death in quiescent young human fibroblasts, the process takes as long as 2 weeks. Repeated attempts at the same serum withdrawal with cultures of senescent human fibroblasts show that phenotypic signs of apoptosis, such as DNA fragmentation and loss of cell viability, are not observed for up to 4 weeks; I suggest that in vitro aged human fibroblasts are resistant to undergoing programmed cell death. I have investigated the level of bcl2 presence as a possible protector of senescent human fibroblasts from apoptotic death; biochemical characterization shows that in mouse as well as human fibroblasts, bcl2 is present as an easily extractable (0.1% Triton) cytoplasmic protein. bcl2 level is in inverse relationship with the ease of induction of apoptotic death between young and senescent human fibroblasts. Immunofluorescence staining shows that, in senescent human fibroblasts, bcl2 is present not only in the cytoplasmic punctate spots seen in both mouse and young human fibroblasts but also in the nuclei as well as large granules surrounding the nuclei. Upon serum deprivation, the bcl2 level is reduced to undetectable in mouse 3T3 fibroblasts within 24 h and in young and intermediate aged human fibroblasts within 2 weeks; however, it remains unchanged in senescent human fibroblasts after the deprivation of serum for 2 weeks. These findings lead me to conclude that senescent fibroblasts are resistant to the induction of apoptotic death by serum deprivation. Furthermore, I suggest that repeated serial passaging during the in vitro aging process has inadvertently instituted a molecular mechanism whereby the bcl2 level cannot be repressed upon serum deprivation, which may subsequently allow senescent fibroblasts to be long-lived and protected from self-destruction.