Wempe F, Yang J Y, Hammann J, von Melchner H
Laboratory for Molecular Hematology, University of Frankfurt Medical School, 60590 Frankfurt am Main, Germany.
Genome Biol. 2001;2(7):RESEARCH0023. doi: 10.1186/gb-2001-2-7-research0023. Epub 2001 Jun 27.
The existence of a constitutively expressed machinery for death in individual cells has led to the notion that survival factors repress this machinery and, if such factors are unavailable, cells die by default. In many cells, however, mRNA and protein synthesis inhibitors induce apoptosis, suggesting that in some cases transcriptional activity might actually impede cell death. To identify transcriptional mechanisms that interfere with cell death and survival, we combined gene trap mutagenesis with site-specific recombination (Cre/loxP system) to isolate genes from cells undergoing apoptosis by growth factor deprivation.
From an integration library consisting of approximately 2 x 106 unique proviral integrations obtained by infecting the interleukin-3 (IL-3)-dependent hematopoietic cell line - FLOXIL3 - with U3Cre gene trap virus, we have isolated 125 individual clones that converted to factor independence upon IL-3 withdrawal. Of 102 cellular sequences adjacent to U3Cre integration sites, 17% belonged to known genes, 11% matched single expressed sequence tags (ESTs) or full cDNAs with unknown function and 72% had no match within the public databases. Most of the known genes recovered in this analysis encoded proteins with survival functions.
We have shown that hematopoietic cells undergoing apoptosis after withdrawal of IL-3 activate survival genes that impede cell death. This results in reduced apoptosis and improved survival of cells treated with a transient apoptotic stimulus. Thus, apoptosis in hematopoietic cells is the end result of a conflict between death and survival signals, rather than a simple death by default.
单个细胞中存在一种组成性表达的死亡机制，这导致了一种观点，即存活因子会抑制这种机制，如果这些因子缺失，细胞就会默认死亡。然而，在许多细胞中，mRNA和蛋白质合成抑制剂会诱导细胞凋亡，这表明在某些情况下，转录活性实际上可能会阻碍细胞死亡。为了确定干扰细胞死亡和存活的转录机制，我们将基因陷阱诱变与位点特异性重组（Cre/loxP系统）相结合，从因生长因子剥夺而发生凋亡的细胞中分离基因。
通过用U3Cre基因陷阱病毒感染依赖白细胞介素-3（IL-3）的造血细胞系——FLOXIL3，我们获得了一个由大约2×10⁶个独特的原病毒整合组成的整合文库。从中我们分离出了125个单个克隆，这些克隆在IL-3撤除后转变为因子非依赖性。在与U3Cre整合位点相邻的102个细胞序列中，17%属于已知基因，11%与功能未知的单个表达序列标签（EST）或全长cDNA匹配，72%在公共数据库中没有匹配项。在该分析中回收的大多数已知基因编码具有存活功能的蛋白质。
我们已经表明，在撤除IL-3后发生凋亡的造血细胞会激活阻碍细胞死亡的存活基因。这导致凋亡减少，并且在用短暂凋亡刺激处理的细胞中存活率提高。因此，造血细胞中的凋亡是死亡信号与存活信号之间冲突的最终结果，而不是简单的默认死亡。
