Lilly M, Sandholm J, Cooper J J, Koskinen P J, Kraft A
Department of Medicine, University of Washington, Veterans Affairs Medical Center, Seattle, USA.
Oncogene. 1999 Jul 8;18(27):4022-31. doi: 10.1038/sj.onc.1202741.
We have examined potential mechanisms by which the Pim-1 kinase acts as a hematopoietic cell survival factor. Enforced expression of the wild type 33 kd (FD/hpim33) and 44 kd (FD/mpim44) Pim-1 proteins in murine factor-dependent FDCP1 cells prolonged survival after withdrawal of IL-3, while expression of a dominant negative Pim-1 protein (FD/pimNT81) shortened survival. Following removal of IL-3 FDCP1 cells exhibited loss of mitochondrial transmembrane potential and production of reactive oxygen species, as determined by flow cytometry analysis. The wild type Pim-1 proteins decreased these changes while the dominant negative protein enhanced mitochondrial dysfunction. The antiapoptotic activity of the kinases could not be attributed to modulation of glutathione, catalase, or superoxide dismutase activities. Both the FD/hpim33 and FD/mpim44 cells maintained expression of bcl-2 mRNA following cytokine removal, while a substantial decrease was seen in FD/neo cells. To modulate Bcl-2 protein levels, a bcl-2 antisense RNA construct was coexpressed with the wild type pim-1 cDNAs. FD/hpim33 cells with low cellular Bcl-2 protein levels had shortened cytokine-independent survival compared with FD/hpim33 clones with high Bcl-2 expression. However survival of FD/mpim44 cells after IL-3 withdrawal was substantially independent of cellular Bcl-2 protein levels. The 33 kd protein delayed, and the 44 kd protein completely prevented enhanced cell death associated with enforced expression of human Bax protein however. Our results suggest that the 33 kd Pim-1 kinase may enhance cell survival through cooperation with and regulation of bcl-2. In addition the 44 kd kinase may regulate the expression or activity of other pro- and anti-apoptotic members of the bcl-2 family.
我们研究了Pim-1激酶作为造血细胞存活因子的潜在机制。在鼠类因子依赖性FDCP1细胞中强制表达野生型33kd（FD/hpim33）和44kd（FD/mpim44）Pim-1蛋白，可延长白细胞介素-3撤除后的存活时间，而表达显性负性Pim-1蛋白（FD/pimNT81）则缩短存活时间。通过流式细胞术分析确定，撤除白细胞介素-3后，FDCP1细胞表现出线粒体跨膜电位丧失和活性氧生成。野生型Pim-1蛋白减少了这些变化，而显性负性蛋白则增强了线粒体功能障碍。激酶的抗凋亡活性不能归因于对谷胱甘肽、过氧化氢酶或超氧化物歧化酶活性的调节。细胞因子撤除后，FD/hpim33和FD/mpim44细胞均维持bcl-2 mRNA的表达，而FD/neo细胞中则显著下降。为了调节Bcl-2蛋白水平，将bcl-2反义RNA构建体与野生型pim-1 cDNA共表达。与Bcl-2高表达的FD/hpim33克隆相比，细胞Bcl-2蛋白水平低的FD/hpim33细胞在无细胞因子情况下存活时间缩短。然而，白细胞介素-3撤除后FD/mpim44细胞的存活基本上与细胞Bcl-2蛋白水平无关。不过，33kd蛋白延迟了，44kd蛋白完全阻止了与强制表达人Bax蛋白相关的细胞死亡增强。我们的结果表明33kd Pim-1激酶可能通过与bcl-2合作及调节bcl-2来增强细胞存活。此外44kd激酶可能调节bcl-2家族其他促凋亡和抗凋亡成员的表达或活性。
