Koma Y-I, Ito A, Watabe K, Kimura S H, Kitamura Y
Department of Pathology and Department of Internal Medicine and Molecular Science, Osaka University Medical School, Suita, Osaka, Japan and Department of Pharmacology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan.
Histol Histopathol. 2004 Apr;19(2):391-400. doi: 10.14670/HH-19.391.
F10, a subline of the B16 mouse melanoma cell line, is itself the parent of the more metastatic BL6 line. BL6 cells differ from F10 cells by an alteration of the gene encoding the B56gamma regulatory subunit of protein phosphatase 2A (PP2A), which results in mRNA encoding a truncated variant of the subunit (deltagamma1). Expression of deltagamma 1 protein is detectable only when BL6 cells are transplanted into mice and then gamma-irradiated. Recently, B56gamma subunit-containing PP2A holoenzymes have shown to dephosphorylate Mdm2, a negative regulator of p53. Thus, we assessed whether the expression of deltagamma1 affects irradiation-induced phosphorylation of Mdm2 and radioresistance of melanoma cells by perturbing the regulation of p53. Western blot analyses revealed that irradiated COS-7 and NIH3T3 cells stably expressing deltagamma1 showed significantly less irradiation-induced Mdm2 phosphorylation. Mdm2 phosphorylation reduces the ability of Mdm2 to target p53 for degradation, which probably explained why p53 protein levels in NIH3T3 cells expressing deltagamma1 were not significantly elevated by irradiation, unlike in wild-type cells. This was also true for F10 cells transfected with deltagamma1 (F10deltagamma1) when the cells expressed deltagamma1 after being irradiated in vivo. p53 mRNA levels in irradiated wild-type and deltagamma 1-expressing cells were both only slightly elevated, suggesting that Mdm2 regulates p53 levels by a post-transcriptional mechanism. p53-mediated induction of the pro-apoptotic gene encoding Bax was also significantly lower in F10deltagamma1 cells irradiated in vivo. Moreover, F10deltagamma1 and BL6 cells were less apoptotic than F10 cells when the cells were irradiated in vivo. The p53 in F10 cells appears to be as functional as that in NIH3T3 cells because irradiation-induced expression of p53-target genes was comparable in both cells. Collectively, deltagamma1 appears to reduce irradiation-induced Mdm2 phosphorylation, which then blocks irradiation-stimulated p53 accumulation. Defects, such as deltagamma1, in PP2A may thus contribute to melanoma cell radioresistance.
F10是B16小鼠黑色素瘤细胞系的一个亚系，它本身又是转移性更强的BL6细胞系的亲本。BL6细胞与F10细胞的不同之处在于，编码蛋白磷酸酶2A（PP2A）的B56γ调节亚基的基因发生了改变，这导致mRNA编码该亚基的一个截短变体（δγ1）。只有当BL6细胞被移植到小鼠体内然后进行γ射线照射时，才能检测到δγ1蛋白的表达。最近，含有B56γ亚基的PP2A全酶已被证明可使Mdm2去磷酸化，Mdm2是p53的一个负调节因子。因此，我们评估了δγ1的表达是否通过干扰p53的调节来影响照射诱导的Mdm2磷酸化和黑色素瘤细胞的放射抗性。蛋白质印迹分析显示，稳定表达δγ1的经照射的COS-7和NIH3T3细胞显示出明显较少的照射诱导的Mdm2磷酸化。Mdm2磷酸化降低了Mdm2将p53靶向降解的能力，这可能解释了为什么与野生型细胞不同，表达δγ1的NIH3T3细胞中p53蛋白水平不会因照射而显著升高。当F10细胞（F10δγ1）在体内照射后表达δγ1时也是如此。照射后的野生型细胞和表达δγ1的细胞中p53 mRNA水平都只是略有升高，这表明Mdm2通过转录后机制调节p53水平。在体内照射的F10δγ1细胞中，p53介导的促凋亡基因Bax的诱导也显著降低。此外，当在体内照射时，F10δγ1和BL6细胞比F10细胞凋亡更少。F10细胞中的p53似乎与NIH3T3细胞中的p53功能一样，因为照射诱导的p53靶基因的表达在两种细胞中相当。总的来说，δγ1似乎减少了照射诱导的Mdm2磷酸化，进而阻止了照射刺激的p53积累。因此，PP2A中的缺陷，如δγ1，可能导致黑色素瘤细胞的放射抗性。
