Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Proc Natl Acad Sci U S A. 2011 Jan 4;108(1):391-6. doi: 10.1073/pnas.1016962108. Epub 2010 Dec 20.
Discodermolide is a microtubule-stabilizing agent that induces accelerated cell senescence. A discodermolide-resistant cell line, AD32, was generated from the human lung cancer cell line A549. We hypothesize that the major resistance mechanism in these cells is escape from accelerated senescence. AD32 cells have decreased levels of 4E-BP1 mRNA and protein, relative to the parental discodermolide-sensitive A549 cells. Lentiviral-mediated re-expression of wild-type 4E-BP1 in AD32 cells increased the proliferation rate and reverted resistance to discodermolide via restoration of discodermolide-induced accelerated senescence. Consistent with this, cell growth and response to discodermolide was confirmed in vivo using tumor xenograft models. Furthermore, reintroduction of a nonphosphorylatable mutant (Thr-37/46 Ala) of 4E-BP1 was able to partially restore sensitivity and enhance proliferation in AD32 cells, suggesting that these effects are independent of phosphorylation by mTORC1. Microarray profiling of AD32-resistant cells versus sensitive A549 cells, and subsequent unbiased gene ontology analysis, identified molecular pathways and functional groupings of differentially expressed mRNAs implicated in overcoming discodermolide-induced senescence. The most statistically significant classes of differentially expressed genes included p53 signaling, G2/M checkpoint regulation, and genes involved in the role of BRCA1 in the DNA damage response. Consistent with this, p53 protein expression was up-regulated and had increased nuclear localization in AD32 cells relative to parental A549 cells. Furthermore, the stability of p53 was enhanced in AD32 cells. Our studies propose a role for 4E-BP1 as a regulator of discodermolide-induced accelerated senescence.
碟内酯是一种微管稳定剂，能诱导细胞加速衰老。我们从人肺癌细胞系 A549 中生成了一种碟内酯耐药细胞系 AD32。我们假设这些细胞的主要耐药机制是逃避加速衰老。与亲本碟内酯敏感的 A549 细胞相比，AD32 细胞中 4E-BP1 mRNA 和蛋白水平降低。通过慢病毒介导的野生型 4E-BP1 在 AD32 细胞中的重新表达，增加了增殖率，并通过恢复碟内酯诱导的加速衰老，使 AD32 细胞重新对碟内酯敏感。与此一致，我们在肿瘤异种移植模型中证实了细胞生长和对碟内酯的反应。此外，重新引入非磷酸化突变体（Thr-37/46 Ala）的 4E-BP1 能够部分恢复 AD32 细胞的敏感性并增强其增殖能力，表明这些作用独立于 mTORC1 的磷酸化。AD32 耐药细胞与敏感的 A549 细胞的微阵列分析，以及随后的无偏基因本体分析，鉴定了克服碟内酯诱导衰老的差异表达 mRNA 涉及的分子途径和功能分组。差异表达基因中最显著的统计学类别包括 p53 信号、G2/M 检查点调节以及 BRCA1 在 DNA 损伤反应中的作用相关基因。与此一致，与亲本 A549 细胞相比，AD32 细胞中 p53 蛋白表达上调，核定位增加。此外，AD32 细胞中 p53 的稳定性增强。我们的研究提出了 4E-BP1 作为碟内酯诱导加速衰老的调节剂的作用。
