Induction of Senescence by Doxorubicin Is Associated with Upregulated Mir-375 and Induction of Autophagy In CML Cell Line K562

Abstract 1843 Senescence is a specialized form of growth arrest that it is generally irreversible and can be induced by telomere attrition, oxidative stress, oncogene expression and DNA damage signaling. Senescent cells display a typical upregulated senescence-associated (SA)-b-galactosidase activity and novel changes in chromatin architecture, the formation of SA heterochromatic foci (SAHF). Changes in gene expression, such as upregulated p16, p53, and p21 expression and silencing of E2F target genes, have been characterized to promote the establishment of senescence. Besides, by the actions of HP1g, HMGA, and DNMT proteins to produce a repressive chromatin environment, the transcription of proliferation-associated genes can be suppressed. Therefore, senescence has been suggested to functions as a natural brake to tumor development. In this study, we first sought to establish an in vitro senescence model using doxorubicin (DOX) and paclitaxel to treat CML cell line K562. We found that 50 nM DOX induced senescence, but did not induce apoptosis. In contrast, 10 nM and 100 nM paclitaxel induced apoptosis but did not induce senescence, and lower doses of paclitaxel (1 nM and 5 nM) had no effect on the cells. p53 and p16-pRb are the two major senescence pathways. Since p53 and p16 are homozygously deleted in the K562 cells, the DOX-induced senescence in K562 cells is supposed to be established by a pathway independent of p53 and p16-pRb pathways. Indeed, the expression of the typical SA-premalignant cell markers (CDC6, Ki67, p19, p38, PU1, DNMT1, HMGA1, HP1g) did not change in the DOX-induced senescent K562 cells although the typical SA-b-galactosidase staining and SAHF were apparent. MicroRNA profiling revealed that miR-375 was upregulated in DOX-induced senescent K562 cells. Treatment with miR-375 inhibitor could rescue the proliferation ability suppressed by DOX (p < 0.05). The identification of miR-375 targets should help us to elucidate the substitution pathway that is responsible for the DOX-induced senescence in the absence of both p16 and p53 genes. With the observation that DOX treatment induced cells entering senescence but eventually lead to cell death, we also investigated if the alternative mode of cell death, autophagy, was involved. By examining the expression patterns of the 26 human autophagy-related (ATG) genes, a 12-fold increase of ATG9B at day 4 and a 20-fold increase of ATG18 at day 2 after DOX treatment were noted. Both ATG9 and ATG18 are crucial for the formation of autophagosome. Hence, in addition to the upregulation of miR-375, our results also demonstrated that senescence induced by DOX in K562 cells is associated with the initiation of autophagy. Updated results on the identification of miR-375 targets and the regulation of senescence and autophagy pathway will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.