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The FAS ligand (FASLG) is expressed on lymphocytes, which employ it to activate death receptors on target cells. Cancer cells are generally resistant to apoptosis triggered by FASLG. In this work, we found a way to circumvent this resistance by treatment with actinomycin D (ActD) and nutlin-3a (Nut3a). We selected this drug combination based on our transcriptomic data showing strong activation of proapoptotic genes, including those for receptor-mediated apoptosis, in cells exposed to actinomycin D and nutlin-3a. To test our hypothesis, we pre-exposed cancer cell lines to this drug combination for 45 h and then treated them with recombinant FASLG. This almost instantaneously killed most cells. Actinomycin D and nutlin-3a strongly cooperated in the sensitization because the effect of the drugs acting solo was not as spectacular as the drug combination, which together with FASLG killed more than 99% of cells. Based on the caspase activation pattern (caspase-8, caspase-9, caspase-10), we conclude that both extrinsic and intrinsic pro-apoptotic pathways were engaged. In engineered p53-deficient cells, this pro-apoptotic effect was completely abrogated. Therefore, the combination of ActD + Nut3a activates p53 in an extraordinary way, which overcomes the resistance of cancer cells to apoptosis triggered by FASLG. Interestingly, other combinations of drugs, e.g., etoposide + nutlin-3a, actinomycin D + RG7112, and actinomycin D + idasanutlin had a similar effect. Moreover, normal human fibroblasts are less sensitive to death induced by ActD + Nut3a + FASLG. Our findings create the opportunity to revive the abandoned attempts of cancer immunotherapy employing the recombinant FAS ligand.

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The p53 tumor suppressor protein activates various sets of genes depending on its covalent modifications, which are controlled by the nature and intensity of cellular stress. We observed that actinomycin D and nutlin-3a (A + N) collaborate in inducing activating phosphorylation of p53. Our recent transcriptomic data demonstrated that these substances strongly synergize in the upregulation of DUSP13, a gene with an unusual pattern of expression, coding for obscure phosphatase having two isoforms, one expressed in the testes and the other in skeletal muscles. In cancer cells exposed to A + N, DUSP13 is expressed from an alternative promoter in the intron, resulting in the expression of an isoform named TMDP-L1. Luciferase reporter tests demonstrated that this promoter is activated by both endogenous and ectopically expressed p53. We demonstrated for the first time that mRNA expressed from this promoter actually produces the protein, which can be detected with Western blotting, in all examined cancer cell lines with wild-type p53 exposed to A + N. In some cell lines, it is also induced by clinically relevant camptothecin, by nutlin-3a acting alone, or by a combination of actinomycin D and other antagonists of p53-MDM2 interaction-idasanutlin or RG7112. This isoform, fused with green fluorescent protein, localizes in the perinuclear region of cells.

RESUMEN

Transcriptomic analyses have revealed hundreds of p53-regulated genes; however, these studies used a limited number of cell lines and p53-activating agents. Therefore, we searched for candidate p53-target genes by employing stress factors and cell lines never before used in a high-throughput search for p53-regulated genes. We performed RNA-Seq on A549 cells exposed to camptothecin, actinomycin D, nutlin-3a, as well as a combination of actinomycin D and nutlin-3a (A + N). The latter two substances synergise upon the activation of selected p53-target genes. A similar analysis was performed on other cell lines (U-2 OS, NCI-H460, A375) exposed to A + N. To identify proteins in cell lysates or those secreted into a medium of A549 cells in control conditions or treated with A + N, we employed mass spectrometry. The expression of selected genes strongly upregulated by A + N or camptothecin was examined by RT-PCR in p53-deficient cells and their controls. We found that p53 participates in the upregulation of: ACP5, APOL3, CDH3, CIBAR2, CRABP2, CTHRC1, CTSH, FAM13C, FBXO2, FRMD8, FRZB, GAST, ICOSLG, KANK3, KCNK6, KLRG2, MAFB, MR1, NDRG4, PTAFR, RETSAT, TMEM52, TNFRSF14, TRANK1, TYSND1, WFDC2, WFDC5, WNT4 genes. Twelve of these proteins were detected in the secretome and/or proteome of treated cells. Our data generated new hypotheses concerning the functioning of p53. Many genes activated by A + N or camptothecin are also activated by interferons, indicating a noticeable overlap between transcriptional programs of p53 and these antiviral cytokines. Moreover, several identified genes code for antagonists of WNT/ß-catenin signalling pathways, which suggests new connections between these two cancer-related signalling systems. One of these antagonists is DRAXIN. Previously, we found that its gene is activated by p53. In this study, using mass spectrometry and Western blotting, we detected expression of DRAXIN in a medium of A549 cells exposed to A + N. Thus, this protein functions not only in the development of the nervous system, but it may also have a new cancer-related function.

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Immunosenescence in monocytes has been shown to be associated with several biochemical and functional changes, including development of senescence-associated secretory phenotype (SASP), which may be inhibited by klotho protein. To date, it was believed that SASP activation is associated with accumulating DNA damage. However, some literature data suggest that endoplasmic reticulum and Golgi stress pathways may be involved in SASP development. Thus, the aim of this study was to investigate the role of klotho protein in the regulation of immunosenescence-associated Golgi apparatus and ER stress response induced by bacterial antigens in monocytes. We provide evidence that initiation of immunosenescent-like phenotype in monocytes is accompanied by activation of CREB34L and TFE3 Golgi stress response and ATF6 and IRE1 endoplasmic reticulum stress response, while klotho overexpression prevents these changes. Further, these changes are followed by upregulated secretion of proinflammatory cytokines, which final modification takes place exclusively in the Golgi apparatus. In conclusion, we provide for the first time evidence of klotho involvement in the crosstalk on the line ER-Golgi, which may, in turn, affect activation of SASP. This data may be useful for a novel potential target for therapy in age-related and chronic inflammatory conditions.

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In the previous paper of our group, we have demonstrated that one of the crucial factors involved in the crosstalk between autophagy and apoptosis is klotho protein. We have shown that klotho silencing in normal human fibroblasts intensifies lipopolysaccharide (LPS)-induced p-eIF2a-mediated stress of endoplasmic reticulum and thus leads to retardation of prosurvival autophagy and induction of apoptotic cell death. In this study, we have performed a detailed step-by-step analysis of autophagy flux-related genes' expression and endoplasmic reticulum and Golgi stress related pathways in order to determine the exact mechanistic event when autophagy is inhibited in klotho-deficient cells on account of apoptosis initiation. We provide evidence that klotho-silencing in LPS-treated cells results in differential course of ER- and Golgi-mediated stress response. Further, we show that in klotho-deficient cells formation of ULK1 complex is inhibited and thus autophagy initiation is blocked on the account of apoptosis activation, while in the control cells cytoprotective autophagy is activated. Finally, in klotho-deficient cells formation of ULK1 complex is prevented by downregulated expression of Atg13. Thus, this study suggests a novel targeting pathway for efficient elimination of autophagy-deficient cells.

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