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.

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.

The FAS ligand (FASLG) is expressed on lymphocytes, which employ it to activated death receptors on targets 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 the ones 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 45h and next we treated them with recombinant FASLG. This almost instantaneously killed most cells. Actinomycin D and nutlin-3a strongly synergized 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 lost. 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 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.

The p53 tumor suppressor protein is an activator of transcription. Diverse stress factors lead to various sets of posttranslational modifications of p53 what results in different sets of upregulat-ed genes. We noticed that actinomycin D and nutlin-3a (A+N) synergize in inducing activating phosphorylations of p53 and upregulation of selected p53-target genes. Here we found that one of these genes is DUSP13, which codes for poorly-studied, dual-specificity phosphatase having at least two isoforms, one expressed in testis and the other in skeletal muscles. In cancer cells ex-posed to A+N, DUSP13 is expressed from an alternative promoter in intron, what results in ex-pression of isoform named TMDP-L1. The luciferase reporter tests demonstrated that this pro-moter is activated by both endogenous and ectopically expressed p53. We showed for the first time that mRNA expressed from this promoter actually produces the protein, which can be de-tected by 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 or by nutlin-3a acting alone. This isoform, fused with green fluorescent protein localizes in perinuclear region of cells. Thus, TMDP-L1 isoform may be an important element of p53-regulated stress response system.

The p53 tumor suppressor protein shows antiviral activity. The replication of viruses is also antagonized by interferons (IFNs) – cytokines, which control immunity genes by the STAT transcription factors. The best studied interferons belong to type I (e.g. IFNα) and type II (IFNγ) groups. IFNα and IFNγ both induce activating phosphorylation of STAT1 on Tyr701. Previously, we noticed that p53 activates SOCS1, which is a negative regulator of STAT1 phosphorylation. Thus, we hypothesized that p53 by activating SOCS1 expression reduces phosphorylation of STAT1 and attenuates activation of genes stimulated either by IFNα or IFNγ. To test this hypothesis, we exposed p53-proficient and p53-deficient cells to strong p53 activators, and either to IFNα or IFNγ. Subsequently, we estimated the phosphorylation status of STAT1 and the expression of genes regulated by either of these cytokines. Strong activation of p53 reduced the phosphorylation of STAT1 on Tyr701, however surprisingly it did not lead to reduced expression of most tested interferon-stimulated genes. On contrary, p53 and IFNγ synergized in the activation of CASP1, IFIT1 and IFIT3 genes. We conclude that the interactions between p53 and interferon-activated pathways are more complicated than initially expected, but the apparent cooperation between p53 and IFNγ deserves further studies.