Research Paper Volume 4, Issue 12 pp 932—951
IL1- and TGFβ-Nox4 signaling, oxidative stress and DNA damage response are shared features of replicative, oncogene-induced, and drug-induced paracrine ‘Bystander senescence’
Figure 5. TGFβ- and IL1-dependent expression of Nox4 in bystander senescent cells. (A) Nox4 mRNA levels quantified by real time qRT-PCR in BJ cells treated with senescent medium from drug-induced (DSM), oncogene-induced (OSM) or replicative (RSM) senescent BJ cells for 20 days or (B) treated with recombinant TGFβ1 (1μM) for 4 days. BJ cells treated with medium from non-senescent BJ cells (CM) or non-treated cells (ctrl) were used as a control. The mRNA values represent average of two independent experiments and are shown as a fold induction relative to control BJ cells; error bars represent standard error. β-actin was used as a reference gene. (C) Detection of ROS production by 2',7'-dichlorofluorescein (DCF) staining in BJ cells in presence or absence of recombinant TGFβ, protein (4 days). (D) Nox4 mRNA levels quantified by real time qRT-PCR in BJ cells treated with replicative senescent cell medium (RSM) in presence or absence of TGFβ receptor inhibitor (TBRi; 10 μM) or IL1 receptor antagonist (IL1R ant.; 25 μM). The mRNA values represent average of two independent experiments and are shown as a fold induction relative to RSM BJ cells; error bars represent standard error. β-actin was used as a reference gene. (E) Detection of cytokines in medium of different bystander senescent cells 20 days after treatment using FACS beads assay. Fresh medium was added 24 hours before harvest to allow measurement of cytokines produced by bystander cells. The values are shown as a fold induction relative to BJ cells treated with medium from non-senescent BJ cells (CM). (F) Schematic representation of the IL1- and TGFβ-dependent induction of DNA damage and secondary (bystander) senescence common to three forms of primary (parental) senescence: senescence-associated secretome (SASP), especially IL1β and TGFβ, produced by three forms of senescent cells is able to induce DNA damage and bystander senescence in neighboring cells. Induction of secondary SASP in bystander cells indicates a possibility to spread DNA damage and senescence in surrounding tissue.