The DNA-methylating drug temozolomide, which induces cell death through apoptosis, is used for the treatment of malignant glioma. Here, we investigate the mechanisms underlying the ability of temozolomide to induce senescence in glioblastoma cells. Temozolomide-induced senescence was triggered by the specific DNA lesion O 6methylguanine (O 6 MeG) and characterized by arrest of cells in the G 2 -M phase. Inhibitor experiments revealed that temozolomide-induced senescence was initiated by damage recognition through the MRN complex, activation of the ATR/CHK1 axis of the DNA damage response pathway, and mediated by degradation of CDC25c. Temozolomideinduced senescence required functional p53 and was dependent on sustained p21 induction. p53-deficient cells, not expressing p21, failed to induce senescence, but were still able to induce a G 2 -M arrest. p14 and p16, targets of p53, were silenced in our cell system and did not seem to play a role in temozolomide-induced senescence. In addition to p21, the NF-kB pathway was required for senescence, which was accompanied by induction of the senescence-associated secretory phenotype. Upon temozolomide exposure, we found a strong repression of the mismatch repair proteins MSH2, MSH6, and EXO1 as well as the homologous recombination protein RAD51, which was downregulated by disruption of the E2F1/DP1 complex. Repression of these repair factors was not observed in G 2 -M arrested p53deficient cells and, therefore, it seems to represent a specific trait of temozolomide-induced senescence.Significance: These findings reveal a mechanism by which the anticancer drug temozolomide induces senescence and downregulation of DNA repair pathways in glioma cells.
Interleukin-6 (IL-6) and ciliary neurotrophic factor (CNTF) are "4 -helical bundle" cytokines of the IL-6 type family of neuropoietic and hematopoietic cytokines. IL-6 signals by induction of a gp130 homodimer (e.g. IL-6), whereas CNTF and leukemia inhibitory factor (LIF) signal via a heterodimer of gp130 and LIF receptor (LIFR). Despite binding to the same receptor component (gp130) and a similar protein structure, IL-6 and CNTF share only 6% sequence identity. Using molecular modeling we defined a putative LIFR binding epitope on CNTF that consists of three distinct regions (C-terminal A-helix/N-terminal AB loop, BC loop, C-terminal CDloop/N-terminal D-helix).A corresponding gp130-binding site on IL-6 was exchanged with this epitope. The resulting IL-6/CNTF chimera lost the capacity to signal via gp130 on cells without LIFR, but acquired the ability to signal via the gp130/LIFR heterodimer and STAT3 on responsive cells. Besides identifying a specific LIFR binding epitope on CNTF, our results suggest that receptor recognition sites of cytokines are organized as modules that are exchangeable even between cytokines with limited sequence homology.
Cells respond to genotoxic stress with the induction of DNA damage defence functions. Aimed at identifying novel players in this response, we analysed the genotoxic stress-induced expression of DNA repair genes in mouse fibroblasts proficient and deficient for c-Fos or c-Jun. The experiments revealed a clear up-regulation of the three prime exonuclease I (trex1) mRNA following ultraviolet (UV) light treatment. This occurred in the wild-type but not c-fos and c-jun null cells, indicating the involvement of AP-1 in trex1 induction. Trex1 up-regulation was also observed in human cells and was found on promoter, RNA and protein level. Apart from UV light, TREX1 is induced by other DNA damaging agents such as benzo(a)pyrene and hydrogen peroxide. The mouse and human trex1 promoter harbours an AP-1 binding site that is recognized by c-Fos and c-Jun, and its mutational inactivation abrogated trex1 induction. Upon genotoxic stress, TREX1 is not only up-regulated but also translocated into the nucleus. Cells deficient in TREX1 show reduced recovery from the UV and benzo(a)pyrene-induced replication inhibition and increased sensitivity towards the genotoxins compared to the isogenic control. The data revealed trex1 as a novel DNA damage-inducible repair gene that plays a protective role in the genotoxic stress response.
Although regulated ectodomain shedding affects a large panel of structurally and functionally unrelated proteins, little is known about the mechanisms controlling this process. Despite a lack of sequence similarities around cleavage sites, most proteins are shed in response to the stimulation of protein kinase C by phorbol esters. The signal-transducing receptor subunit gp130 is not a substrate of the regulated shedding machinery. We generated several chimaeric proteins of gp130 and the proteins tumour necrosis factor alpha (TNF-alpha), transforming growth factor alpha (TGF-alpha) and interleukin 6 receptor (IL-6R), which are known to be subject to shedding. By exchanging small peptide sequences of gp130 for cleavage-site peptides of TNF-alpha, TGF-alpha and IL-6R we showed that these short sequences conferred susceptibility to spontaneous and phorbol-ester-induced shedding of gp130. Importantly, these chimaeric gp130 proteins were functional, as shown by the phosphorylation of gp130 and the activation of signal transduction and activators of transcription 3 ('STAT3') on stimulation with cytokine. To investigate minimal requirements for shedding, truncated cleavage-site peptides of IL-6R were inserted into gp130. The resulting chimaeras were susceptible to shedding and showed the same cleavage pattern as observed in the chimaeras containing the complete IL-6R cleavage site. Surprisingly, we could also generate cleavable chimaeras by exchanging the juxtamembrane sequence of gp130 for the corresponding region of leukaemia inhibitory factor ('LIF') receptor, a protein that like gp130 is not subject to regulated or spontaneous shedding. Thus it seems that there is no minimal consensus shedding sequence. We speculate that structural changes allow the access of the protease to a membrane-proximal region, leading to shedding of the protein.
Cells deficient in c-Fos are hypersensitive to ultraviolet (UV-C) light. Here we demonstrate that mouse embryonic fibroblasts lacking c-Fos (fos−/−) are defective in the repair of UV-C induced DNA lesions. They show a decreased rate of sealing of repair-mediated DNA strand breaks and are unable to remove cyclobutane pyrimidine dimers from DNA. A search for genes responsible for the DNA repair defect revealed that upon UV-C treatment the level of xpf and xpg mRNA declined but, in contrast to the wild type (wt), did not recover in fos−/− cells. The observed decline in xpf and xpg mRNA is due to impaired re-synthesis, as shown by experiments using actinomycin D. Block of xpf transcription resulted in a lack of XPF protein after irradiation of fos−/− cells, whereas the XPF level normalized quickly in the wt. Although the xpg mRNA level was reduced, the amount of XPG protein was not altered in c-Fos-deficient cells after UV-C, due to higher stability of the XPG protein. The data suggest a new role for c-Fos in cells exposed to genotoxic stress. Being part of the transcription factor AP-1, c-Fos stimulates NER via the upregulation of xpf and thus plays a central role in the recovery of cells from UV light induced DNA damage.
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