Carrol Terleth

Induction of ATF3 by ionizing radiation is mediated via a signaling pathway that includes ATM, Nibrin1, stress-induced MAPkinases and ATF-2

Exposure of human cells to genotoxic agents induces various signaling pathways involved in the execution of stress- and DNA-damage responses. Inappropriate functioning of the DNA-damage response to ionizing radiation (IR) is associated with the human diseases ataxia-telangiectasia (A-T) and Nijmegen Breakage syndrome (NBS). Here, we show that IR efficiently induces Jun/ATF transcription factor activity in normal human diploid fibroblasts, but not in fibroblasts derived from A-T and NBS patients. IR was found to enhance the expression of c-Jun and, in particular, ATF3, but, in contrast to various other stress stimuli, did not induce the expression of c-Fos. Using specific inhibitors, we found that the ATM- and Nibrin1-dependent activation of ATF3 does neither require p53 nor reactive oxygen species, but is dependent on the p38 and JNK MAPkinases. Via these kinases, IR activates ATF-2, one of the transcription factors acting on the atf3 promoter. The activation of ATF-2 by IR resembles ATF-2 activation by certain growth factors, since IR mainly induced the second step of ATF-2 phosphorylation via the stress-inducible MAPkinases, phosphorylation of Thr69. As IR does not enhance ATF-2 phosphorylation in ATM and Nibrin1-deficient cells, both ATF-2 and ATF3 seem to play an important role in the protective response of human cells to IR.

DNA damage in transcribed genes induces apoptosis via the JNK pathway and the JNK-phosphatase MKP-1

The nucleotide excision repair (NER) system consists of two subpathways, global genome repair (GGR) and transcription-coupled repair (TCR), which exhibit distinct functions in the cellular response to genotoxic stress. Defects in TCR result in prolonged UV light-induced stalling of RNA polymerase II and hypersensitivity to apoptosis induced by UV and certain chemotherapeutic drugs. Here, we show that low doses of UV trigger delayed activation of the stress-induced MAPkinase JNK and its proapoptotic targets c-Jun and ATF-3 in TCR-deficient primary human fibroblasts from Xeroderma Pigmentosum (XP) and Cockayne syndrome (CS) patients. This delayed activation of the JNK pathway is not observed in GGR-deficient TCR-proficient XP cells, is independent of functional p53, and is established through repression of the JNK-phosphatase MKP-1 rather than by activation of the JNK kinases MKK4 and 7. Enzymatic reversal of UV-induced cyclobutane pyrimidine dimers (CPDs) by CPD photolyase abrogated JNK activation, MKP-1 repression, and apoptosis in TCR-deficient XPA cells. Ectopic expression of MKP-1 inhibited DNA-damage-induced JNK activity and apoptosis. These results identify both MKP-1 and JNK as sensors and downstream effectors of persistent DNA damage in transcribed genes and suggest a link between the JNK pathway and UV-induced stalling of RNApol II.

The novel MMS-inducible gene Mif1/KIAA0025 is a target of the unfolded protein response pathway

In a search for genes induced by DNA‐damaging agents, we identified two genes that are activated by methyl methanesulfonate (MMS). Expression of both genes is regulated after endoplasmic reticulum (ER) stress via the unfolded protein response (UPR) pathway. The first gene of those identified is the molecular chaperone BiP/GRP78. The second gene, Mif1, is identical to the anonymous cDNA KIAA0025. Treatment with the glycosylation inhibitor tunicamycin both enhances the synthesis of Mif1 mRNA and protein. The Mif1 5′ flanking region contains a functional ER stress‐responsive element which is sufficient for induction by tunicamycin. MMS, on the other hand, activates Mif1 via an UPR‐independent pathway. The gene encodes a 52 kDa protein with homology to the human DNA repair protein HHR23A and contains an ubiquitin‐like domain. Overexpressed Mif1 protein is localized in the ER.

A role for Rad23 proteins in 26S proteasome-dependent protein degradation?

Treatment of cells with genotoxic agents affects protein degradation in both positive and negative ways. Exposure of S. cerevisiae to the alkylating agent MMS resulted in activation of genes that are involved in ubiquitin- and 26S proteasome-dependent protein degradation. This process partially overlaps with the activation of the ER-associated protein degradation pathway. The DNA repair protein Rad23p and its mammalian homologues have been shown to inhibit degradation of specific substrates in response to DNA damage. Particularly the recently identified inhibition of degradation by mouse Rad23 protein (mHR23) of the associated nucleotide excision repair protein XPC was shown to stimulate DNA repair.Recently, it was shown that Rad23p and the mouse homologue mHR23B also associate with Png1p, a deglycosylation enzyme. Png1p-mediated deglycosylation plays a role in ER-associated protein degradation after accumulation of malfolded proteins in the endoplasmic reticulum. Thus, if stabilization of proteins that are associated with the C-terminus of Rad23p is a general phenomenon, then Rad23 might be implicated in the stimulation of ER-associated protein degradation as well. Interestingly, the recently identified HHR23-like protein Mif1 is also thought to play a role in ER-associated protein degradation. The MIF1 gene is strongly activated in response to ER-stress. Mif1 contains a ubiquitin-like domain which is most probably involved in binding to S5a, a subunit of the 19S regulatory complex of the 26S proteasome. On the basis of its localization in the ER-membrane, it is hypothesized that Mif1 could play a role in the translocation of the 26S proteasome towards the ER-membrane, thereby enhancing ER-associated protein degradation.

Mif1: A Missing Link between the Unfolded Protein Response Pathway and ER-Associated Protein Degradation?

Eukaryotic cells have three different mechanisms to deal with the accumulation of unfolded proteins in the endoplasmic reticulum: (1) In cells in which unfolded polypeptides accumulate, translation initiation is inhibited to prevent further accumulation of unfolded proteins. (2) Expression of proteins involved in polypeptide folding is strongly enhanced by a process called the Unfolded Protein Response (UPR). (3) Proteins missing the proper tertiary structure are degraded by the ER-Associated protein Degradation (ERAD) mechanism. Recent studies in S. cerevisiae have shown that the processes of UPR and ERAD are functionally linked to each other. Cells lacking a functional ERAD show a constitutive activation of UPR. In addition, many of the components of ERAD are under the direct transcriptional control of UPR. Finally, while neither UPR nor ERAD are essential for cell viability, deletion of both pathways results in severe growth impairment. UPR and ERAD are conserved between yeast and mammalian cells. One of the components of mammalian UPR is the protease presenilin-1. Mutations in the gene for presenilin-1 cause early-onset familial Alzheimer disease. Interestingly, inhibition of proteolysis by the ubiquitin-26S proteasome system has also been described for Alzheimer s disease. This suggests a link between UPR and ERAD in mammalian cells. The recently identified gene Mif1 is a possible candidate to form a direct link between UPR and ERAD in mammalian cells. The Mif1 gene is under the direct control of UPR. Mif1 is a trans-ER-membrane protein, with both the N- and the C-termini facing the cytoplasmic side of the ER membrane. It contains an N-terminal ubiquitin-like domain. It is anticipated that Mif1 may associate through its ubiquitin-like domain with the 26S proteasome, in this way connecting the protein degradation machinery to the ER membrane and resulting in an efficient ERAD.

Induction of the SAPK activator MIG‐6 by the alkylating agent methyl methanesulfonate

The alkylating agent methylmethanesulfonate (MMS) activates the c‐jun N‐terminal kinase (JNK)/stress‐activated protein kinase (SAPK) and the p38 mitogen‐activated protein kinase (p38MAPK) pathways via different mechanisms of action. Activation of p38MAPK by MMS involves the pp125 focal adhesion kinase–related tyrosine kinase RAFTK and the MAPK kinase 3. The way in which MMS can activate JNK/SAPK has not been elucidated. Here we describe the identification by differential display of human mitogen‐activated gene‐6 (MIG‐6) as a novel MMS‐inducible gene. Induction of MIG‐6 by MMS was found in human diploid skin fibroblasts and in simian virus 40–transformed skin fibroblasts, indicating that the enhanced expression of MIG‐6 after MMS‐treatment did not require p53. The signal leading to activation of MIG‐6 appeared to be independent of DNA damage. High MIG‐6 expression was found in the liver, lung, and placenta. MIG‐6 is an adapter protein that binds to the activated form of cdc42Hs and to 14‐3‐3 proteins, thereby activating JNK/SAPKs. Our results suggest that activation of JNK/SAPKs by MMS may involve the induction of MIG‐6.

Impaired DNA repair capacity in skin fibroblasts from various hereditary cancer-prone syndromes

Host-cell reactivation (HCR) of UV-C-irradiated herpes simplex virus type 1 (HSV-1) has been determined in skin fibroblasts from the following hereditary cancer-prone syndromes: aniridia (AN), dysplastic nevus syndrome (DNS), Von Hippel-Lindau syndrome (VHL), Li-Fraumeni syndrome (LFS) and a family with high incidence of breast and ovarian cancer. Cells from AN, DNS or VHL patients were found to exhibit heterogeneity in HCR. Cells from individuals belonging to an LFS family show reduced HCR in all cases where the cells were derived from persons carrying one mutated p53 allele, whereas cells derived from members with two wild-type alleles show normal HCR. LFS cells with reduced HCR also reveal reduced genome overall repair, and a slower gene-specific repair of the active adenosine deaminase (ADA) gene, but little if any repair of the inactive 754 gene. In the breast/ovarian cancer family, reduced HCR is observed in skin fibroblasts derived from both afflicted and unaffected individuals. In addition, these cells display lower survival after exposure to UV-C and exhibit higher levels of SCEs than those in normal cells. These observations indicate that various hereditary cancer-prone syndromes, carrying mutations in different tumor-suppressor genes, exhibit an unexplained impairment of the capacity to repair UV-damaged DNA.

Different regulation of p53 stability in UV-irradiated normal and DNA repair deficient human cells

The stabilization of p53 protein was studied after UV exposure of normal human skin fibroblasts and cells derived from patients suffering from xeroderma pigmentosum (XP) and trichothiodystrophy (TTD). The data show that p53 is transiently stabilized both in UV-irradiated normal and repair deficient cells. However, particularly at later times after UV irradiation, stabilization of p53 persists much longer in repair deficient XP and TTD cells than in normal cells. The stabilization of p53 was found to be dose-dependent in normal and XP cells. These results indicate that unremoved DNA damage could possibly be responsible for the induction of transient stabilization of p53.

Temperature-sensitive mutant p53 (ala143) interferes transiently with DNA-synthesis and cell-cycle progression in Saos-2 cells.

It has been demonstrated that temperature-sensitive mutant p53 (val-->ala143) inhibits cell-proliferation at the permissive temperature, albeit to a lesser extent than wild-type p53 (Zhang et al.: EMBO J 13:2535-2544, 1994). We have studied its effect on the cell-cycle by dual-parameter flow cytometry, extended pulse-labeling, and pulse-chase experiments. p53ala143 interferes in Saos-2 cells at three levels with cell-cycle progression at permissive temperatures: it caused a G1-arrest, a reduced rate of DNA synthesis during S, and a prolonged G2/M. Strikingly, all these effects are transient. Continued culturing at 32 degrees C resulted in normal cell-cycle progression. Abrogation of the G1-block occurred even in the presence of high p21Waf1 protein levels, a negative cell-cycle regulator of which the expression is induced by wild-type p53.