The activation of the ataxia telangiectasia mutated (ATM) and ATM/Rad3-related (ATR) kinases triggers a diverse cellular response including the initiation of DNA damage-induced cell cycle checkpoints. Mediator of DNA Damage Checkpoint protein, MDC1, and H2AX are chromatin remodeling factors required for the recruitment of DNA repair proteins to the DNA damage sites. We identified a novel mediator protein, Cep164 (KIAA1052), that interacts with both ATR and ATM. Cep164 is phosphorylated upon replication stress, ultraviolet radiation (UV), and ionizing radiation (IR). Ser186 of Cep164 is phosphorylated by ATR/ATM in vitro and in vivo. The phosphorylation of Ser186 is not affected by RPA knockdown but is severely hampered by MDC1 knockdown. siRNA-mediated silencing of Cep164 significantly reduces DNA damage-induced phosphorylation of RPA, H2AX, MDC1, CHK2, and CHK1, but not NBS1. Analyses of Cep164 knockdown cells demonstrate a critical role of Cep164 in G2/M checkpoint and nuclear divisions. These findings reveal that Cep164 is a key player in the DNA damage-activated signaling cascade.[Keywords: DNA damage signal pathways; ATR; CEP164; RPA; MDC1; CHK1; H2AX] Supplemental material is available at http://www.genesdev.org. The genome is subjected to continuous damage and repair. Free radicals generated during cellular metabolism, DNA replication errors, and exogenous carcinogens can all lead to DNA damage, which triggers multiple signal transduction pathways to slow down cell cycle progression, allowing for the repair of the damaged DNA. It has been proposed that these pathways sense DNA damage, activate cell cycle checkpoints, and recruit repair proteins to the damaged DNA Stucki and Jackson 2006). ATM (ataxia telangiectasia mutated) and ATR (ATM-and Rad3-related gene) are two evolutionarily conserved phosphatidylinositol kinase-related proteins that play critical roles in checkpoint activation (Shiloh 2003). Mutations in ATM lead to AT (ataxia telangiectasia) disorder (Savitsky et al. 1995), which is characterized by neuronal degeneration and cancer predisposition, while reduced ATR expression leads to Seckel syndrome (O'Driscoll et al. 2003), which is characterized by retarded development. Knockout of ATM and ATR in mice, respectively, demonstrates that ATR, but not ATM, is essential for embryogenesis and cell survival (Barlow et al. 1996;Brown and Baltimore 2000;Cortez et al. 2001).ATM is activated by DNA double-strand break (DSB)-causing agents including ionizing radiation (IR), while ATR is activated by ultraviolet radiation (UV) as well as replication block (Abraham 2001). Interestingly, IR also activates ATR in an ATM-and Mre11-NBS1-Rad50 (MRN)-dependent manner (Jazayeri et al. 2005;Zhong et al. 2005;Myers and Cortez 2006). ATM and ATR share many substrates; i.e., Ser15 of p53 (Siliciano et al. 1997), and Ser1423 and Ser1524 of BRCA1 (Fabbro et al. 2004). In addition, phosphorylation of Ser139 of H2AX by ATM/ATR is well documented (Burma et al. 2001). Mediator of DNA damage checkpoint protein 1, MDC1, is a c...

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SummaryMutations in the lamin A/C gene cause the rare genetic disorder Hutchinson-Gilford progeria syndrome (HGPS). The prevalent mutation results in the production of a mutant lamin A protein with an internal 50 amino acid deletion which causes a cellular aging phenotype characterized by growth defects, limited replicative lifespan, and nuclear membrane abnormalities. However, the relevance of these findings to normal human aging is unclear. In this study, we demonstrate that increased levels of wild-type lamin A in normal human cells result in decreased replicative lifespan and nuclear membrane abnormalities that lead to apoptotic cell death and senescence in a manner that is strongly reminiscent of the phenotype shown by HGPS cells. In contrast to the accelerated aging defects observed in HGPS cells, the progeroid phenotype resulting from increased expression of wild-type lamin A can be rescued by overexpression of ZMPSTE24, the metalloproteinase responsible for the removal of the farnesylated carboxyl terminal region of lamin A. Furthermore, farnesyltransferase inhibitors also serve to reverse the progeroid phenotype resulting from increased lamin A expression. Significantly, cells expressing elevated levels of lamin A display abnormal lamin A localization and similar alterations in the nuclear distribution of lamin A are also observed in cells from old-age individuals. These data demonstrate that the metabolism of wild-type lamin A is delicately poised and even in the absence of diseaselinked mutations small perturbations in this system are sufficient to cause prominent nuclear defects and result in a progeroid phenotype.

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We report here that earthworm can be used as an in vivo system to prescreen antimitotic compounds. The known antimitotic compound colchicine and the aqueous extract of Acorus calamus L. rhizome were used to examine this system. The antimitotic activity of colchicine and the rhizome extract was confirmed by methods with Allium cepa root tip, earthworm regeneration, and MTT (3-(4,5-dimethyl thiazolyl-2-yl)-2,5-diphenyltetrazolium bromide). An earthworm regeneration assay examined the regeneration ability of tissues from amputated regions of the earthworm. All 3 assays showed that cell division is inhibited with colchicine and the aqueous extract of A. calamus rhizome. Histology studies with amputated earthworms confirmed that the development of the blastema was observed only in control worms. Immunohistochemistry analysis with proliferation cell nuclear antigen and phospho serine 10 histone H3 antibodies showed that amputated worms treated with colchicine or the aqueous extract of A. calamus did not have active cell division. MTT assay with the MCF-7 cell line (human breast carcinoma) further confirmed that the aqueous extract of rhizome affected cell proliferation. Our results suggest that the earthworm could be used to prescreen the antimitotic potential of plant extracts or other unknown compounds. It is simple, reproducible, and cost-efficient compared to animal cell line-based methods.

Lamin A is an intermediate filament protein found in most of the differentiated vertebrate cells but absent in stem cells. It shapes the skeletal frame structure beneath the inner nuclear membrane of the cell nucleus. As there are few studies of the expression of lamin A in invertebrates, in the present work, we have analyzed the sequence, immunochemical conservation and expression pattern of lamin A protein in the earthworm Eudrilus eugeniae, a model organism for tissue regeneration. The expression of lamin A has been confirmed in E. eugeniae by immunoblot. Its localization in the nuclear membrane has been observed by immunohistochemistry using two different rabbit anti-sera raised against human lamin A peptides, which are located at the C-terminus of the lamin A protein. These two antibodies detected 70 kDa lamin A protein in mice and a single 65 kDa protein in the earthworm. The Oct-4 positive undifferentiated blastemal tissues of regenerating earthworm do not express lamin A, while the Oct-4 negative differentiated cells express lamin A. This pattern was also confirmed in the earthworm prostate gland. The present study is the first evidence for the immunochemical identification of lamin A and Oct-4 in the earthworm. Along with the partial sequence obtained from the earthworm genome, the present results suggest that lamin A protein and its expression pattern is conserved from the earthworm to humans.

TCTP (Translationally Controlled Tumour Protein) is a multifunctional protein that plays a role in the development, immune system, tumour reversion, and maintenance of stem cells. The mRNA of the Tpt1 gene is over-expressed during liver regeneration. But, the function of the protein in regeneration is not known. To study the role of the protein in regeneration, the earthworm Eudrilus eugeniae was chosen. First, the full length cDNA of the Tpt1 gene was sequenced. The size of the cDNA is 504 bp and the protein has 167 amino acids. The highest level of TCTP expression was documented in the worm after three days of regeneration. The protein was found to be expressed specifically in the epithelial layer of the skin. During regeneration, the protein expression was found to be the highest at the tip of blastema. The pharmacological suppression of TCTP using nutlin-3 and TCTP RNAi experiments resulted in the failure of the regeneration process. The suppression of TCTP caused the arrest of proliferation in posterior amputated worms. The severe cell death was documented in the amputated region of nutlin-3 injected worm. The silencing of TCTP has blocked the modification of clitellar segments. The experiments confirm that TCTP has major functions in the upstream signalling of cell proliferation in the early regeneration process in E. eugeniae.