The interaction of ataxia-telangiectasia mutated (ATM) and the Mre11/Rad50/Nbs1 (MRN) complex is critical for the response of cells to DNA double-strand breaks; however, little is known of the role of these proteins in response to DNA replication stress. Here, we report a mutant allele of MRE11 found in a colon cancer cell line that sensitizes cells to agents causing replication fork stress. The mutant Mre11 weakly interacts with Rad50 relative to wild type and shows little affinity for Nbs1. The mutant protein lacks 3-5 exonuclease activity as a result of loss of part of the conserved nuclease domain; however, it retains binding affinity for single-stranded DNA (ssDNA), double-stranded DNA with a 3 singlestrand overhang, and fork-like structures containing ssDNA regions. In cells, the mutant protein shows a time-and dose-dependent accumulation in chromatin after thymidine treatment that corresponds with increased recruitment and hyperphosphorylation of replication protein A. ATM autophosphorylation, Mre11 foci, and thymidine-induced homologous recombination are suppressed in cells expressing the mutant allele. Together, our results suggest that the mutant Mre11 suppresses the cellular response to replication stress by binding to ssDNA regions at disrupted forks and impeding replication restart in a dominant negative manner. INTRODUCTIONThe MRN complex, consisting of Mre11, Rad50 and NBS1, has diverse functions in DNA damage recognition (Petrini and Stracker, 2003), DNA replication (Costanzo et al., 2001), cell cycle checkpoint activation (Grenon et al., 2001), nonhomolgous end joining (Paull and Gellert, 2000), and telomere maintenance (Wu et al., 2007). The Mre11 complex binds DNA double-strand breaks (DSBs) soon after they are formed implicating it in DNA damage detection . Furthermore, the complex can tether linear duplex molecules (de Jager et al., 2001), and it is able to bridge broken DNA ends or sister chromatids (van den Bosch et al., 2003). Mre11 has 3Ј-5Ј exonuclease activity and endonuclease activity (Paull and Gellert, 1999), suggesting a role in the processing of DNA ends into forms that can be recognized by cell cycle checkpoint and DNA repair proteins (Paull and Gellert, 1999;Lee and Paull, 2005;Jazayeri et al., 2006). However, precise cellular roles of the Mre11 complex have been difficult to establish, because null mutations of all components of the complex are lethal to vertebrate cells (Luo et al., 1999;Yamaguchi-Iwai et al., 1999;Zhu et al., 2001).There are several lines of evidence implicating the MRN complex in DNA replication. The complex associates with chromatin and colocalizes with proliferating cell nuclear antigen (PCNA) throughout S phase (Maser et al., 2001). In addition, chromatin loading of Mre11 is enhanced by fork stalling, suggesting that the complex is loaded at the replication fork (Mirzoeva and Petrini, 2003). Depletion of Mre11 from DT40 or Xenopus leads to increased chromosomal breaks and accumulation of DSBs during DNA replication (Yamaguchi-Iwai et al., 1999;Costanzo et...

Interleukin 1 (IL-1)-mediated gene regulation is dependent on cell-matrix interactions. Both IL-1-activated pathways, nuclear factor kappaB (NF-kappaB) and the stress-activated protein kinase (SAPK), can be regulated by cell adhesion and changes in the cytoskeleton, suggesting that cell-matrix effects on IL-1 responses are initiated in part though effects on signal transduction. Here we show that IL-1-induced transient alterations in cell shape and in the cytoskeleton in fibronectin attached cells are correlated with effects on peak activity of NF-kappaB and SAPK. Cells on fibronectin showed a 1.5-2-fold enhancement in IL-1-induced NF-kappaB activity compared with levels in cells on poly(l-lysine) or bare tissue culture plates. The effect was increased with increasing concentrations of fibronectin and was most prominent at lower concentrations of IL-1. In contrast, fibronectin attachment caused an approx. 50% decrease in the IL-1 activation of SAPK, eliminating the peak activity after 15 min of stimulation with IL-1. IL-1-induced NF-kappaB activity showed a successive, substratum-independent increase during 4 h of attachment and spreading, whereas the inhibitory effect of fibronectin on the SAPK pathway was induced at the initial stages of attachment. Further, the addition of a peptide containing the motif RGD resulted in a 40% decrease in NF-kappaB activity in cells on fibronectin, largely accounted for by an effect on the p50/p65 heterodimer. Similarly, blocking of integrin aggregation by RGD-containing peptide resulted in a total abrogation of the fibronectin effect on IL-1-induced SAPK activity. The results demonstrate disparate effects of cell adhesion on the activation by IL-1 of the NF-kappaB and SAPK pathways. Thus fibronectin attachment causes an up-regulation of NF-kappaB activity in the presence of IL-1, whereas in contrast it results in a pronounced decrease in IL-1-induced SAPK activity.

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We recently demonstrated that a fragment of human fibrinogen, fibrinogen E fragment, inhibits the migration and differentiation of human endothelial cells in vitro. Here we show that it exerts similar effects on murine endothelial cells in vitro, and selectively disrupts tumour endothelium in vivo, causing widespread intravascular thrombosis and retarding the growth of CT26 tumours in a syngeneic murine model.