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Lynch syndrome is an inherited disease leading to the development predominantly of colorectal cancer (CRC). The crucial cause is malfunction of DNA mismatch repair that is characterized by high level of microsatellite instability; however, new knowledge of two MSI modes (types A and B) suggests a more complex molecular basis of this syndrome. To investigate, whether the extensive alterations in individual MSI markers (type B) can indicate the potential deficiency of DNA double-strand break (DSB) repair in Lynch-syndrome-related tumours, we evaluated the MSI type and alterations in the MRE11 and RAD50 repeats that are associated with the reduced protein expression and functional impairment of the MRE11-RAD50-NBS1 (MRN) complex. Of 27 CRCs, 21 samples manifested type B in at least one MSI+ marker. From type B tumours, the genetic alterations were identified in 16 (76%) samples; seven, one and eight cases manifested mutations in MRE11, RAD50 and both genes, respectively. However, predominantly biallelic MRE11 alterations with simultaneously developed RAD50 mutations impaired the protein expressions with different intensity and location in tumour. Of six tumours presenting changes

Background and Aims The objective of this study was to analyse the role of nitric oxide (NO) in relation to reactive oxygen species (ROS) production and root growth inhibition in the early response of barley root tip to cadmium. Methods We used cell-permeable NO-binding dye DAF-2DA for the NO localization and cellimpermeable dye DAR-4M for the quantification of NO in barley root apex. Results A considerable increase in NO production was detected in the transition zone of root tips already 30 min after the immersion of barley roots into 15 μM Cd. This Cd-induced NO generation further increased during the recovery period and reached its maximum level 2 h after the short-term treatment. At this stage an enhanced Cd-induced NO generation was observed in the meristematic, transition and in the beginning of elongation zone in comparison with control roots. Inhibitor of NO synthase activity markedly reduced root growth in control seedlings and had a synergistic growth inhibitory effect with Cd. On the contrary, this Cdinduced root growth inhibition was markedly alleviated by the NO donor. Exposure of roots to Cd markedly increased the production of ROS in the proximal elongation zone of root. While NO did not affect ROS generation, inhibition of its synthesis slightly decreased ROS generation in both control and Cd-treated roots. Conclusions Our results revealed that enhanced NO production is a very early response of barley root to Cd stress and it is involved in the regulation of root growth mitigating Cd-induced root growth inhibition.

Morphogenic responses, ROS generation and changes in enzymes activity induced by the short-term exposure of barley roots to Cd were very similar to those of triiodobenzoic acid (TIBA) treatment. Both Cd and TIBA markedly increased the amount of IAA in root apex. In addition, the treatment of roots with TIBA before Cd treatment had an additive effect on morphogenic responses, changes in enzymes activity and ROS generation. In turn, the application of auxin efflux/influx inhibitor 1-naphthoxyacetic acid (1-NOA) before Cd treatment significantly reduced the Cd-induced ROS generation, root growth inhibition and enzymes activity in spite of the elevated level of IAA in the root apex. The auxin signalling inhibitor p-chlorophenoxyisobutyric acid (PCIB) nearly fully eliminated some of these Cd-induced responses. These results indicated that the short-term Cd treatment-induced ROS generation, morphogenic responses and activity of some enzymes can be attributed to the disturbance of IAA homeostasis in root apex. While auxin efflux inhibitor TIBA evoked similar response to Cd, auxin efflux/influx inhibitor 1-NOA attenuated and auxin signalling inhibitor PCIB blocked these Cd-induced responses suggesting that apart from the elevated level of IAA in root apex its enter and accumulation inside of until closely undetermined cells or tissues is a prerequisite for the induction of characteristic root responses to short-term Cd exposure.

Lynch syndrome is an inherited disease resulting predominantly in colorectal cancer (CRC). The crucial cause is DNA mismatch repair (MMR) malfunction that is associated mostly with MLH1 or MSH2 germline mutations. A significant hallmark of repair defects is a high level of instability in microsatellites (MSI-H). In many sporadic unstable CRCs, the MLH1 gene is inactivated by promoter hypermethylation in addition to extensive promoter methylation in many tumor-suppressor genes known as CpG island methylation phenotype (CIMP). To investigate the possible role of epigenetic alterations in causing MMR deficiency and thereby Lynch syndrome, we evaluated the MLH1 specific and global hypermethylation in hereditary CRCs. Of 22 Lynch-syndrome-related CRCs, 18 (81.8%) demonstrated various levels of DNA methylation; of these, 14 (63.6%) and 4 (18.2%) were methylated in distal and both distal and proximal regions of the MLH1 promoter, respectively. However, only 7/18 (38.9%) of results were confirmed by bisulfite sequencing. Similar methylation patterns in tumors and frequently in matched normal DNA were found in twelve and four patients with MLH1 and MSH2 alterations documented by the absence of protein or presence of germline mutation, respectively. Moreover, the same results were observed in five stable CRCs. None of 22 Lynch-syndrome-related tumors presented CIMP in contrast to 3/10 (30%) stable carcinomas. The rather randomly distributed weak methylation patterns in hereditary CRCs indicate that epigenetic events are redundant in Lynch-syndrome etiology, in contrast to the widespread DNA methylation in sporadic unstable CRCs. These methylation-profile differences can lead to more effective molecular diagnosis of Lynch syndrome.

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Exposure of roots to low Cd concentration induced morphogenic responses including the inhibition of root growth and the radial swelling of root tip. High Cd concentrations within a few minutes caused a robust induction of superoxide generation leading to the cell death and root growth arrest. This toxic superoxide generation blocked the development of low Cd concentration-activated morphogenic responses. While the morphogenic responses of roots to low Cd concentration are induced very rapidly and probably due to the interaction of Cd with the apoplast of root tissue, high Cd concentration-induced superoxide production required the entry of Cd into the symplast. Auxin signaling is involved in the activation of Cd-induced morphogenic defence responses but not in the Cd-induced toxic superoxide generation. These results suggest that oxidative stress is not a primary cause for the Cd-induced morphogenic responses such as growth reduction and radial cell expansion in barley root tips.

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