[Colorectal cancer susceptibility genetic variants in tumor free and colorectal carcinoma bearing Hungarian population. Individual predisposition to cancer]. / Vastagbélrák-hajlamosító DNS-szekvencia-variációk tumormentes és vastagbél-daganatos populációban Magyarországon. Az egyedi daganathajlam becslése.

Genome-wide association studies (GWAS) using population-based designs have identified many genetic loci, at which common variants can influence the risk of developing the sporadic colon cancers. These are single nucleotide polymorphisms (SNPs) located on different chromosomes, close to genes involved in cancer developing process, and the SNPs modify their functions, and as a consequence the cancer risk is increased. Our aim was to provide frequency distributions data of variable (risk) allele of six independent SNPs in patients with colorectal cancers and in control Hungarian population, predicting the increased risk effect of sequence variant of SNPs. We also investigated the frequency distribution of tumor localization between right or left half of large bowel as well as the RAS mutation status. 47 non-tumorous patients and 47 patients with colorectal cancer were given oral mucosa cells or blood samples for SNP analysis. After DNA isolation, an LC480 (Roche) type PCR instrument, asymmetric LATE PCR method and melting point analysis were used for detection of sequence variations, by the assistance of two SNP specific primers, unlabeled specific probe and intercalating fluorescent dye. Genomic frequency distribution of variable alleles of SNPs predisposed to tumor development have been investigated in colorectal cancer carrier patients and the results have been compared with the same allele frequency distribution data obtained from the non-tumorous control patients and from CEU population stored in SNPnexus data base. The homozygous risk alleles of SNPs showed a 1.5-2.3-time increase in colorectal cancer carrier patients then in control and CEU patients, but the heterozygous risk allele distribution was identical in tumorous and control population. The frequency distribution of homozygous risk alleles of six SNPs was also investigated in the same time and some patients. Among 47 patients with colorectal cancer, in 3 patients carrying 3 SNPs with homozygous risk alleles, in additional 5 tumor samples two and 24 samples contain only one SNP's homozygous risk alleles, and in 15 patients, SNPs with homozygous risk alleles do not occur. In 47 control patients, only 3 samples contain two SNPs with homozygous risk alleles and 17 samples contain only one SNP with homozygous risk alleles. Significant differences of the tumorous and the control population can be seen detected. NRAS mutation was not found. Our results showed a real increased risk effect of several newly recognized low-penetrance colorectal cancer susceptibility genetic variants by influence of the regulation of neighboring genes, however, the degree of cancer risk is individual, and influenced by others environmental factors, such as dietary factors. Orv Hetil. 2018; 159(40): 1614-1623.

Microcystin-LR induces mitotic spindle assembly disorders in Vicia faba by protein phosphatase inhibition and not reactive oxygen species induction.

We aimed to reveal the mechanisms of mitotic spindle anomalies induced by microcystin-LR (MCY-LR), a cyanobacterial toxin in Vicia faba, a well-known model in plant cell and molecular biology. MCY-LR inhibits type 1 and 2A phosphoserine/threonine specific protein phosphatases (PP1 and PP2A) and induces reactive oxygen species (ROS) formation. The cytoskeleton is one of the main targets of the cyanotoxin during cytopathogenesis. Histochemical-immunohistochemical and biochemical methods were used. A significant number of MCY-LR induced spindle alterations are described for the first time. Disrupted, multipolar spindles and missing kinetochore fibers were detected both in metaphase and anaphase cells. Additional polar microtubule (MT) bundles, hyperbundling of spindle MTs, monopolar spindles, C-S- shaped, additional and asymmetric spindles were detected in metaphase, while midplane kinetochore fibers were detected in anaphase cells only. Several spindle anomalies induced mitotic disorders, i.e. they occurred concomitantly with altered sister chromatid separation. Alterations were dependent on the MCY-LR dose and exposure time. Under long-term (2 and mainly 6 days') exposure they were detected in the concentration range of 0.1-20µgmL(-1) MCY-LR that inhibited PP1 and PP2A significantly without significant ROS induction. Elevated peroxidase/catalase activities indicated that MCY-LR treated V. faba plants showed efficient defense against oxidative stress. Thus, although the elevation of ROS is known to induce cytoskeletal aberrations in general, this study shows that long-term protein phosphatase inhibition is the primary cause of MCY-LR induced spindle disorders.

Cytotoxic effects of cylindrospermopsin in mitotic and non-mitotic Vicia faba cells.

Cylindrospermopsin (CYN) is a cyanobacterial toxin known as a eukaryotic protein synthesis inhibitor. We aimed to study its effects on growth, stress responses and mitosis of a eukaryotic model, Vicia faba (broad bean). Growth responses depended on exposure time (3 or 6d), cyanotoxin concentration, culture conditions (dark or continuous light) and V. faba cultivar ("Standard" or "ARC Egypt Cross"). At 6d of exposure, CYN had a transient stimulatory effect on root system growth, roots being possibly capable of detoxification. The toxin induced nucleus fragmentation, blebbing and chromosomal breaks indicating double stranded DNA breaks and programmed cell death. Root necrotic tissue was observed at 0.1-20 µg mL(-1) CYN that probably impeded toxin uptake into vascular tissue. Growth and cell death processes observed were general stress responses. In lateral root tip meristems, lower CYN concentrations (0.01-0.1 µg mL(-1)) induced the stimulation of mitosis and distinct mitotic phases, irrespective of culture conditions or the cultivar used. Higher cyanotoxin concentrations inhibited mitosis. Short-term exposure of hydroxylurea-synchronized roots to 5 µg mL(-1) CYN induced delay of mitosis that might have been related to a delay of de novo protein synthesis. CYN induced the formation of double, split and asymmetric preprophase bands (PPBs), in parallel with the alteration of cell division planes, related to the interference of cyanotoxin with protein synthesis, thus it was a plant- and CYN specific alteration.

Microcystin-LR, a protein phosphatase inhibitor, induces alterations in mitotic chromatin and microtubule organization leading to the formation of micronuclei in Vicia faba.

BACKGROUND AND AIMS: Microcystin-LR (MCY-LR) is a cyanobacterial toxin, a specific inhibitor of type 1 and 2A protein phosphatases (PP1 and PP2A) with significant impact on aquatic ecosystems. It has the potential to alter regulation of the plant cell cycle. The aim of this study was improved understanding of the mitotic alterations induced by cyanotoxin in Vicia faba, a model organism for plant cell biology studies. METHODS: Vicia faba seedlings were treated over the long and short term with MCY-LR purified in our laboratory. Short-term treatments were performed on root meristems synchronized with hydroxylurea. Sections of lateral root tips were labelled for chromatin, phosphorylated histone H3 and ß-tubulin via histochemical and immunohistochemical methods. Mitotic activity and the occurrence of mitotic alterations were detected and analysed by fluorescence microscopy. The phosphorylation state of histone H3 was studied by Western blotting. KEY RESULTS: Long-term MCY-LR exposure of lateral root tip meristems increased the percentage of either early or late mitosis in a concentration-dependent manner. We observed hypercondensed chromosomes and altered sister chromatid segregation (lagging chromosomes) leading to the formation of micronuclei, accompanied by the formation of disrupted, multipolar and monopolar spindles, disrupted phragmoplasts and the hyperphosphorylation of histone H3 at Ser10. Short-term MCY-LR treatment of synchronized cells showed that PP1 and PP2A inhibition delayed the onset of anaphase at 1 µg mL(-1) MCY-LR, accelerated cell cycle at 10 µg mL(-1) MCY-LR and induced the formation of lagging chromosomes. In this case mitotic microtubule alterations were not detected, but histone H3 was hyperphosphorylated. CONCLUSIONS: MCY-LR delayed metaphase-anaphase transition. Consequently, it induced aberrant chromatid segregation and micronucleus formation that could be associated with both H3 hyperphosphorylation and altered microtubule organization. However, these two phenomena seemed to be independent. The toxin may be a useful tool in the study of plant cell cycle regulation.