Pifithrin-alpha, an inhibitor of p53, enhances the genetic instability induced by etoposide (VP16) in human lymphoblastoid cells treated in vitro

Recent studies indicate that p53-dependent apoptosis induced in normal tissues during chemo- and radiotherapy can cause severe side effects of anti-cancer treatments that limit their efficiency.The aim of the present work was to further characterise the role of p53 in maintaining genomic stability and to verify whether the inhibition of p53 function in normal cells by pifithrin-alpha (PFT-alpha) may contribute in reducing the side effects of cancer therapy. Two human lymphoblastoid cell lines, derived from the same donor, TK6 (p53 wild type) and WTK1 (p53 mutated) have been treated with an anti-neoplastic drug, the etoposide (VP16), an inhibitor of DNA topoisomerase II in presence or in absence of the p53 inhibitor PFT-alpha. Following treatments with VP16 on TK6 and WTK1, we observed a higher induction of chromosome aberrations in WTK1 (p53 mutated) and of apoptosis in TK6 (p53 wild-type) cells. The p53 inhibition by PFT-alpha in VP16 treated TK6 cells produced an increase of chromosomal aberrations and a reduction of apoptosis. Therefore, the temporary suppression of the function of p53 by PFT-alpha, increasing the survival of the normal cells, could be a promising approach to reduce the side-effects of cancer therapy but it is important to consider that the surviving cells could be genetically modified and consequently the risk of secondary tumours could be increased.     

Karyotypic studies on species of Phaseolus (Fabaceae: Phaseolinae)

Ten karyotypes of Phaseolus were analyzed. Possible directions of karyological evolution of those species are suggested. All species studied have karyotypes composed exclusively of metacentric and submetacentric chromosomes, except P. filiformis with all metacentrics, and P. chiapasanus with one pair of subtelocentric chromosomes. Secondary constrictions and satellites are common among species studied. Phaseolus leptostachyus, P. micranthus, and P. macvaughii are considered as aneuploids with 2n= 20, whereas the rest of the species have 2n= 22.     

Evolutionary rescue by compensatory mutations is constrained by genomic and environmental backgrounds

Since deleterious mutations may be rescued by secondary mutations during evolution, compensatory evolution could identify genetic solutions leading to therapeutic targets. Here, we tested this hypothesis and examined whether these solutions would be universal or would need to be adapted to one's genetic and environmental makeups. We performed experimental evolutionary rescue in a yeast disease model for the Wiskott–Aldrich syndrome in two genetic backgrounds and carbon sources. We found that multiple aspects of the evolutionary rescue outcome depend on the genotype, the environment, or a combination thereof. Specifically, the compensatory mutation rate and type, the molecular rescue mechanism, the genetic target, and the associated fitness cost varied across contexts. The course of compensatory evolution is therefore highly contingent on the initial conditions in which the deleterious mutation occurs. In addition, these results reveal biologically favored therapeutic targets for the Wiskott–Aldrich syndrome, including the target of an unrelated clinically approved drug. Our results experimentally illustrate the importance of epistasis and environmental evolutionary constraints that shape the adaptive landscape and evolutionary rate of molecular networks.     

In vitro and in vivo characterization of alkyl hydroperoxide reductase mutant strains of Helicobacter hepaticus

Mutant strains in the tsaA gene encoding alkyl hydroperoxide reductase were more sensitive to O₂ and to oxidizing agents (paraquat, cumene hydroperoxide and t-butylhydroperoxide) than the wild type, but were markedly more resistant to hydrogen peroxide. The mutant strains resistance phenotype could be attributed to a 4-fold and 3-fold increase in the catalase protein amount and activity, respectively compared to the parent strain. The wild type did not show an increase in catalase expression in response to sequential increases in O₂ exposure or to oxidative stress reagents, so an adaptive compensatory mutation has probably occurred in the mutants. In support of this, chromosomal complementation of tsaA mutants restored alkyl hydroperoxide reductase, but catalase was still up-expressed in all complemented strains. The katA promoter sequence was the same in all mutant strains and the wild type. Like its Helicobacter pylori counterpart strain, a H. hepaticus tsaA mutant contained more lipid hydroperoxides than the wild type strain. Hepatic tissue from mice inoculated with a tsaA mutant had lesions similar to those inoculated with the wild type, and included coagulative necrosis of hepatocytes. The liver and cecum colonizing abilities of the wild type and tsaA mutant were comparable. Up-expression of catalase in the tsaA mutants likely permits the bacterium to compensate (in colonization and virulence attributes) for the loss of an otherwise important oxidative stress-combating enzyme, alkyl hydroperoxide reductase. The use of erythromycin resistance insertion as a facile way to screen for gene-targeted mutants, and the chromosomal complementation of those mutants are new genetic procedures for studying H. hepaticus.     

三株光合细菌的毒性试验研究

以昆明小白鼠为实验动物,对类球红细菌Z1、沼泽红假单胞菌Z2及光合细菌分离株C2采用急性毒性试验、骨髓细胞微核试验和精子畸形试验进行安全性评定。结果表明,急性毒性试验:实验组小白鼠的一般状况、器官含水量、器官系数及血常规检查,与对照组差异均无显著性(P>0.05);骨髓细胞微核试验和精子畸形试验实验组与阴性对照组(生理盐水)差异均无显著性(P>0.05),与阳性对照组(环磷酰胺40 mg/kg)比较,骨髓细胞微核试验差异有极显著性(P<0.01),精子畸形率试验差异有显著性(P<0.05)。以上结果说明,三株光合细菌均不具有毒性。     

Aneuploidy and cancer

The cell's euploid status is influenced by, amongst other mechanisms, an intact spindle assembly checkpoint (SAC), an accurate centrosome cycle, and proper cytokinesis. Studies in mammalian cells suggest that dysregulated SAC function, centrosome cycle, and cytokinesis can all contribute significantly to aneuploidy. Of interest, human cancers are frequently aneuploid and show altered expression in SAC genes. The SAC is a multi-protein complex that monitors against mis-segregation of sister chromatids. Several recent experimental mouse models have suggested a link between weakened SAC and in vivo tumorigenesis. Here, we review in brief some mechanisms which contribute to cellular aneuploidy and offer a perspective on the relationship between aneuploidy and human cancers.     

Cytogenetic effects of extremely low frequency magnetic field on Wistar rat bone marrow

In this study, the genotoxic and cytotoxic potential of extremely low frequency magnetic fields (ELF-MF) was investigated in Wistar rat tibial bone marrow cells, using the chromosomal aberration (CA) and micronucleus (MN) test systems. In addition to these test systems, we also investigated the mitotic index (MI), and the ratio of polychromatic erythrocytes (PCEs) to normochromatic erythrocytes (NCEs). Wistar rats were exposed to acute (1 day for 4 h) and long-term (4 h/day for 45 days) to a horizontal 50 Hz, 1 mT uniform magnetic field generated by a Helmholtz coil system. Mitomycin C (MMC, 2 mg/kg BW) was used as positive control. Results obtained by chromosome analysis do not show any statistically significant differences between the negative control and both acute and long-term ELF-MF exposed samples. When comparing the group mean CA of long-term exposure with the negative control and acute exposure, the group mean of the long-term exposed group was higher, but this was not statistically significant. However, the mean micronucleus frequency of the longer-term exposed group was considerably higher than the negative control and acutely exposed groups. This difference was statistically significant (p < 0.01). The results of the MI in bone marrow showed that the averages of both A-MF and L-MF groups significantly decreased when compared to those in the negative control (p < 0.001 and p < 0.01, respectively). No significant differences were found between the group mean MI of A-MF exposure with L-MF. We found that the average of PCEs/NCEs ratios of A-MF exposed group was significantly lower than the negative control and L-MF exposed groups (p < 0.001 and p < 0.01, respectively). In addition, the group mean of the PCEs/NCEs ratios of L-MF was significantly lower than negative control (p < 0.01). We also found that the MMC treated group showed higher the number of CA and the frequency of MN formation when compared to those in all other each groups (p-values of all each groups <0.01) and also MMC treated group showed lower MI and the PCEs/NCEs ratios when compared to those in all other each groups (p-values of all groups <0.01). These observations indicate the in vivo suspectibility of mammals to the genotoxicity potential of ELF-MF.     

Allelic Variation,Aneuploidy, and Nongenetic Mechanisms Suppress a Monogenic Trait in Yeast

Clinically relevant features of monogenic diseases, including severity of symptoms and age of onset, can vary widely in response to environmental differences as well as to the presence of genetic modifiers affecting the trait’s penetrance and expressivity. While a better understanding of modifier loci could lead to treatments for Mendelian diseases, the rarity of individuals harboring both a disease-causing allele and a modifying genotype hinders their study in human populations. We examined the genetic architecture of monogenic trait modifiers using a well-characterized yeast model of the human Mendelian disease classic galactosemia. Yeast strains with loss-of-function mutations in the yeast ortholog (GAL7) of the human disease gene (GALT) fail to grow in the presence of even small amounts of galactose due to accumulation of the same toxic intermediates that poison human cells. To isolate and individually genotype large numbers of the very rare (∼0.1%) galactose-tolerant recombinant progeny from a cross between two gal7Δ parents, we developed a new method, called “FACS-QTL.” FACS-QTL improves upon the currently used approaches of bulk segregant analysis and extreme QTL mapping by requiring less genome engineering and strain manipulation as well as maintaining individual genotype information. Our results identified multiple distinct solutions by which the monogenic trait could be suppressed, including genetic and nongenetic mechanisms as well as frequent aneuploidy. Taken together, our results imply that the modifiers of monogenic traits are likely to be genetically complex and heterogeneous.