Maxim Sorokin

Mitochondrial signaling in Saccharomyces cerevisiae pseudohyphae formation induced by butanol.

Yeasts growing limited for nitrogen source or treated with fusel alcohols form elongated cells--pseudohyphae. Absence of mitochondrial DNA or anaerobic conditions inhibits this process, but the precise role of mitochondria is not clear. We found that a significant percentage of pseudohyphal cells contained mitochondria with different levels of membrane potential within one cell. An uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), but not the ATP-synthase inhibitor oligomycin D, prevented pseudohyphal growth. Interestingly, repression of the MIH1 gene encoding phosphatase activator of the G2/M transition partially restores the ability of yeast to form pseudohyphal cells in the presence of FCCP or in the absence of mitochondrial DNA. At the same time, retrograde signaling (the one triggered by dysfunctional mitochondria) appeared to be a positive regulator of butanol-induced pseudohyphae formation: the deletion of any of the retrograde signaling genes (RTG1, RTG2, or RTG3) partially suppressed pseudohyphal growth. Together, our data suggest that two subpopulations of mitochondria are required for filamentous growth: one with high and another with low transmembrane potential. These mitochondria-activated signaling pathways appear to converge at Mih1p level.

Acquired resistance to tyrosine kinase inhibitors may be linked with the decreased sensitivity to X-ray irradiation

Acquired resistance to chemotherapy and radiation therapy is one of the major obstacles decreasing efficiency of treatment of the oncologic diseases. In this study, on the two cell lines (ovarian carcinoma SKOV-3 and neuroblastoma NGP-127), we modeled acquired resistance to five target anticancer drugs. The cells were grown on gradually increasing concentrations of the clinically relevant tyrosine kinase inhibitors (TKIs) Sorafenib, Pazopanib and Sunitinib, and rapalogs Everolimus and Temsirolimus, for 20 weeks. After 20 weeks of culturing, the half-inhibitory concentrations (IC50) increased by 25 – 186% for the particular combinations of the drugs and cell types. We next subjected cells to 10 Gy irradiation, a dose frequently used in clinical radiation therapy. For the SKOV-3, but not NGP-127 cells, for the TKIs Sorafenib, Pazopanib and Sunitinib, we noticed statistically significant increase in capacity to repair radiation-induced DNA double strand breaks compared to naïve control cells not previously treated with TKIs. These peculiarities were linked with the increased activation of ATM DNA repair pathway in the TKI-treated SKOV-3, but not NGP-127 cells. Our results provide a new cell culture model for studying anti-cancer therapy efficiency and evidence that there may be a tissue-specific radioresistance emerging as a side effect of treatment with TKIs.

Gene expression and molecular pathway activation signatures of MYCN-amplified neuroblastomas

Neuroblastoma is a pediatric cancer arising from sympathetic nervous system. Remarkable heterogeneity in outcomes is one of its widely known features. One of the traits strongly associated with the unfavorable subtype is the amplification of oncogene MYCN. Here, we performed cross-platform biomarker detection by comparing gene expression and pathway activation patterns from the two literature reports and from our experimental dataset, combining profiles for the 761 neuroblastoma patients with known MYCN amplification status. We identified 109 / 25 gene expression / pathway activation biomarkers strongly linked with the MYCN amplification. The marker genes/pathways are involved in the processes of purine nucleotide biosynthesis, ATP-binding, tetrahydrofolate metabolism, building mitochondrial matrix, biosynthesis of amino acids, tRNA aminoacylation and NADP-linked oxidation-reduction processes, as well as in the tyrosine phosphatase activity, p53 signaling, cell cycle progression and the G1/S and G2/M checkpoints. To connect molecular functions of the genes involved in MYCN-amplified phenotype, we built a new molecular pathway using known intracellular protein interaction networks. The activation of this pathway was highly selective in discriminating MYCN-amplified neuroblastomas in all three datasets. Our data also suggest that the phosphoinositide 3-kinase (PI3K) inhibitors may provide new opportunities for the treatment of the MYCN-amplified neuroblastoma subtype.

Early stage of cytomegalovirus infection suppresses host microRNA expression regulation in human fibroblasts

ABSTRACT Responses to human cytomegalovirus (HCMV) infection are largely individual and cell type specific. We investigated molecular profiles in 2 primary cell cultures of human fibroblasts, which are highly or marginally sensitive to HCMV infection, respectively. We screened expression of genes and microRNAs (miRs) at the early (3 hours) stage of infection. To assess molecular pathway activation profiles, we applied bioinformatic algorithms OncoFinder and MiRImpact. In both cell types, pathway regulation properties at mRNA and miR levels were markedly different. Surprisingly, in the infected highly sensitive cells, we observed a “freeze” of miR expression profiles compared to uninfected controls. Our results evidence that in the sensitive cells, HCMV blocks intracellular regulation of microRNA expression already at the earliest stage of infection. These data suggest somewhat new functions for HCMV products and demonstrate dependence of miR expression arrest on the host-encoded factors.

The contribution of Saccharomyces cerevisiae replicative age to the variations in the levels of Trx2p, Pdr5p, Can1p and Idh isoforms

Asymmetrical division can be a reason for microbial populations heterogeneity. In particular, budding yeast daughter cells are more vulnerable to stresses than the mothers. It was suggested that yeast mother cells could also differ from each other depending on their replicative age. To test this, we measured the levels of Idh1-GFP, Idh2-GFP, Trx2-GFP, Pdr5-GFP and Can1-GFP proteins in cells of the few first, most represented, age cohorts. Pdr5p and Can1p were selected because of the pronounced mother-bud asymmetry for these proteins distributions, Trx2p as indicator of oxidative stress. Isocitrate dehydrogenase subunits Idh1p and Idh2p were assessed because their levels are regulated by mitochondria. We found a small negative correlation between yeast replicative age and Idh1-GFP or Idh2-GFP but not Trx2-GFP levels. Mitochondrial network fragmentation was also confirmed as an early event of replicative aging. No significant difference in the membrane proteins levels Pdr5p and Can1p was found. Moreover, the elder mother cells showed lower coefficient of variation for Pdr5p levels compared to the younger ones and the daughters. Our data suggest that the levels of stress-response proteins Pdr5p and Trx2p in the mother cells are stable during the first few cell cycles regardless of their mother-bud asymmetry.

Molecular pathway activation features of pediatric acute myeloid leukemia (AML) and acute lymphoblast leukemia (ALL) cells

Acute lymphoblast leukemia (ALL) is characterized by overproduction of immature white blood cells in the bone marrow. ALL is most common in the childhood and has high (>80%) cure rate. In contrast, acute myeloid leukemia (AML) has far greater mortality rate than the ALL and is most commonly affecting older adults. However, AML is a leading cause of childhood cancer mortality. In this study, we compare gene expression and molecular pathway activation patterns in three normal blood, seven pediatric ALL and seven pediatric AML bone marrow samples. We identified 172/94 and 148/31 characteristic gene expression/pathway activation signatures, clearly distinguishing pediatric ALL and AML cells, respectively, from the normal blood. The pediatric AML and ALL cells differed by 139/34 gene expression/pathway activation biomarkers. For the adult 30 AML and 17 normal blood samples, we found 132/33 gene expression/pathway AML-specific features, of which only 7/2 were common for the adult and pediatric AML and, therefore, age-independent. At the pathway level, we found more differences than similarities between the adult and pediatric forms. These findings suggest that the adult and pediatric AMLs may require different treatment strategies.

Prooxidants prevent yeast cell death induced by genotoxic stress

It was shown earlier that DNA damage induced by alkylating agent MMS (methyl methanesulfonate) results in formation of ROS (reactive oxygen species) in yeast cells. Here, we asked whether this ROS generation is favourable for the cells. It appeared that prooxidants rather than antioxidants stimulate the survival after MMS treatment. We found that positively charged detergents increase the survival via induction of H2O2 formation in the cells. Interestingly, prooxidants protected yeast cells from the moderate doses of MMS and enhanced the toxicity of relatively high ones. Prooxidants also protect the cells arrested in mitosis (nocodazole treatment), indicating that the protection is mostly due to ROS‐mediated transcriptional stress—response rather than due to enrichment of cell culture with highly MMS‐resistant G2/M cells. The comparison of the published expression profile responses to prooxidant and MMS treatments identifies a set of ROS‐activated genes, which are likely to protect cells from the genotoxic stress.

Oncobox Bioinformatical Platform for Selecting Potentially Effective Combinations of Target Cancer Drugs Using High-Throughput Gene Expression Data

Sequential courses of anticancer target therapy lead to selection of drug-resistant cells, which results in continuous decrease of clinical response. Here we present a new approach for predicting effective combinations of target drugs, which act in a synergistic manner. Synergistic combinations of drugs may prevent or postpone acquired resistance, thus increasing treatment efficiency. We cultured human ovarian carcinoma SKOV-3 and neuroblastoma NGP-127 cancer cell lines in the presence of Tyrosine Kinase Inhibitors (Pazopanib, Sorafenib, and Sunitinib) and Rapalogues (Temsirolimus and Everolimus) for four months and obtained cell lines demonstrating increased drug resistance. We investigated gene expression profiles of intact and resistant cells by microarrays and analyzed alterations in 378 cancer-related signaling pathways using the bioinformatical platform Oncobox. This revealed numerous pathways linked with development of drug resistant phenotypes. Our approach is based on targeting proteins involved in as many as possible signaling pathways upregulated in resistant cells. We tested 13 combinations of drugs and/or selective inhibitors predicted by Oncobox and 10 random combinations. Synergy scores for Oncobox predictions were significantly higher than for randomly selected drug combinations. Thus, the proposed approach significantly outperforms random selection of drugs and can be adopted to enhance discovery of new synergistic combinations of anticancer target drugs.