Anna Deregowska

Diosmin-induced senescence, apoptosis and autophagy in breast cancer cells of different p53 status and ERK activity.

Relatively low bioavailability of plant-derived nutraceuticals with anticancer properties may limit their usefulness for prevention and therapy of cancer. In the present study, we have screened for nutraceuticals (n=30) that would act at low micromolar range against phenotypically distinct breast cancer cell lines, namely MCF-7 (ER+, PR+/-, HER2-), MDA-MB-231 (ER-, PR-, HER2-) and SK-BR-3 (ER-, PR-, HER2+), and diosmin, a citrus fruit flavonoid belonging to a flavone subclass, was selected. MCF-7 cell line was found to be the most sensitive to diosmin treatment. Diosmin caused G2/M cell cycle arrest, elevation in p53, p21 and p27 levels and stress-induced premature senescence when used at lower concentrations (5 and 10μM). Diosmin (20μM) also promoted apoptosis that was not observed in normal human mammary epithelial cells (HMEC). Diosmin stimulated oxidative and nitrosative stress, DNA damage and changes in global DNA methylation patterns. The status of p53 (wild type versus mutant) and the levels of phosphorylated ERK1/2 in a steady state, and diosmin-induced autophagy may reflect diverse response to diosmin treatment in MCF-7, MDA-MB-231 and SK-BR-3 cells, which in turn results in different cell fates. Taken together, diosmin that acts at low micromolar range against breast cancer cells may be considered as a promising candidate for anticancer therapy.

Affected chromosome homeostasis and genomic instability of clonal yeast cultures.

Yeast cells originating from one single colony are considered genotypically and phenotypically identical. However, taking into account the cellular heterogeneity, it seems also important to monitor cell-to-cell variations within a clone population. In the present study, a comprehensive yeast karyotype screening was conducted using single chromosome comet assay. Chromosome-dependent and mutation-dependent changes in DNA (DNA with breaks or with abnormal replication intermediates) were studied using both single-gene deletion haploid mutants (bub1, bub2, mad1, tel1, rad1 and tor1) and diploid cells lacking one active gene of interest, namely BUB1/bub1, BUB2/bub2, MAD1/mad1, TEL1/tel1, RAD1/rad1 and TOR1/tor1 involved in the control of cell cycle progression, DNA repair and the regulation of longevity. Increased chromosome fragility and replication stress-mediated chromosome abnormalities were correlated with elevated incidence of genomic instability, namely aneuploid events—disomies, monosomies and to a lesser extent trisomies as judged by in situ comparative genomic hybridization (CGH). The tor1 longevity mutant with relatively balanced chromosome homeostasis was found the most genomically stable among analyzed mutants. During clonal yeast culture, spontaneously formed abnormal chromosome structures may stimulate changes in the ploidy state and, in turn, promote genomic heterogeneity. These alterations may be more accented in selected mutated genetic backgrounds, namely in yeast cells deficient in proper cell cycle regulation and DNA repair.

Sulforaphane-Induced Cell Cycle Arrest and Senescence are accompanied by DNA Hypomethylation and Changes in microRNA Profile in Breast Cancer Cells

Cancer cells are characterized by genetic and epigenetic alterations and phytochemicals, epigenetic modulators, are considered as promising candidates for epigenetic therapy of cancer. In the present study, we have investigated cancer cell fates upon stimulation of breast cancer cells (MCF-7, MDA-MB-231, SK-BR-3) with low doses of sulforaphane (SFN), an isothiocyanate. SFN (5-10 µM) promoted cell cycle arrest, elevation in the levels of p21 and p27 and cellular senescence, whereas at the concentration of 20 µM, apoptosis was induced. The effects were accompanied by nitro-oxidative stress, genotoxicity and diminished AKT signaling. Moreover, SFN stimulated energy stress as judged by decreased pools of ATP and AMPK activation, and autophagy induction. Anticancer effects of SFN were mediated by global DNA hypomethylation, decreased levels of DNA methyltransferases (DNMT1, DNMT3B) and diminished pools of N6-methyladenosine (m6A) RNA methylation. SFN (10 µM) also affected microRNA profiles, namely SFN caused upregulation of sixty microRNAs and downregulation of thirty two microRNAs, and SFN promoted statistically significant decrease in the levels of miR-23b, miR-92b, miR-381 and miR-382 in three breast cancer cells. Taken together, we show for the first time that SFN is an epigenetic modulator in breast cancer cells that results in cell cycle arrest and senescence, and SFN may be considered to be used in epigenome-focused anticancer therapy.

Shifts in rDNA levels act as a genome buffer promoting chromosome homeostasis

The nucleolus is considered to be a stress sensor and rDNA-based regulation of cellular senescence and longevity has been proposed. However, the role of rDNA in the maintenance of genome integrity has not been investigated in detail. Using genomically diverse industrial yeasts as a model and array-based comparative genomic hybridization (aCGH), we show that chromosome level may be balanced during passages and as a response to alcohol stress that may be associated with changes in rDNA pools. Generation- and ethanol-mediated changes in genes responsible for protein and DNA/RNA metabolism were revealed using next-generation sequencing. Links between redox homeostasis, DNA stability, and telomere and nucleolus states were also established. These results suggest that yeast genome is dynamic and chromosome homeostasis may be controlled by rDNA.

Identification of dermatophyte species using genomic in situ hybridization (GISH).

A genomic in situ hybridization (GISH)-based method for dermatophyte identification has been developed. Using specific GISH probes, discrimination between Trichophyton interdigitale, Trichophyton rubrum and Microsporum canis has been conducted. Moreover, GISH has been found particularly helpful when proper dermatophyte identification was difficult due to ambiguous PCR-RFLP patterns.

Reduced levels of methyltransferase DNMT2 sensitize human fibroblasts to oxidative stress and DNA damage that is accompanied by changes in proliferation-related miRNA expression

Methyltransferase DNMT2 is suggested to be involved in the regulation of numerous processes, however its biological significance and underlying molecular mechanisms remain elusive. In the present study, we have used WI-38 and BJ human fibroblasts as an in vitro model system to investigate the effects of siRNA-based DNMT2 silencing. DNMT2-depleted cells were found to be sensitive to oxidative stress conditions as judged by increased production of reactive oxygen species and susceptible to DNA damage that resulted in the inhibition of cell proliferation. DNMT2 silencing promoted upregulation of proliferation-related and tumor suppressor miRNAs, namely miR-28-3p, miR-34a-3p, miR-30b-5p, miR-29b-3p, miR-200c-3p, miR-28-5p, miR-379-5p, miR-382-5p, miR-194-5p, miR-193b-3p and miR-409-3p. Moreover, DNMT2 silencing induced cellular senescence and DNMT2 levels were elevated in replicatively senescent cells. Taken together, we found that DNMT2 may take part in the regulation of cell proliferation and longevity in human fibroblasts and speculate that the manipulation of DNMT2 levels that limits cell proliferation may be potentially useful anticancer strategy.

Adaptive response to chronic mild ethanol stress involves ROS, sirtuins and changes in chromosome dosage in wine yeasts

Industrial yeast strains of economic importance used in winemaking and beer production are genomically diverse and subjected to harsh environmental conditions during fermentation. In the present study, we investigated wine yeast adaptation to chronic mild alcohol stress when cells were cultured for 100 generations in the presence of non-cytotoxic ethanol concentration. Ethanol-induced reactive oxygen species (ROS) and superoxide signals promoted growth rate during passages that was accompanied by increased expression of sirtuin proteins, Sir1, Sir2 and Sir3, and DNA-binding transcription regulator Rap1. Genome-wide array-CGH analysis revealed that yeast genome was shaped during passages. The gains of chromosomes I, III and VI and significant changes in the gene copy number in nine functional gene categories involved in metabolic processes and stress responses were observed. Ethanol-mediated gains of YRF1 and CUP1 genes were the most accented. Ethanol also induced nucleolus fragmentation that confirms that nucleolus is a stress sensor in yeasts. Taken together, we postulate that wine yeasts of different origin may adapt to mild alcohol stress by shifts in intracellular redox state promoting growth capacity, upregulation of key regulators of longevity, namely sirtuins and changes in the dosage of genes involved in the telomere maintenance and ion detoxification.

Genetic profiling of yeast industrial strains using in situ comparative genomic hybridization (CGH).

The genetic differences and changes in genomic stability may affect fermentation processes involving bakers, brewers and wine yeast strains. Thus, it seems worthwhile to monitor the changes in genomic DNA copy number of industrial strains. In the present study, we developed an in situ comparative genomic hybridization (CGH) to investigate the ploidy and genetic differences between selected industrial yeast strains. The CGH-based system was validated using the laboratory Saccharomyces cerevisiae yeast strains (haploid BY4741 and diploid BY4743). DNA isolated from BY4743 cells was considered a reference DNA. The ploidy and DNA gains and losses of bakers, brewers and wine strains were revealed. Taken together, the in situ CGH was shown a helpful molecular tool to identify genomic differences between yeast industrial strains. Moreover, the in situ CGH-based system may be used at the single-cell level of analysis to supplement array-based techniques and high-throughput analyses at the population scale.