Jan Fronk

Induction of senescence with doxorubicin leads to increased genomic instability of HCT116 cells

Induction of senescence has been proposed as a possible in vivo tumor response to anticancer treatment. Senescent cancer cells are often polyploid, however, their route to polyploidy is poorly recognized (endoreduplication versus aberrant mitoses). We showed that after treatment of HCT116 cells with a low dose of doxorubicin most of them stopped proliferation as documented by SA-beta-galactosidase activity and the lack of Ki67 expression. Increased expression of other common senescence markers, p53, p21 and cyclin D1, was also observed. The cells became giant, polyploid and polymorphic, with multinucleated cells comprising a substantial fraction. The vast majority of the doxorubicin-treated cells did not enter mitoses, as evidenced by mitotic index analysis, as well as by the predominantly cytoplasmic localization of cyclin B1 and a lack of separation of multiplied centrosomes. This allowed us to conclude that doxorubicin-treated HCT116 cells underwent endoreduplication. However, the rare events of aberrant mitoses of polyploid cells observed by us led to aneuploid progeny as was documented by cytogenetic analysis of survivors. Thus, a senescence-inducing treatment of HCT116 cancer cells had a dual effect-it stopped the proliferation of the majority of the cells, but also led to the appearance of proliferating aneuploid ones.

Mutation in dystrophin-encoding gene affects energy metabolism in mouse myoblasts

Duchenne Muscular Dystrophy is characterized by severe defects in differentiated muscle fibers, including abnormal calcium homeostasis and impaired cellular energy metabolism. Here we demonstrate that myoblasts derived from dystrophic (mdx) mouse exhibit reduced oxygen consumption, increased mitochondrial membrane potential, enhanced reactive oxygen species formation, stimulated glycolysis but unaffected total cellular ATP content. Moreover, reduced amounts of specific subunits of the mitochondrial respiratory complexes and ATP-synthase as well as disorganized mitochondrial network were observed. Both the dystrophic and control myoblasts used were derived from a common inbred mouse strain and the only difference between them is a point mutation in the dystrophin-encoding gene, thus these data indicate that this mutation results in multiple phenotypic alterations demonstrating as early as in undifferentiated myoblasts. This finding sheds new light on the molecular mechanisms of Duchenne Muscular Dystrophy pathogenesis.

Tunicamycin desensitizes store-operated Ca2+ entry to ATP and mitochondrial potential.

Tunicamycin effect on thapsigargin-induced store-operated calcium entry was investigated. Ca2+ influx was stimulated by 50% upon exposure of Jurkat cells to tunicamycin. Moreover, tunicamycin efficiently prevented the inhibition of store-operated calcium entry caused by dissipation of mitochondrial membrane potential. Protective action of tunicamycin on store-operated Ca2+ entry was also partially preserved in Jurkat cells depleted of ATP, while Ca2+ entry into ATP-deprived cells grown in tunicamycin-free medium was almost completely inhibited. Tunicamycin-evoked changes in cellular Ca2+ fluxes coincided with decreased glycosylation of STIM1 protein. Although the latter observation is correlative and needs additional confirmation it may suggest that deglycosylation of STIM1 protein deprives store-operated calcium entry system of an important regulatory mechanism. This study suggests a novel mechanism of modulation of the activity of store-operated calcium channels in lymphoidal cells.

Yeast transcription factor Oaf1 forms homodimer and induces some oleate-responsive genes in absence of Pip2

Genes encoding peroxisomal proteins in the yeast Saccharomyces cereviasiae are induced in the presence of oleate in growth medium. This induction is known to be mediated by the binding of a heterodimer of transcription factors Oaf1 and Pip2 to an upstream activating sequence called ORE (oleate response element). By analyzing expression of nine ORE-containing genes we show that the presence of an ORE sequence is not sufficient to confer oleate inducibility, as three such genes were in fact expressed constitutively. Moreover, some of the oleate-inducible genes undergo activation even in the absence of Pip2. Using coimmunoprecipitation we show that, when Pip2 is missing, Oaf1 may form homodimers which apparently substitute for the Oaf1-Pip2 heterodimer.

Identification of a ras gene in the slime mold Physarum polycephalum

A ras homologue was identified in the cDNA library from the slime mold Physarum polycephalum. The cDNA codes for a protein of 189 amino acids, showing high homology to ras genes from other organisms, especially to these from Dictyostelium discoideum. Amino acid sequence at the C-terminus of the putative protein suggests that unlike most other ras proteins, it is not palmitoylated and bears a geranylgeranyl rather than farnesyl chain.

Rhythmic Diel Pattern of Gene Expression in Juvenile Maize Leaf

Background Numerous biochemical and physiological parameters of living organisms follow a circadian rhythm. Although such rhythmic behavior is particularly pronounced in plants, which are strictly dependent on the daily photoperiod, data on the molecular aspects of the diurnal cycle in plants is scarce and mostly concerns the model species Arabidopsis thaliana. Here we studied the leaf transcriptome in seedlings of maize, an important C4 crop only distantly related to A. thaliana, throughout a cycle of 10 h darkness and 14 h light to look for rhythmic patterns of gene expression. Results Using DNA microarrays comprising ca. 43,000 maize-specific probes we found that ca. 12% of all genes showed clear-cut diel rhythms of expression. Cluster analysis identified 35 groups containing from four to ca. 1,000 genes, each comprising genes of similar expression patterns. Perhaps unexpectedly, the most pronounced and most common (concerning the highest number of genes) expression maxima were observed towards and during the dark phase. Using Gene Ontology classification several meaningful functional associations were found among genes showing similar diel expression patterns, including massive induction of expression of genes related to gene expression, translation, protein modification and folding at dusk and night. Additionally, we found a clear-cut tendency among genes belonging to individual clusters to share defined transcription factor-binding sequences. Conclusions Co-expressed genes belonging to individual clusters are likely to be regulated by common mechanisms. The nocturnal phase of the diurnal cycle involves gross induction of fundamental biochemical processes and should be studied more thoroughly than was appreciated in most earlier physiological studies. Although some general mechanisms responsible for the diel regulation of gene expression might be shared among plants, details of the diurnal regulation of gene expression seem to differ between taxa.

Expression of murine DNA methyltransferases Dnmt1 and Dnmt3a in the yeast Saccharomyces cerevisiae

Murine DNA methyltransferases Dnmt1 and Dnmt3a were expressed in the yeast Saccharomyces cerevisiae. Adjustment to yeast preferences of the nucleotide sequences upstream and downstream of the translation initiation sites of both cDNAs was needed to obtain significant levels of the methyltransferases. Both proteins were correctly localized to the nucleus and their presence had no measurable influence on the functioning of yeast cells. Both Dnmt1 and Dnmt3a expressed in yeast cells were enzymatically active in vitro, and in vivo in the genomic DNA of the transgenic S. cerevisiae ca. 0.06% and 0.4%, respectively, of cytosines became methylated. This level of DNA methylation is about 100‐ to 10‐fold less than that observed in mammalian cells. The constructed system may be used to investigate the in vivo specificity of individual mammalian DNA methyltransferases and to search for additional factors needed to allow more efficient in vivo methylation of chromatin‐contained DNA and to study their mechanism of action. Copyright

Nuclear import and export signals of human cohesins SA1/STAG1 and SA2/STAG2 expressed in Saccharomyces cerevisiae.

Background Human SA/STAG proteins, homologues of the yeast Irr1/Scc3 cohesin, are the least studied constituents of the sister chromatid cohesion complex crucial for proper chromosome segregation. The two SA paralogues, SA1 and SA2, show some specificity towards the chromosome region they stabilize, and SA2, but not SA1, has been shown to participate in transcriptional regulation as well. The molecular basis of this functional divergence is unknown. Methodology/Principal Findings In silico analysis indicates numerous putative nuclear localization (NLS) and export (NES) signals in the SA proteins, suggesting the possibility of their nucleocytoplasmic shuttling. We studied the functionality of those putative signals by expressing fluorescently tagged SA1 and SA2 in the yeast Saccharomyces cerevisiae. Only the N-terminal NLS turned out to be functional in SA1. In contrast, the SA2 protein has at least two functional NLS and also two functional NES. Depending on the balance between these opposing signals, SA2 resides in the nucleus or is distributed throughout the cell. Validation of the above conclusions in HeLa cells confirmed that the same N-terminal NLS of SA1 is functional in those cells. In contrast, in SA2 the principal NLS functioning in HeLa cells is different from that identified in yeast and is localized to the C-terminus. Conclusions/Significance This is the first demonstration of the possibility of non-nuclear localization of an SA protein. The reported difference in the organization between the two SA homologues may also be relevant to their partially divergent functions. The mechanisms determining subcellular localization of cohesins are only partially conserved between yeast and human cells.

Chromatin reorganization during early differentiation of Physarum polycephalum

Abstract When microplasmodia of Physarum polycephalum are induced to differentiate by starvation, the rate of RNA synthesis measured in vitro with endogenous RNA polymerase B drops rapidly, decreasing after 12 h of starvation to 30% of the initial value. Transcriptional inactivation is accompanied by: (a) 60% decrease in the amount of the chromatin fraction solubilizable by light DNAase I digestion; (b) increase in the content of histone H1 and several nonhistone proteins in the DNAase-I-solubilized chromatin; and (c) formation of a nucleosome-like structure that is absent from this fraction during normal growth. No marked change in the structure and composition of the total chromatin on starvation is evident. These data suggest that the repression of transcription during differentiation in Physarum is correlated with structural reorganization of active chromatin regions.

The F658G substitution in Saccharomyces cerevisiae cohesin Irr1/Scc3 is semi-dominant in the diploid and disturbs mitosis, meiosis and the cell cycle

The sister chromatid cohesion complex of Saccharomyces cerevisiae includes chromosomal ATPases Smc1p and Smc3p, the kleisin Mcd1p/Scc1p, and Irr1p/Scc3p, the least studied component. We have created an irr1-1 mutation (F658G substitution) which is lethal in the haploid and semi-dominant in the heterozygous diploid irr1-1/IRR1. The mutated Irr1-1 protein is present in the nucleus, its level is similar to that of wild-type Irr1p/Scc3p and it is able to interact with chromosomes. The irr1-1/IRR1 diploid exhibits mitotic and meiotic chromosome segregation defects, irregularities in mitotic divisions and is severely affected in meiosis. These defects are gene-dosage dependent, and experiments with synchronous cultures suggest that they may result from the malfunctioning of the spindle assembly checkpoint. The partial structure of Irr1p/Scc3p was predicted and the F658G substitution was found to induce marked changes in the general shape of the predicted protein. Nevertheless, the mutant protein retains its ability to interact with Scc1p, another component of the cohesin complex, as shown by coimmunoprecipitation.