Dai Ayusawa

Induction of senescence-associated genes by 5-bromodeoxyuridine in HeLa cells.

5-Bromodeoxyuridine (BrdU) universally induces a senescence-like phenomenon in mammalian cells. To assess this phenomenon at the level of gene expression, we constructed a PCR-based subtractive cDNA library enriched for mRNA species that immediately increase by administration of BrdU to HeLa cells. Candidate cDNA clones were isolated by differential colony hybridization, and then positive clones were identified by Northern blot analysis. Sequencing analysis revealed that the identified cDNA species were classified into three groups: widely used senescence-markers, known species whose relevance to senescence is yet to be reported, and known or novel ESTs. As expected, the majority of them showed an increase in expression in senescent human diploid fibroblasts. These results suggest that similar mechanisms operate in the regulation of BrdU-induced genes and senescence-associated genes.

Downregulation of hnRNP C1/C2 by siRNA sensitizes HeLa cells to various stresses.

The heterogeneous nuclear ribonucleoprotein C1/C2 is one of the most abundant proteins in the nucleus, and shown to have roles in cellular differentiation and proliferation through post-transcriptional regulations of certain mRNA species. We studied its role in stress response using siRNA mediated knockdown approach in HeLa cells. Upon transient transfection with plasmid encoding siRNA, the cells showed increased sensitivities to various chemical agents, namely H2O2, paraquat, camptothecin, ICRF-193 and halogenated deoxyuridines. These results demonstrate that hnRNP C1/C2 is involved in maintenance of cellular homeostasis besides cellular differentiation and proliferation.

Expression of the human cGMP‐dependent protein kinase II gene is lost upon introduction of SV40 T antigen or immortalization in human cells

We have cloned a human cGMP‐dependent protein kinase type II cDNA to examine its gene expression in terms of cellular senescence and/or immortalization. The genetic locus was mapped to band 4q21 by FISH. Northern blot analysis revealed that expression of the type II gene was markedly decreased or lost in mortal or immortal human fibroblasts producing SV40 T antigen. Also in various immortalized cell lines tested, the gene was not expressed. In normal diploid fibroblasts, the gene was constitutively expressed during cell‐cycle and population doubling levels (PDLs).

[FeFe]-hydrogenase-like gene is involved in the regulation of sensitivity to oxygen in yeast and nematode

Oxygen is essential for the life of aerobic organisms, but reactive oxygen species (ROS) derived from oxygen can be a threat for it. Many genes are involved in generation of ROS, but not much attention has been focused on the reactions from which ROS are generated. We therefore screened for mutants that showed an increased sensitivity to oxidative stress in the nematode Caenorhabditis elegans, and isolated a novel mutant, oxy‐4(qa5001). This mutant showed an increased sensitivity to a high concentration of oxygen, and decreased longevity at 20 °C but not at 26 °C. The genetic analysis has revealed that oxy‐4 had a causative mutation in an [FeFe]‐hydrogenase‐like gene (Y54H5A.4). In the yeast Saccharomyces cerevisiae, a deletion of NAR1, a possible homologue of oxy‐4, also caused a similar increased sensitivity to oxygen. [FeFe]‐hydrogenases are enzymes that catalyze both the formation and the splitting of molecular hydrogen, and function in anaerobic respiration in anaerobes. In contrast, [FeFe]‐hydrogenase‐like genes identified in aerobic eukaryotes do not generate hydrogen, and its functional roles are less understood. Our results suggested that [FeFe]‐hydrogenase‐like genes were involved in the regulation of sensitivity to oxygen in S. cerevisiae and C. elegans.

5-Bromouracil disrupts nucleosome positioning by inducing A-form-like DNA conformation in yeast cells

5-Bromodeoxyuridine (BrdU) modulates expression of particular genes associated with cellular differentiation and senescence. Our previous studies have suggested an involvement of chromatin structure in this phenomenon. Here, we examined the effect of 5-bromouracil on nucleosome positioning in vivo using TALS plasmid in yeast cells. This plasmid can stably and precisely be assembled nucleosomes aided by the alpha2 repressor complex bound to its alpha2 operator. Insertion of AT-rich sequences into a site near the operator destabilized nucleosome positioning dependent on their length and sequences. Addition of BrdU almost completely disrupted nucleosome positioning through specific AT-tracts. The effective AT-rich sequences migrated faster on polyacrylamide gel electrophoresis, and their mobility was further accelerated by substitution of thymine with 5-bromouracil. Since this property is indicative of a rigid conformation of DNA, our results suggest that 5-bromouracil disrupts nucleosome positioning by inducing A-form-like DNA.

5-Bromo-2′-deoxyuridine Efficiently Suppresses Division Potential of the Yeast Saccharomyces cerevisiae

We established a thymidine-auxotrophic strain of the yeast Saccharomyces cerevisiae to examine biological effects of thymidine analogues. 5-Bromo-2′-deoxyuridine efficiently suppressed the division potential of yeast showing morphology similar to senescent cells.

Overexpression of HAM1 gene detoxifies 5-bromodeoxyuridine in the yeast Saccharomyces cerevisiae

Abstract5-Bromodeoxyuridine (BrdU) is known to modulate expression of particular genes, and eventually arrest cell division in mammalian and yeast cells. To study a molecular basis for these phenomena, we adopted a genetic approach with a yeast cell system. We screened multicopy suppressor genes that confer resistance to BrdU with a thymidine-auxotrophic strain of the yeast Saccharomyces cerevisiae. One of such genes was found to encode Ham1 protein, which was originally identified as a possible triphosphatase for N-6-hydroxylaminopurine triphosphate. Consistent with this, overexpression of the HAM1 gene reversed growth arrest caused by BrdU, and blocked incorporation of BrdU into genomic DNA. On the contrary, disruption of the gene sensitized cells to BrdU. A crude extract from Ham1-overproducing cells showed a high activity to hydrolyze BrdUTP to BrdUMP and pyrophosphate in addition to abnormal purine nucleotides. Purified recombinant Ham1 protein showed the same activity. These results demonstrate that Ham1 protein detoxifies abnormal pyrimidine as well as purine nucleotides.

Accumulation of multiple forms of lamin A with down-regulation of FACE-1 suppresses growth in senescent human cells.

5‐Bromodeoxyuridine (BrdU) clearly induces a senescence‐like phenomenon in every cell type. Proteome analysis revealed that lamin A and C were most highly increased in the nuclei of HeLa cells upon addition of BrdU. Immunoblot analysis also revealed marked accumulation of nuclear prelamin A. Consistently, farnesylated‐proteins converting enzyme 1 (FACE‐1) was markedly down‐regulated in the same cells. Similar phenomena were also observed in normal human fibroblasts undergoing replicative senescence. Immunochemical analysis confirmed the above results. Lamin A is a major component of lamina and responsible for several genetic diseases. Thus, we ectopically expressed a wild‐type, a mature type and a premature type of lamin in HeLa cells. All of these forms similarly inhibited colony formation and delayed cell cycle progression mainly through G2 phase. These results suggest that a change in the amount of lamin A, rather than appearance of its truncated form, is responsible for growth retardation in affected cells.

5-bromodeoxyuridine induces transcription of repressed genes with disruption of nucleosome positioning.

5‐Bromodeoxyuridine (BrdU) modulates the expression of particular genes associated with cellular differentiation and senescence when incorporated into DNA instead of thymidine (dThd). To date, a molecular mechanism for this phenomenon remains a mystery in spite of a large number of studies. Recently, we have demonstrated that BrdU disrupts nucleosome positioning on model plasmids mediated by specific AT‐tracts in yeast cells. Here we constructed a cognate plasmid that can form an ordered array of nucleosomes determined by an α2 operator and contains the BAR1 gene as an expression marker gene to examine BAR1 expression in dThd‐auxotrophic MATα cells under various conditions. In medium containing dThd, BAR1 expression was completely repressed, associated with the formation of the stable array of nucleosomes. Insertion of AT‐tracts into a site of the promoter region slightly increased BAR1 expression and slightly destabilized nucleosome positioning dependent on their sequence specificity. In medium containing BrdU, BAR1 expression was further enhanced, associated with more marked disruption of nucleosome positioning on the promoter region. Disruption of nucleosome positioning seems to be sufficient for full expression of the marker gene if necessary transcription factors are supplied. Incorporation of 5‐bromouracil into the plasmid did not weaken the binding of the α2/Mcm1 repressor complex to its legitimate binding site, as revealed by an inu2003vivo UV photofootprinting assay. These results suggest that BrdU increases transcription of repressed genes by disruption of nucleosome positioning around their promoters.

ERK1/2 mediates unbalanced growth leading to senescence induced by excess thymidine in human cells

Excess thymidine induces unbalanced growth by delaying DNA replication and subsequently induces senescence in every human cell type. Our previous studies with use of inhibitors suggested that ERK1/2 has a major role in these processes. Here we directly assessed the roles of ERK1 and ERK2 in unbalanced growth induced by excess thymidine. Knockdown of ERK2 and ERK1 by vector-based RNA interference prevented loss of colony forming ability and appearance of senescence markers induced by excess thymidine in HeLa and TIG-7 cells, respectively. Such cells continued growing in the presence of excess thymidine. Double knockdown of ERK1 and ERK2 did not improve the effects of single knockdowns of ERK1 and ERK2 in either cell types. These results demonstrate that ERK1 or ERK2 has a major role in manifestation of unbalanced growth in human cells.