Objective
To develop a mouse artificial chromosome (MAC) carrying the mouse Xist gene (X-inactive specific transcript; Xist-MAC) as a systematic in vitro approach for investigating Xist RNA-mediated ...chromosome inactivation.
Results
Ectopic expression of the Xist gene in CHO cells led to the accumulation of Xist RNA in
cis
on the MAC. In addition, the introduction of Xist-MAC to embryonic stem cells from male mice via microcell-mediated chromosome transfer resulted in the accumulation of Xist RNA in
cis
on the MAC. Chromosomal inactivation was observed in the differentiated state. Moreover, this phenomenon was accompanied by the epigenetic modification of H3K27 trimethylation.
Conclusions
We successfully generated a novel chromosome inactivation model, Xist-MAC, which will provide a valuable tool for the screening and functional analysis of X chromosome inactivation-related genes and proteins.
How ploidy is determined in organisms is an important issue in bioscience. Polyploidy is believed to be relevant to useful traits of domesticated plants and microorganisms. As such, polyploidy is ...central to many applications in biotechnology. However, studies of polyploidy are poorly advanced because no methodologies to construct desired polyploid have been developed for any organism. Herein we describe the development of a novel breeding technology, matα2-PBT, to generate polyploid strains of Saccharomyces cerevisiae. S. cerevisiae has two mating types, a and α, determined by MATa and MATα gene each of which consists of a1 and a2 and α1 and α2 cistrons. This novel technology exploits an interesting feature of a specific mutation, matα2-102, in the MATα2 gene. Unlike the MATα wild-type strain, which gives a non-mating phenotype when mated with MATa cells, the matα2-102 strain confers an α mating-type to a-type strains when mated with a-type strains. We constructed plasmid with the cloned matα2-102 mutant gene. An a-type cells harboring this plasmid displayed an α mating-type and mated with a-type cells. Because the resultant hybrid displays an α mating-type, it can mate again with a-type cells. By repeating this procedure, we have constructed an isogenic series of haploid to tetraploid of S. cerevisiae. Although whether even higher polyploid than tetraploid can be constructed by using this technology remains to be determined in the future, we believe that it became possible for the first time with matα2-PBT method to investigate whether higher polyploid than tetraploid can be constructed.
The mammalian HSP105/110 family consists of four members, including Hsp105 and Apg‐1, which function as molecular chaperones. Recently, we reported that Hsp105 knockdown increases sensitivity to the ...DNA‐damaging agent Adriamycin but decreases sensitivity to the microtubule‐targeting agent paclitaxel. However, whether the other Hsp105/110 family proteins have the same functional property is unknown. Here, we show that Apg‐1 has different roles from Hsp105 in cell proliferation, cell division, and drug sensitivity. We generated the Apg‐1‐knockdown HeLa S3 cells by lentiviral expression of Apg‐1‐targeting short hairpin RNA. Knockdown of Apg‐1 but not Hsp105 decreased cell proliferation. Apg‐1 knockdown increased cell death upon Adriamycin treatment without affecting paclitaxel sensitivity. The cell synchronization experiment suggests that Apg‐1 functions in mitotic progression at a different mitotic subphase from Hsp105, which cause difference in paclitaxel sensitivity. Since Apg‐1 is overexpressed in certain types of tumors, Apg‐1 may become a potential therapeutic target for cancer treatment without causing resistance to the microtubule‐targeting agents.
In this study, we show that Apg‐1 has different roles from Hsp105 in cell proliferation, cell division, and drug sensitivity
The nonreceptor tyrosine kinase v-Src is an oncogene first identified in Rous sarcoma virus. The oncogenic effects of v-Src have been intensively studied; however, its effects on chromosomal ...integrity are not fully understood. Here, using HeLa S3/v-Src cells having inducible v-Src expression, we found that v-Src causes mitotic slippage in addition to cytokinesis failure, even when the spindle assembly checkpoint is not satisfied because of the presence of microtubule-targeting agents. v-Src's effect on mitotic slippage was also observed in cells after a knockdown of C-terminal Src kinase (Csk), a protein-tyrosine kinase that inhibits Src-family kinases and was partially inhibited by PP2, an Src-family kinase inhibitor. Proteomic analysis and in vitro kinase assay revealed that v-Src phosphorylates cyclin-dependent kinase 1 (Cdk1) at Tyr-15. This phosphorylation attenuated Cdk1 kinase activity, resulting in a decrease in the phosphorylation of Cdk1 substrates. Furthermore, v-Src–induced mitotic slippage reduced the sensitivity of the cells to microtubule-targeting agents, and cells that survived the microtubule-targeting agents exhibited polyploidy. These results suggest that v-Src causes mitotic slippage by attenuating Cdk1 kinase activity via direct phosphorylation of Cdk1 at Tyr-15. On the basis of these findings, we propose a model for v-Src–induced oncogenesis, in which v-Src–promoted mitotic slippage due to Cdk1 phosphorylation generates genetic diversity via abnormal cell division of polyploid cells and also increases the tolerance of cancer cells to microtubule-targeting agents.
Thermotolerance is a phenomenon in which cells become resistant to stress by prior exposure to heat shock, and its development is associated with the induction of heat shock proteins (Hsps), ...including Hsp70. We previously showed that the expression of Hsp70 is regulated by the cytokine signaling transcription factor Stat3, but the role of Stat3 in thermotolerance is not known. In this study, we examined the possible involvement of Stat3 in the acquisition of thermotolerance. We found that severe heat shock-induced morphological changes and decreases in cell viability, which were suppressed by exposure to non-lethal mild heat shock prior to severe heat shock. This thermotolerance development was accompanied by Stat3 phosphorylation and the induction of Hsps such as Hsp105, Hsp70, and Hsp27. Stat3 phosphorylation and Hsp induction were inhibited by AG490, an inhibitor of JAK tyrosine kinase. Consistent with this, we found that mild heat shock-induced thermotolerance was partially suppressed by AG490 or knockdown of Hsp105. We also found that the Stat3 inhibitor Stattic suppresses the acquisition of thermotolerance by inhibiting the mild heat shock-induced Stat3 phosphorylation and Hsp105 expression. These results suggest that the mild heat shock-dependent stimulation of the JAK-Stat signaling pathway contributes to the development of thermotolerance via the induction of Hsps including Hsp105. This signaling pathway may be a useful target for hyperthermia cancer therapy.
Anaplastic lymphoma kinase (ALK), a receptor-type tyrosine kinase, is involved in the pathogenesis of several cancers. ALK has been targeted with small molecule inhibitors for the treatment of ...different cancers, but absolute success remains elusive. In the present study, the effects of ALK inhibitors on M phase progression were evaluated. Crizotinib, ceritinib, and TAE684 suppressed proliferation of neuroblastoma SH-SY5Y cells in a concentration-dependent manner. At approximate IC
concentrations, these inhibitors caused misorientation of spindles, misalignment of chromosomes and reduction in autophosphorylation. Similarly, knockdown of ALK caused M phase delay, which was rescued by re-expression of ALK. Time-lapse imaging revealed that anaphase onset was delayed. The monopolar spindle 1 (MPS1) inhibitor, AZ3146, and MAD2 knockdown led to a release from inhibitor-induced M phase delay, suggesting that spindle assembly checkpoint may be activated in ALK-inhibited cells. H2228 human lung carcinoma cells that express EML4-ALK fusion showed M phase delay in the presence of TAE684 at about IC
concentrations. These results suggest that ALK plays a role in M phase regulation and ALK inhibition may contribute to the suppression of cell proliferation in ALK-expressing cancer cells.
An efficient method for the synthesis of N‐(hetero)arylcarbazoles, useful compounds for functional materials, is reported. Various (hetero)aryl chlorides reacted with N‐carbazolylmagnesium chloride ...in the presence of a palladium catalyst (0.05 to 0.2 mol%) prepared from allylpalladium(II) chloride dimer {PdCl(allyl)2} and di‐tert‐butyl(2,2‐diphenyl‐1‐methylcyclopropan‐1‐yl)phosphine (cBRIDP) under mild conditions (110 °C) in a short period of time (15 min to 2 h) to give N‐(hetero)arylcarbazoles in high yields. The reactions of bromochlorobenzenes proceeded in favour of the bromo group to afford N‐(chlorophenyl)carbazoles in a highly selective manner. Functional materials for use in organic light‐emitting diodes, such as mCP, 26mcPy, CBP and TCB, were also obtained in high yields within 15 min by the reaction of (hetero)aryl polyhalides. Optimization of the reaction conditions and a postulated catalytic cycle for the reaction are also discussed.
An interaction between the Rad17-RFC2-5 and 9-1-1 complexes is essential for ATR-Chk1 signaling, which is one of the major DNA damage checkpoints. Recently, we showed that the polyanionic C-terminal ...tail of human Rad17 and the embedded conserved sequence iVERGE are important for the interaction with 9-1-1 complex. Here, we show that Rad17-S667 in the C-terminal tail is constitutively phosphorylated in vivo in a casein kinase 2-dependent manner, and the phosphorylation is important for 9-1-1 interaction. The serine phosphorylation of Rad17 could be seen in the absence of exogenous genotoxic stress, and was mostly abolished by S667A substitution. Rad17-S667 was also phosphorylated when the C-terminal tail was fused with EGFP, but the phosphorylation was inhibited by two casein kinase 2 inhibitors. Furthermore, interaction between Rad17 and the 9-1-1 complex was inhibited by the casein kinase 2 inhibitor CX-4945/Silmitasertib, and the effect was dependent on the Rad17-S667 residue, indicating that S667 phosphorylation is the only role of casein kinase 2 in the 9-1-1 interaction. Our data raise the possibility that the C-terminal tail of vertebrate Rad17 regulates ATR-Chk1 signaling through multi-site phosphorylation in the iVERGE.
•Rad17-S667 in the C-terminal tail is constitutively phosphorylated in vivo.•Rad17-S667 phosphorylation is dependent on casein kinase 2 (CK2) activity.•CK2-dependent Rad17-S667 phosphorylation is important for the 9-1-1 interaction.•The Rad17-S667 phosphorylation is the only role of CK2 in the 9-1-1 interaction.
The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ...ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3-96.8%. Additionally, we observed homogenous gene expression in 77.3-87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
v-Src, an oncogene found in Rous sarcoma virus, is a constitutively active variant of c-Src. Activation of Src is observed frequently in colorectal and breast cancers, and is critical in tumor ...progression through multiple processes. However, in some experimental conditions, v-Src causes growth suppression and apoptosis. In this review, we highlight recent progress in our understanding of cytokinesis failure and the attenuation of the tetraploidy checkpoint in v-Src-expressing cells. v-Src induces cell cycle changes-such as the accumulation of the 4N cell population-and increases the number of binucleated cells, which is accompanied by an excess number of centrosomes. Time-lapse analysis of v-Src-expressing cells showed that cytokinesis failure is caused by cleavage furrow regression. Microscopic analysis revealed that v-Src induces delocalization of cytokinesis regulators including Aurora B and Mklp1. Tetraploid cell formation is one of the causes of chromosome instability; however, tetraploid cells can be eliminated at the tetraploidy checkpoint. Interestingly, v-Src weakens the tetraploidy checkpoint by inhibiting the nuclear exclusion of the transcription coactivator YAP, which is downstream of the Hippo pathway and its nuclear exclusion is critical in the tetraploidy checkpoint. We also discuss the relationship between v-Src-induced chromosome instability and growth suppression in v-Src-induced oncogenesis.
PDF
