Development of a novel co-culture system using human pancreatic cancer cells and human iPSC-derived stellate cells to mimic the characteristics of pancreatic ductal adenocarcinoma in vitro.

Pancreatic ductal adenocarcinoma (PDAC) is a serious disease with poor prognosis and prone to chemotherapy resistance. It is speculated that the tumor microenvironment (TME) of PDAC contributes to these characteristics. However, the detailed mechanisms of interactions between pancreatic cancer cells and stroma in the TME are unclear. Therefore, the aim of this study was to establish a co-culture system that mimics the TME, using cancer cells derived from PDAC patient specimens and stellate cells from human induced pluripotent stem cells as stromal cells. We succeeded in observing the interaction between cancer cells and stellate cells and reproduced some features of PDAC in vitro using our co-culture systems. In addition, we demonstrated the applicability of our co-culture system for drug treatment in vitro. To conclude, we propose our co-culture system as a novel method to analyze cell-cell interactions, especially in the TME of PDAC.

Differential regulation of p27Kip1 depending on culture conditions and its correlation with status of p14ARF and p53.

p27Kip1 is known as a major cyclin-dependent kinase inhibitor and a tumor suppressor, and often functionally hampered at protein level. p27 protein expression levels are frequently low in various cancers and negatively correlated with malignancy of cancer. However, in our previous study, we discovered that p27 overexpression does not inhibit the proliferation of two cancer cell lines due to a functional suppression of p27 by nucleophosmin isoform 1 (NPM1); that is, a qualitative, not quantitative, suppression of p27 function occurs in these cancer cell lines. To clarify the regulation of p27 in several types of cancer, we investigated p27 function in other cancer cell lines, based on proliferation assays in those cell lines carrying doxycycline-inducible p27, and found that MDAH041 cells which express p14ARF, an antagonist of NPM1, show growth inhibition depending on p27 induction. Moreover, to investigate p27 function under anchorage-independent culture conditions, we performed soft agar colony formation assay and observed that the colony formation of some cell lines carrying wild-type p53, a major tumor suppressor, was inhibited depending on p27 induction. These results suggest that p27 function is regulated differentially among cancer cell types under anchorage-dependent and anchorage-independent culture conditions.

Photosynthetic 1,8-cineole production using cyanobacteria.

Terpenoid is an important group of compounds not only as biocomponents but also as useful secondary metabolites. A volatile terpenoid 1,8-cineole, which is used as a food additive, flavoring agent, cosmetic, etc., is also attracting attention from a medical perspective due to its antiinflammation and antioxidation. The 1,8-cineole fermentation using a recombinant Escherichia coli strain has been reported, although a carbon source supplement is necessary for a high-yield 1,8-cineole production. We constructed the 1,8-cineole-producing cyanobacteria toward a carbon-free and sustainable 1,8-cineole production. cnsA, the 1,8-cineole synthase gene in Streptomyces clavuligerus ATCC 27064, was introduced and overexpressed in the cyanobacterium Synechococcus elongatus PCC 7942. We succeeded in producing an average of 105.6 µg g-1 wet cell weight of 1,8-cineole in S. elongatus 7942 without supplementing any carbon source. Using the cyanobacteria expression system is an efficient approach to producing 1,8-cineole by photosynthesis.

Evolution of Ribosomal Protein S14 Demonstrated by the Reconstruction of Chimeric Ribosomes in Bacillus subtilis.

Ribosomal protein S14 can be classified into three types. The first, the C+ type has a Zn2+ binding motif and is ancestral. The second and third are the C- short and C- long types, neither of which contain a Zn2+ binding motif and which are ca. 90 residues and 100 residues in length, respectively. In the present study, the C+ type S14 from Bacillus subtilis ribosomes (S14BsC+) were completely replaced by the heterologous C- long type of S14 from Escherichia coli (S14Ec) or Synechococcus elongatus (S14Se). Surprisingly, S14Ec and S14Se were incorporated fully into 70S ribosomes in B. subtilis However, the growth rates as well as the sporulation efficiency of the mutants harboring heterologous S14 were significantly decreased. In these mutants, the polysome fraction was decreased and the 30S and 50S subunits accumulated unusually, indicating that cellular translational activity of these mutants was decreased. In vitro analysis showed a reduction in the translational activity of the 70S ribosome fraction purified from these mutants. The abundance of ribosomal proteins S2 and S3 in the 30S fraction in these mutants was reduced while that of S14 was not significantly decreased. It seems likely that binding of heterologous S14 changes the structure of the 30S subunit, which causes a decrease in the assembly efficiency of S2 and S3, which are located near the binding site of S14. Moreover, we found that S3 from S. elongatus cannot function in B. subtilis unless S14Se is present.IMPORTANCE S14, an essential ribosomal protein, may have evolved to adapt bacteria to zinc-limited environments by replacement of a zinc-binding motif with a zinc-independent sequence. It was expected that the bacterial ribosome would be tolerant to replacement of S14 because of the previous prediction that the spread of C- type S14 involved horizontal gene transfer. In this study, we completely replaced the C+ type of S14 in B. subtilis ribosome with the heterologous C- long type of S14 and characterized the resulting chimeric ribosomes. Our results suggest that the B. subtilis ribosome is permissive for the replacement of S14, but coevolution of S3 might be required to utilize the C- long type of S14 more effectively.

Novel (p)ppGpp0 suppressor mutations reveal an unexpected link between methionine catabolism and GTP synthesis in Bacillus subtilis.

In bacteria, guanosine (penta)tetra-phosphate ([p]ppGpp) is essential for controlling intracellular metabolism that is needed to adapt to environmental changes, such as amino acid starvation. The (p)ppGpp0 strain of Bacillus subtilis, which lacks (p)ppGpp synthetase, is unable to form colonies on minimal medium. Here, we found suppressor mutations in the (p)ppGpp0 strain, in the purine nucleotide biosynthesis genes, prs, purF and rpoB/C, which encode RNA polymerase core enzymes. In comparing our work with prior studies of ppGpp0 suppressors, we discovered that methionine addition masks the suppression on minimal medium, especially of rpoB/C mutations. Furthermore, methionine addition increases intracellular GTP in rpoB suppressor and this effect is decreased by inhibiting GTP biosynthesis, indicating that methionine addition activated GTP biosynthesis and inhibited growth under amino acid starvation conditions in (p)ppGpp0 backgrounds. Furthermore, we propose that the increase in intracellular GTP levels induced by methionine is due to methionine derivatives that increase the activity of the de novo GTP biosynthesis enzyme, GuaB. Our study sheds light on the potential relationship between GTP homeostasis and methionine metabolism, which may be the key to adapting to environmental changes.

Hyperactivation of cyclin A-CDK induces centrosome overduplication and chromosome tetraploidization in mouse cells.

Mammalian cyclin A-CDK (cyclin-dependent kinase) activity during mitotic exit is regulated by two redundant pathways, cyclin degradation and CDK inhibitors (CKIs). Ectopic expression of a destruction box-truncated (thereby stabilized) mutant of cyclin A in the mouse embryonic fibroblasts nullizygous for three CKIs (p21, p27, and p107) results in constitutive activation ("hyperactivation") of cyclin A-CDK and induces rapid tetraploidization, suggesting loss of the two redundant pathways causes genomic instability. To elucidate the mechanism underlying teraploidization by hyperactive cyclin A-CDK, we first examined if the induction of tetraploidization depends on specific cell cycle stage(s). Arresting the cell cycle at either S phase or M phase blocked the induction of tetraploidization, which was restored by subsequent release from the arrest. These results suggest that both S- and M-phase progressions are necessary for the tetraploidization by hyperactive cyclin A-CDK and that the tetraploidization is not caused by chromosome endoreduplication but by mitotic failure. We also observed that the induction of tetraploidization is associated with excessive duplication of centrosomes, which was suppressed by S-phase but not M-phase block, suggesting that hyperactive cyclin A-CDK promotes centrosome overduplication during S phase. Time-lapse microscopy revealed that hyperactive cyclin A-CDK can lead cells to bypass cell division and enter pseudo-G1 state. These observations implicate that hyperactive cyclin A-CDK causes centrosome overduplication, which leads to mitotic slippage and subsequent tetraploidization.

Analysis of E1A domains involved in the enhancement of CDK2 activity.

E1A is an adenoviral protein which is expressed at the early phase after viral infection and contains four conserved regions (CR1, CR2, CR3 and CR4). Our previous work suggests that E1A facilitates the formation of cyclin A-CDK2 complex and thereby enhances CDK2 activity. However, the molecular function of E1A in CDK2 activation has been unclear. Here, we studied the mechanism of enhancement of CDK2 activity by E1A, using the E1A variant forms which selectively contain CR domains. We isolated four E1A variant forms, i.e. 13S (containing CR1, CR2, CR3, CR4), 12S (CR1, CR2, CR4), 10S (CR2, CR4) and 9S (CR4), derived from HEK293 cells which express E1A. 13S promoted G2/M-phase arrest, upon CDK2 hyper-activation by co-expressing a stabilized cyclin A mutant, most strongly among those E1A variant forms. Concomitantly, the specific activity of the 13S-associated CDK2 was highest among them. 10S exhibited lower affinity for CDK2 than the 13S while the affinity for CDK2 was comparable between 13S and 12S. Nonetheless, 12S did not enhance the CDK2 specific activity. On the other hand, a mutation in CR2 domain, which is essential for binding to p107, suppressed both the binding and activation of CDK2. These results suggest that CR1 domain, in addition to CR2 domain via p107 interaction, is important for binding to CycA-CDK2 complex while CR3 domain facilitates CDK2 activation. Since the function of CR3 in cell cycle regulation has been relatively unknown, we propose the enhancement of CDK2 activity as a novel function of CR3 domain.

ParA-like protein influences the distribution of multi-copy chromosomes in cyanobacterium Synechococcus elongatus PCC 7942.

While many bacteria, such as Escherichia coli and Bacillus subtilis, harbour a single-copy chromosome, freshwater cyanobacteria have multiple copies of each chromosome per cell. Although it has been reported that multi-copy chromosomes are evenly distributed along the major axis of the cell in cyanobacterium Synechococcus elongatus PCC 7942, the distribution mechanism of these chromosomes remains unclear. In S. elongatus, the carboxysome, a metabolic microcompartment for carbon fixation that is distributed in a similar manner to the multi-copy chromosomes, is regulated by ParA-like protein (hereafter ParA). To elucidate the role of ParA in the distribution of multi-copy chromosomes, we constructed and analysed ParA disruptant and overexpressing strains of S. elongatus. Our fluorescence in situ hybridization assay revealed that the parA disruptants displayed an aberrant distribution of their multi-copy chromosomes. In the parA disruptant the multiple origin and terminus foci, corresponding to the intracellular position of each chromosomal region, were aggregated, which was compensated by the expression of exogenous ParA from other genomic loci. The parA disruptant is sensitive to UV-C compared to the WT strain. Additionally, giant cells appeared under ParA overexpression at the late stage of growth indicating that excess ParA indirectly inhibits cell division. Screening of the ParA-interacting proteins by yeast two-hybrid analysis revealed four candidates that are involved in DNA repair and cell membrane biogenesis. These results suggest that ParA is involved in the pleiotropic cellular functions with these proteins, while parA is dispensable for cell viability in S. elongatus.

Essentiality of WalRK for growth in Bacillus subtilis and its role during heat stress.

WalRK is an essential two-component signal transduction system that plays a central role in coordinating cell wall synthesis and cell growth in Bacillus subtilis. However, the physiological role of WalRK and its essentiality for growth have not been elucidated. We investigated the behaviour of WalRK during heat stress and its essentiality for cell proliferation. We determined that the inactivation of the walHI genes which encode the negative modulator of WalK, resulted in growth defects and eventual cell lysis at high temperatures. Screening of suppressor mutations revealed that the inactivation of LytE, an dl-endopeptidase, restored the growth of the ΔwalHI mutant at high temperatures. Suppressor mutations that reduced heat induction arising from the walRK regulon were also mapped to the walK ORF. Therefore, we hypothesized that overactivation of LytE affects the phenotype of the ΔwalHI mutant. This hypothesis was corroborated by the overexpression of the negative regulator of LytE, IseA and PdaC, which rescued the growth of the ΔwalHI mutant at high temperatures. Elucidating the cause of the temperature sensitivity of the ΔwalHI mutant could explain the essentiality of WalRK. We proved that the constitutive expression of lytE or cwlO using a synthetic promoter uncouples these expressions from WalRK, and renders WalRK nonessential in the pdaC and iseA mutant backgrounds. We propose that the essentiality of WalRK is derived from the coordination of cell wall metabolism with cell growth by regulating dl-endopeptidase activity under various growth conditions.

Variety of DNA Replication Activity Among Cyanobacteria Correlates with Distinct Respiration Activity in the Dark.

Cyanobacteria exhibit light-dependent cell growth since most of their cellular energy is obtained by photosynthesis. In Synechococcus elongatus PCC 7942, one of the model cyanobacteria, DNA replication depends on photosynthetic electron transport. However, the critical signal for the regulatory mechanism of DNA replication has not been identified. In addition, conservation of this regulatory mechanism has not been investigated among cyanobacteria. To understand this regulatory signal and its dependence on light, we examined the regulation of DNA replication under both light and dark conditions among three model cyanobacteria, S. elongatus PCC 7942, Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120. Interestingly, DNA replication activity in Synechocystis and Anabaena was retained when cells were transferred to the dark, although it was drastically decreased in S. elongatus. Glycogen metabolism and respiration were higher in Synechocystis and Anabaena than in S. elongatus in the dark. Moreover, DNA replication activity in Synechocystis and Anabaena was reduced to the same level as that in S. elongatus by inhibition of respiratory electron transport after transfer to the dark. These results demonstrate that there is disparity in DNA replication occurring in the dark among cyanobacteria, which is caused by the difference in activity of respiratory electron transport.

Defective interaction between p27 and cyclin A-CDK complex in certain human cancer cell lines revealed by split YFP assay in living cells.

Cyclin-cyclin dependent kinase (CDK) complex is negatively regulated by interaction with CDK inhibitors (CKIs). p27 protein is a major CKI in mammals and its down-regulation correlates with malignant transformation. However, some cancer cells express p27 at normal level, suggesting not only quantitative but qualitative control of p27, although little is known about such control. We analyzed the interaction between p27 and cyclin A (CycA)-CDK complex in living human cell lines, using a split yellow fluorescent protein (YFP) system in which the YFP fluorescence solely depends on p27-CycA binding. Introduction of this system into various cancer cell lines revealed that certain cell lines show no detectable YFP fluorescence. Furthermore, these cell lines exhibited reduced p27-CycA interaction as evaluated by immunoprecipitation, while they showed normal co-localization of both proteins. These results suggest that some cancer cells are defective for efficient interaction between p27 and CycA-CDK complex due to qualitative alteration(s).

An essential enzyme for phospholipid synthesis associates with the Bacillus subtilis divisome.

The mechanism by which the membrane synthetic machinery might be co-organized with the cell-division architecture during the bacterial cell cycle remains to be investigated. We characterized a key enzyme of phospholipid and fatty acid synthesis in Bacillus subtilis, the acyl-acyl carrier protein phosphate acyltransferase (PlsX), and identified it as a component of the cell-division machinery. Comprehensive interaction analysis revealed that PlsX interacts with FtsA, the FtsZ-anchoring protein. PlsX mainly localized at the potential division site independent of FtsA and FtsZ and then colocalized with FtsA. By multidirectional approaches, we revealed that the Z-ring stabilizes the association of PlsX at the septum and pole. The localization of PlsX is also affected by the progression of DNA replication. PlsX is needed for cell division and its inactivation leads to aberrant Z-ring formation. We propose that PlsX localization is prior to Z-ring formation in the hierarchy of septum formation events and that PlsX is important for co-ordinating membrane synthesis with cell division in order to properly complete septum formation.

5-Aminolevulinic acid enhances cell death under thermal stress in certain cancer cell lines.

5-aminolevulinic acid (5-ALA) is contained in all organisms and a starting substrate for heme biosynthesis. Since administration of 5-ALA specifically leads cancer cells to accumulate protoporphyrin IX (PpIX), a potent photosensitizer, we tested if 5-ALA also serves as a thermosensitizer. 5-ALA enhanced heat-induced cell death of cancer cell lines such as HepG2, Caco-2, and Kato III, but not other cancer cell lines including U2-OS and normal cell lines including WI-38. Those 5-ALA-sensitive cancer cells, but neither U2-OS nor WI-38, accumulated intracellular PpIX and exhibited an increased reactive oxygen species (ROS) generation under thermal stress with 5-ALA treatment. In addition, blocking the PpIX-exporting transporter ABCG2 in U2-OS and WI-38 cells enhanced their cell death under thermal stress with 5-ALA. Finally, a ROS scavenger compromised the cell death enhancement by 5-ALA. These suggest that 5-ALA can sensitize certain cancer cells, but not normal cells, to thermal stress via accumulation of PpIX and increase of ROS generation.

Regulation of the response regulator gene degU through the binding of SinR/SlrR and exclusion of SinR/SlrR by DegU in Bacillus subtilis.

Bacillus subtilis DegU is a response regulator of the DegS-DegU two-component regulatory system. Phosphorylated DegU (DegU-P) controls many genes and biological processes, such as exoprotease and γ-polyglutamic acid production, in addition to the degU gene, by binding to target gene promoters. Nonphosphorylated DegU and low levels of DegU-P are required for swarming motility and genetic competence. The DNA-binding repressors SinR and SlrR are part of a double-negative feedback loop and comprise the epigenetic switch governing biofilm formation. In this study, we found that SinR repressed degU. Furthermore, SlrR, which interacts with SinR through protein-protein interaction, seems to have an active role in degU expression in in vivo lacZ analysis. An in vitro transcription assay supported this observation. An electrophoretic mobility shift assay (EMSA) showed that SinR bound to the degU promoter and that SlrR formed a complex with SinR on the degU promoter. In EMSA, DegU-P excluded the SinR/SlrR complex but not SinR from the degU promoter in the presence of RNA polymerase. These findings suggest that DegU-P interacts with SlrR. In support of this hypothesis, disruption of the slrR gene resulted in decreased degU expression. This newly identified regulatory mechanism for degU is considered to be sequential transcription factor replacement.

Stable expression of a GFP-reporter gene in the red alga Cyanidioschyzon merolae.

The unicellular red alga Cyanidioschyzon merolae is used as a model organism to investigate the basic architecture of photosynthetic eukaryotes. We established a stable expression system for the green fluorescent protein fused with the phycocyanin-associated rod linker (APCC) protein in C. merolae, which was clearly localized on the plastid. This system should be useful in the genetic engineering of C. merolae.

Light-dependent and asynchronous replication of cyanobacterial multi-copy chromosomes.

While bacteria such as Escherichia coli and Bacillus subtilis harbour a single circular chromosome, some freshwater cyanobacteria have multiple chromosomes p er cell. The detailed mechanism(s) of cyanobacterialreplication remains unclear. To elucidate the replication origin (ori ), form and synchrony of the multi-copy genome in freshwater cyanobacteria Synechococcus elongatus PCC 7942 we constructed strain S. 7942TK that can incorporate 5-bromo-2'- deoxyuridine (BrdU) into genomic DNA and analysed its de novo DNA synthesis. The uptake of BrdU was blocked under dark and resumed after transfer of the culture to light conditions. Mapping analysis of nascent DNA fragments using a next-generation sequencer indicated that replication starts bidirectionally from a single ori, which locates in the upstream region of the dnaN gene. Quantitative analysis of BrdU-labelled DNA and whole-genome sequence analysis indicated that the peak timing of replication precedes that of cell division and that replication is initiated asynchronously not only among cell populations but also among the multi-copy chromosomes. Our findings suggest that replication initiation is regulated less stringently in S. 7942 than in E. coli and B. subtilis.

Characterization of a cyanobacterial rep protein with broad-host range and its utilization for expression vectors.

Owing to their photosynthetic capabilities, cyanobacteria are regarded as ecologically friendly hosts for production of biomaterials. However, compared to other bacteria, tools for genetic engineering, especially expression vector systems, are limited. In this study, we characterized a Rep protein, exhibiting replication activity in multiple cyanobacteria and established an expression vector using this protein. Our comprehensive screening using a genomic library of Synechocystis sp. PCC 6803 revealed that a certain region encoding a Rep-related protein (here named Cyanobacterial Rep protein A2: CyRepA2) exhibits high autonomous replication activity in a heterologous host cyanobacterium, Synechococcus elongatus PCC 7942. A reporter assay using GFP showed that the expression vector pYS carrying CyRepA2 can be maintained in not only S. 6803 and S. 7942, but also Synechococcus sp. PCC 7002 and Anabaena sp. PCC 7120. In S. 7942, GFP expression in the pYS-based system was tightly regulated by IPTG, achieving 10-fold higher levels than in the chromosome-based system. Furthermore, pYS could be used together with the conventional vector pEX, which was constructed from an endogenous plasmid in S. 7942. The combination of pYS with other vectors is useful for genetic engineering, such as modifying metabolic pathways, and is expected to improve the performance of cyanobacteria as bioproduction chassis.

Regulation of RNase E during the UV stress response in the cyanobacterium Synechocystis sp. PCC 6803.

Endoribonucleases govern the maturation and degradation of RNA and are indispensable in the posttranscriptional regulation of gene expression. A key endoribonuclease in Gram-negative bacteria is RNase E. To ensure an appropriate supply of RNase E, some bacteria, such as Escherichia coli, feedback-regulate RNase E expression via the rne 5'-untranslated region (5' UTR) in cis. However, the mechanisms involved in the control of RNase E in other bacteria largely remain unknown. Cyanobacteria rely on solar light as an energy source for photosynthesis, despite the inherent ultraviolet (UV) irradiation. In this study, we first investigated globally the changes in gene expression in the cyanobacterium Synechocystis sp. PCC 6803 after a brief exposure to UV. Among the 407 responding genes 2 h after UV exposure was a prominent upregulation of rne mRNA level. Moreover, the enzymatic activity of RNase E rapidly increased as well, although the protein stability decreased. This unique response was underpinned by the increased accumulation of full-length rne mRNA caused by the stabilization of its 5' UTR and suppression of premature transcriptional termination, but not by an increased transcription rate. Mapping of RNA 3' ends and in vitro cleavage assays revealed that RNase E cleaves within a stretch of six consecutive uridine residues within the rne 5' UTR, indicating autoregulation. These observations suggest that RNase E in cyanobacteria contributes to reshaping the transcriptome during the UV stress response and that its required activity level is secured at the RNA level despite the enhanced turnover of the protein.

Exploration of a possible partnership among orphan two-component system proteins in cyanobacterium Synechococcus elongatus PCC 7942.

To understand the induction of the adaptive response under various stress conditions, it is important to determine the partnership between histidine kinase and response regulators in the bacterial two-component system (TCS). The genes encoding TCS partners are usually comprised of an operon in the genome, but many of them are orphans in the cyanobacterial genome. There is little information on their partnerships in Synechococcus elongatus PCC 7942. Our comprehensive analysis of protein-protein interactions among all 37 full-length proteins and the truncated domains of 24 orphans revealed a number of specific interactions. They involved evolutionarily well-conserved orphan proteins among cyanobacterial species such as Synpcc7942_0453/Ycf29, NblS/RpaB, NblS/SrrA, SasA/RpaA, and SasA/Synpcc7942_2466. Our investigation of the transphosphorylation of interaction partners indicates that orphan TCSs comprise a complex signaling network.

Novel heat shock response mechanism mediated by the initiation nucleotide of transcription.

Among SigA-dependent promoters in Bacillus subtilis, we compared the nucleotide sequences of heat shock responding and non-responding promoters. Chimeric promoter experiments revealed that the heat shock response could be ascribed to the initiation nucleotide (iNTP) of the transcription. Our in vivo reporter assay results indicated that a full response was achieved using GTP, a reduced response was observed using ATP, and no additional expression was observed using UTP or CTP. We then investigated the in vitro transcription assay in more detail. Enhanced transcription that was dependent upon the iNTP was observed when heat treatment was administered during the pre-initiation period. We next analyzed the efficiency of open complex formation using potassium permanganate footprinting, and our results revealed an increase in the ratio of open complex formation at elevated temperatures. Based on this, we suggest that the overall intensification of transcription at high temperatures was derived from the high efficiency of open complex formation together with the high affinity of RNA polymerase (RNAP) for the initiation nucleotide GTP. To determine if this mechanism observed in B. subtilis RNAP is common among bacterial species, we performed similar experiments using Escherichia coli RNAP. Our results indicated that E. coli RNAP also exhibited both temperature- and iNTP-dependent enhancement of transcription. Although the temperature ranges and the ratios of enhancement are somewhat different, the overall heat shock response mechanism mediated by the iNTP of transcription appears to be conserved among bacterial RNAP.