Temperature-sensitive Cell Growth Research Articles

The F-box Protein Rcy1 Is Involved in the Degradation of Histone H3 Variant Cse4 and Genome Maintenance

Cse4, a histone H3-like centromeric protein, plays critical functions in chromosome segregation. Cse4 level is tightly regulated, but the underlying mechanism remains poorly understood. We employed a toxicity-based screen to look for the degradation components involved in Cse4 regulation. Here, we show that the F-box containing protein Rcy1 is required for efficient Cse4 turnover as Cse4 degradation is compromised in yeast cells lacking RCY1 Excessive Cse4 accumulation in rcy1Δ cells leads to growth retardation. Furthermore, the deletion of RCY1 is tied to enhanced chromosome instability and temperature-sensitive cell growth. Our results reveal the involvement of Rcy1 in chromosome regulation and another regulatory pathway controlling the Cse4 level and activity.

Conditionally immortalized syncytiotrophoblast cell lines as new tools for study of the blood-placenta barrier.

Syncytiotrophoblasts play an essential role in restriction of drug delivery through the blood-placenta barrier (BPB). Conditionally immortalized syncytiotrophoblast cell lines, TR-TBTs, were established at gestational day 18 from pregnant transgenic rats (Tg-rats) harboring the temperature-sensitive SV 40 (ts SV40) large T-antigen. TR-TBTs exhibit temperature-sensitive cell growth due to the expression of SV 40 large T-antigen, and thus the cell growth can be regulated by changing the culture temperature. TR-TBTs exhibit typical properties of syncytiotrophoblast cells, such as syncytium-like morphology, the expression of cytokeratins and hormones, and polarized expression of glucose transporter 1 (GLUT1) and GLUT3. TR-TBTs express in vivo influx and efflux transporters, such as taurine transporter (TauT), betaine/GABA transporter (BGT-1), amino acid transporter 2 (ATA2), organic anion transporting polypeptide 2 (oatp2), organic cation/carnitine transporter (OCTN2), P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP/ABCG2). Moreover, TR-TBTs exhibit taurine, GABA, and DHEA-S uptake activity via TauT, BGT-1, and oatp2, respectively. Therefore, TR-TBTs can be used for the analysis of these functions and would be a good in vitro models for investigating carrier-mediated transport functions at the BPB.

細胞治療とＤＤＳ 条件的不死化血液胎盤細胞株の樹立と薬物動態研究への応用

Syncytiotrophoblasts, which form a continuous barrier between the maternal and fetal circulation, play an essential role in restriction of drug delivery through the blood-placental barrier (BPB). In the BPB many influx and efflux transporters for substrates such as neurotransmitters, neuroactive steroids, and nucleosides may play in the development of the fetus and fetal brain. However, the functions of these transporters in BPB, and the molecular and cellular aspects of these transporters remain unclear. The human term placenta and human chorionic carcinoma cell lines, such as BeWo and JAR cells have been mainly used for analysis of transporter and hormone secretion in the placenta. However it is difficult to confirm in vivo evidence using these conventional cell lines. In order to investigate the relationship between in vitro and in vivo results, cell lines from animals as an in vitro model are needed. We have established new syncytiotrophoblast cell lines (TR-TBTs) from the recently developed transgenic rat harboring temperature-sensitive simian virus 40 large T-antigen gene (Tg-rat). TR-TBTs have temperature-sensitive cell growth due to the expression of is SV 40 large T-antigen. These cell lines had a syncytium-like morphology, could be prepared as monolayers, expressed cytokeratins, rat syncytiotrophoblast markers, and in vivo influx and efflux transporters such as oatp2, TauT, GAT-2/BGT-1, and mdr 1 a. Moreover, TR-TBTs exhibited apical or basal GLUT1. localizations and apical GLUT3 localizations. Thus, these results suggest that TR-TBTs could be very useful tool to clarify the BPB functions, including transporter genes and their regulation systems.

再生医療とＤＤＳ 血液網膜関門および血液脳関門の再構築とドラッグデリバリー研究への応用

Blood-brain (BBB) and inner blood-retinal barriers (iBRB) are formed by tight-junction of the brain and retinal capillary endothelial cells, respectively. These barriers are known to restrict the entry of substances from circulating blood to the brain and retina. For the development of new drug targeting system and tissue regeneration of these barrier organs, it would be very important subject to reconstitute barrier organs using in vitro immortalized cell line. Conditionally immortalized brain and retinal capillary endothelial cell lines were established from a transgenic rat (Tg rat) and rnouse (Tg mouse) harboring temperature-sensitive a simian virus 40 (ts SV 40) large T-antigen. These cell lines have temperature-sensitive cell growth due to the expression of is SV 40 large T-antigen and endothelialmarkers. These cell lines have expression of in vivo influx and efflux transporters such as P-glycoprotein, GLUT 1 and MCT 1, demonstrating that these in vitro models would reflect the in vivo conditions. Gene expression of GLUT 1 and enzyme activities of γ-glutamyl transpeptidase and alkaline phosphatase were significantly induced by the co-culture between rat conditionally immortalized brain endothelial cell line (TR-BBB) and conditionally immortalized astrocyte cell line (TR-AST). Our in vitro model system could be very useful tool to clarify the barrier functions, including transporter genes and tight junction proteins and their regulation system.

Conditionally immortalized cell lines as a new in vitro model for the study of barrier functions.

Conditionally immortalized brain and retinal capillary endothelial and choroid plexus epithelial cell lines were established from a transgenic rat (Tg rat) and mouse (Tg mouse) harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen. These cell lines exhibit temperature-sensitive cell growth due to the expression of ts SV 40 large T-antigen. Mouse brain (TM-BBB) and rat brain (TR-BBB) and rat retinal (TR-iBRB) capillary endothelial cell lines appear to have a spindle-fiber shaped morphology and exhibit the typical endothelial markers, such as von Willebrand factor and acetylated low-density lipoprotein uptake. These cell lines express in vivo influx and efflux transporters, such as P-glycoprotein (P-gp) and GLUT1, which is capable of 3-O-methyl-D-glucose transport. TM-BBB cells are able to undergo efflux transport of cyclosporin A, which is a substrate for P-gp transport activity. They may also express oatp2 and exhibit dehydroepiandrosterone sulfate and digoxin uptake activity. TR-BBB cells express the mRNA of multidrug resistance associated protein 1 (MRP1) and a large neutral amino acid transporter, which consists of LAT1 and 4F2hc. TR-iBRB cells exhibit pH-dependent L-lactic acid transport activity and express the mRNA of monocarboxylate transporter (MCT) 1 and 2. The choroid plexus epithelial cell line (TR-CSFB) has polygonal cell morphology, expresses the typical choroid plexus epithelial cell marker, transthyretin, and has Na+, K+-ATPase located on the apical side. TR-CSFB cells also exhibit amino acid transport activity which has been observed in vivo. These barrier cell lines established from the Tg rat and Tg mouse have in vivo transport functions and are good in vitro models for drug transport to the brain and retina and as a screen for drugs which might be capable of delivery to the brain and retina.

The RFC2 gene, encoding the third-largest subunit of the replication factor C complex, is required for an S-phase checkpoint in Saccharomyces cerevisiae.

Replication factor C (RF-C), an auxiliary factor for DNA polymerases delta and epsilon, is a multiprotein complex consisting of five different polypeptides. It recognizes a primer on a template DNA, binds to a primer terminus, and helps load proliferating cell nuclear antigen onto the DNA template. The RFC2 gene encodes the third-largest subunit of the RF-C complex. To elucidate the role of this subunit in DNA metabolism, we isolated a thermosensitive mutation (rfc2-1) in the RFC2 gene. It was shown that mutant cells having the rfc2-1 mutation exhibit (i) temperature-sensitive cell growth; (ii) defects in the integrity of chromosomal DNA at restrictive temperatures; (iii) progression through cell cycle without definitive terminal morphology and rapid loss of cell viability at restrictive temperatures; (iv) sensitivity to hydroxyurea, methyl methanesulfonate, and UV light; and (v) increased rate of spontaneous mitotic recombination and chromosome loss. These phenotypes of the mutant suggest that the RFC2 gene product is required not only for chromosomal DNA replication but also for a cell cycle checkpoint. It was also shown that the rfc2-1 mutation is synthetically lethal with either the cdc44-1 or rfc5-1 mutation and that the restrictive temperature of rfc2-1 mutant cells can be lowered by combining either with the cdc2-2 or pol2-11 mutation. Finally, it was shown that the temperature-sensitive cell growth phenotype and checkpoint defect of the rfc2-1 mutation can be suppressed by a multicopy plasmid containing the RFC5 gene. These results suggest that the RFC2 gene product interacts with the CDC44/RFC1 and RFC5 gene products in the RF-C complex and with both DNA polymerases delta and epsilon during chromosomal DNA replication.

Characterization of a human genomic DNA fragment which rescues defective lipid-linked oligosaccharide synthesis in a mutant G258 cell line isolated from the FM3A mouse mammary carcinoma cell line.

The G258 mutant cell line, isolated from the FM3A mouse mammary carcinoma cell line, is temperature-sensitive for both cell growth and asparagine-linked glycosylation due to mutation at a single location. The biochemical defect in the G258 mutant resides in the formation of lipid-linked oligosaccharide, presumably in one of the steps of GDP-mannose-dependent mannosylation (Y. Nishikawa, J. Cell. Physiol. 119, 260-266, 1984; Y. Nishikawa, Biochim. Biophys. Acta 1091, 135-140, 1991). In the present study, we transfected human genomic DNA fragments into the G258 mutant by the radiation hybrid method and isolated transformants (KK-1, -3 and -4) which showed recovery from both temperature-sensitive cell growth and asparagine-linked glycosylation. These transformants contained a common Alu-containing human DNA fragment (1.3 kb) which will be used as a marker for isolating the gene that complements the defect of lipid-liked oligosaccharide synthesis in the G258 mutant.

Mutations causing high basal level transcription that is independent of transcriptional activators but dependent on chromosomal position in Saccharomyces cerevisiae.

Two single (bel2 and bel4) and two double (bel3 bel7 and bel5 be16) mutations causing enhanced transcription of a gene fusion, consisting of the open reading frame of PHO5 connected to the HIS5 promoter (HIS5p) integrated at the ura3 or leu2 locus, were isolated from a gcn4-disrupted mutant of Saccharomyces cerevisiae. The PHO5 gene, encoding repressible acid phosphatase, in the HIS5p-PHO5 construct was derepressed under amino acid starved conditions by the action of the transcriptional activator Gcn4p. The bel mutants showed temperature-sensitive cell growth and/or cell aggregation. All the mutants except bel4 also showed high levels of transcription of an intact PHO5 DNA integrated at the URA3 locus in the absence of the cognate transcriptional activator, Pho4p, and in the absence of upstream activating sequences of PHO5. The HIS5 and PHO5 genes at their original chromosomal positions were, however, not affected by the bel2 mutation. The BEL2 gene was found to be identical with SIN4/TSF3, mutations in which cause high levels of transcription of the HO and GAL genes in the absence of their respective transcriptional activators, Swi5p and Gal4p. The effect of the bel2/sin4/tsf3 mutation on PHO5 transcription was additive with the Pho4p function. Thus the effect of the bel2/sin4/tsf3 mutation is dependent on the position of PHO5 in the chromosome and independent of Pho4p and Gen4p activation.

Elongation factor 2 mutants deficient in diphthamide formation show temperature-sensitive cell growth.

Protein synthesis elongation factor 2 (EF-2) from eukaryotes contains an unusual modified histidine residue, termed diphthamide. Diphthamide has been shown to be a site of ADP-ribosylation by bacterial toxins, but its function remains obscure. We expressed mutant genes of EF-2 with substitutions of 19 other amino acids for His-699, which is modified to diphthamide, in yeast cells and found that they can be classified into three groups. In the first group (Group 1), replacement of His-699 by the basic amino acid Arg or Lys showed not only loss of EF-2 activity but also inhibitory effects on the growth of cells co-expressing wild-type EF-2. In the second group (Group 2), replacement with Gly, Pro, Ser, or Asp resulted in nonfunctional EF-2, but it did not affect the growth of cells co-expressing wild-type EF-2. In the third group (Group 3), replacement by one of the other 13 amino acids resulted in a functional EF-2. In the Group 3 mutants, EF-2 was not ribosylated by diphtheria toxin, indicating that the mutant EF-2s did not form diphthamide. However, the viable cells grew more slowly than cells expressing wild-type EF-2 and showed temperature sensitivities. This result suggests that diphthamide may confer heat resistance on EF-2, although it still may be active without diphthamide at a normal temperature.

Isolation of mutants showing temperature-sensitive cell growth from embryonal carcinoma cells: Control of stem cell differentiation by incubation temperatures

Embryonal carcinoma(EC) cells, the undifferentiated stem cells of teratocarcinomas, have many properties in common with pluripotent embryonic cells, and thus provide an excellent system for studying the early events involved in embryonic development and stem cell differentiation. We have isolated three novel mutants with temperature-sensitive ( ts ) cell growth that were able to differentiate at a non-permissive temperature for cell growth. These mutations affect the progression of the cell cycle, leading to the transient accumulation of cells in a specific phase, the S phase, of the cell cycle, which is likely to be the primary cause of stem cell differentiation of EC cells at non-permissive temperature. Isolation of these mutants strongly supports the notion that there is a close association between the inhibition of DNA synthesis and EC cell differentiation.

Host function of MAK16: G1 arrest by a mak16 mutant of Saccharomyces cerevisiae.

The MAK16 gene was first defined as a gene whose mutation resulted in loss of M1 double-stranded RNA virus-like particles. The mak16-1 mutation also produces temperature-sensitive cell growth. We report here that mak16-1 cells arrest at the nonpermissive temperature in G1 phase, such that they are mating competent. We sequenced the MAK16 gene and found an open reading frame of 306 amino acids encoding a predicted protein of Mr 35,694. Two typical nuclear localization signal sequences were found. MAK16-LacZ fusion proteins that include one of these putative signals entered the nucleus, while unfused beta-galactosidase did not, as judged by subcellular fractionation experiments. In the C-terminal third of the MAK16 open reading frame is an acidic region in which 25 of 41 residues are either glutamate or aspartate. This region contains potential phosphorylation sites for "casein kinases," protein kinases specific for serine or threonine residues in an acidic environment.

Isolation and characterization of rho mutants of Escherichia coli with increased transcription termination activities.

A novel type of rho mutants, rhos, with increased transcription termination activities have been isolated. A termination defective rho mutation rho-ts702 (formerly designated nitA702), which causes temperature-sensitive cell growth, was found to be dominant over the wild-type allele in relieving mutational polarity. The rhos mutations were derived as temperature-resistant revertants of rho-ts702 carried by lambda transducing phage. They exhibited dominance over rho-ts702 leading to restoration of polarity. When the rhos mutations were introduced into the Escherichia coli chromosome, they caused increased polarity in the trp and lac operons. The rhos mutants were classified into two groups in terms of their terminator specificity: The first group demonstrated increased termination efficiencies against all terminators tested, whereas the second exhibited various efficiencies, either more than or less than the normal level depending on the terminator. The cellular content of p protein in each rhos strain was significantly lower than that in the rho+ strain. Moreover, in an in vitro transcription system, purified ps proteins showed increased termination activities against the trpE pseudoterminators. These results indicate that the rhos phenotype is due to qualitative alterations, rather than quantitative increases, of the p protein. The reduced content of ps enforces the current notion that the rho gene is autogenously regulated by rho-dependent transcriptional attenuation.