Temperature-sensitive Cell Line Research Articles

Genome-Wide Chromatin Landscape Transitions Identify Novel Pathways in Early Commitment to Osteoblast Differentiation.

Bone continuously undergoes remodeling by a tightly regulated process that involves osteoblast differentiation from Mesenchymal Stem Cells (MSC). However, commitment of MSC to osteoblastic lineage is a poorly understood process. Chromatin organization functions as a molecular gatekeeper of DNA functions. Detection of sites that are hypersensitive to Dnase I has been used for detailed examination of changes in response to hormones and differentiation cues. To investigate the early steps in commitment of MSC to osteoblasts, we used a model human temperature-sensitive cell line, hFOB. When shifted to non-permissive temperature, these cells undergo "spontaneous" differentiation that takes several weeks, a process that is greatly accelerated by osteogenic induction media. We performed Dnase I hypersensitivity assays combined with deep sequencing to identify genome-wide potential regulatory events in cells undergoing early steps of commitment to osteoblasts. Massive reorganization of chromatin occurred within hours of differentiation. Whereas ~30% of unique DHS sites were located in the promoters, the majority was outside of the promoters, designated as enhancers. Many of them were at novel genomic sites and need to be confirmed experimentally. We developed a novel method for identification of cellular networks based solely on DHS enhancers signature correlated to gene expression. The analysis of enhancers that were unique to differentiating cells led to identification of bone developmental program encompassing 147 genes that directly or indirectly participate in osteogenesis. Identification of these pathways provided an unprecedented view of genomic regulation during early steps of differentiation and changes related to WNT, AP-1 and other pathways may have therapeutic implications.

Brain stem cell transplantation daring mild hypothermia treatment of patients with traumatic brain injuries

Objective To study the feasibility of stem cell transplantation under mild hypothermia so as to provide a prerequisite for stem cell transplantation in patients with traumatic brain injuries (TBI) during mild hypothermia treatment. Methods After transfecting plasmid containing temperature-sensitive simian virus 40 large T-antigen (tsSV40LT) into temperature-sensitive umbilical cord mes-enchymal stem cells (tsUCMSCs) , the changes of cell morphology, nuclear proliferation index (PIx) and telomerase activity were detested when the tsUCMSCs were cultured at 33℃ and 37℃. After the mouse model with tTBI treated with mild hypothermia was established, tsUCMSCs were transplanted into semi-injury area to detect survival rate, proliferation and apoptosis indices and perform neurological deficit scoring. Results When cultured at 33℃, the tsUCMSCs displayed long spindle-shaped and highly refractive, with higher proliferation index and telomerase activity than those cultured at 37℃. Compared with control group (non-temperature-sensitive UCMSCs transplantation), tsUCMSCs in semi-injury area showed much higher cell survival and proliferating cell nuclear antigen expression ( P < 0.05 ) , with fewer apoptotic cells and better neurological function (P < 0.05). Conclusion The establishment of temperature-sensitive stem cell line enables stem cell transplantation during treatment of TBI with mild hypothermia, as provides us a new direction for treatment of TBI. Key words: Brain injuries; Hypothermia, artificial; Stem cells; Transplantation

Establishment of a temperature-sensitive stem cell line and its application in traumatic brain injury patients during hypothemia treatment

Objective To establish a temperature-sensitive stem cell line for appropriating cells transplantation in injured brain area during patients accepting hypothermia treatment. Method After transfecting plasmid containing temperature-sensitive simian virus 40 large T-antigen (tsSV40LT) into umbilical cord mesenchymal stem cells (UCMSCs), PCR was used to detect gene integration. Then cell growth curves were drawn in 33℃ and 37℃ respectively. Serum-dependent experimental and soft agar colony assay were used to examine the tumorigenesis of the stem cells. After TBI models in thymic mice were established, the stem cells were transplated into injured area. Proliferating cell nuclear antigen expression and in situ apoptosis were detected by immunofluorescence. Results The tsSV40LT gene was integrated into UCMSCs successfully. When cultured at 33℃ incubator, the new cell line displayed high proliferation activity, strong tolerance to low nutrient conditions, and strong cell clone ability. But when cultured at 37℃ incubator, the cell line showed a converse profile. In vivo, the results showed that the brain tissue during hypothermia treatment more highly expressed PCNA, and existed lower cell apoptosis, than normothermia group. Conclusions The stem cell line modified by tsSV40LT is highly temperature-sensitive, and its proliferation activity can be regulated effectively, and that supports the clinical application of stem cells transplantation into injured brain area during hypothermia treatment. Key words: Craniocerebral trauma; Hypothermia; Stem cells; Cell proliferation

Involvement of the IκB Kinase (IKK)-Related Kinases Tank-Binding Kinase 1/IKKi and Cullin-Based Ubiquitin Ligases in IFN Regulatory Factor-3 Degradation

Activation of the innate arm of the immune system following pathogen infection relies on the recruitment of latent transcription factors involved in the induction of a subset of genes responsible for viral clearance. One of these transcription factors, IFN regulatory factor 3 (IRF-3), is targeted for proteosomal degradation following virus infection. However, the molecular mechanisms involved in this process are still unknown. In this study, we show that polyubiquitination of IRF-3 increases in response to Sendai virus infection. Using an E1 temperature-sensitive cell line, we demonstrate that polyubiquitination is required for the observed degradation of IRF-3. Inactivation of NEDD8-activating E1 enzyme also results in stabilization of IRF-3 suggesting the NEDDylation also plays a role in IRF-3 degradation following Sendai virus infection. In agreement with this observation, IRF-3 is recruited to Cullin1 following virus infection and overexpression of a dominant-negative mutant of Cullin1 significantly inhibits the degradation of IRF-3 observed in infected cells. We also asked whether the C-terminal cluster of phosphoacceptor sites of IRF-3 could serve as a destabilization signal and we therefore measured the half-life of C-terminal phosphomimetic IRF-3 mutants. Interestingly, we found them to be short-lived in contrast to wild-type IRF-3. In addition, no degradation of IRF-3 was observed in TBK1(-/-) mouse embryonic fibroblasts. All together, these data demonstrate that virus infection stimulates a host cell signaling pathway that modulates the expression level of IRF-3 through its C-terminal phosphorylation by the IkappaB kinase-related kinases followed by its polyubiquitination, which is mediated in part by a Cullin-based ubiquitin ligase.

Differential Effects of Modification of Membrane Cholesterol and Sphingolipids on the Conformation, Function, and Trafficking of the G Protein-coupled Cholecystokinin Receptor

The lipid microenvironment of receptors can influence their conformation, function, and regulation. Cholecystokinin (CCK)-stimulated signaling is abnormal in some forms of hyperlipidemia, suggesting the possibility of unique sensitivity to its lipid environment. Here we examined the influence of cholesterol and sphingolipids on CCK receptors in model Chinese hamster ovary cell systems having lipid levels modified. Cholesterol was modulated chemically or metabolically, and sphingolipids were modulated using a temperature-sensitive cell line (SPB-1). Receptor conformation was probed with a fluorescent full agonist ligand, Alexa 488-conjugated Gly-[Nle(28,31)]CCK-(26-33), shown previously to decrease in anisotropy and lifetime when occupying a receptor in the active conformation (Harikumar, K. G., Pinon, D. L., Wessels, W. S., Prendergast, F. G., and Miller, L. J. (2002) J. Biol. Chem. 277, 18552-18560). Anisotropy and lifetime of this probe were increased and prolonged with cholesterol enrichment, and decreased and shortened with depletion of cholesterol or sphingolipids. The increase in these parameters with cholesterol enrichment may reflect change in CCK receptor conformation toward its inactive, uncoupled state. Indeed, cholesterol enrichment resulted in nonproductive agonist ligand binding, with affinity of binding higher than normal and calcium signaling in response to this reduced. In cholesterol- and sphingolipid-depleted states, the receptor moved into conformations that were less than optimal. With cholesterol depletion, both ligand binding and signaling were decreased, yet internalization and trafficking were unperturbed. With sphingolipid depletion, ligand binding and signaling were normal, but internalization and trafficking were markedly inhibited. Of note, normal transferrin receptor trafficking through the same clathrin-dependent pathway was maintained under these conditions. Thus, lipid microenvironment of the CCK receptor is particularly important, with different lipids having distinct effects.

Characterization of a temperature-sensitive mouse middle ear epithelial cell line

Conclusion. Temperature-sensitive mouse middle ear epithelial cells have been successfully established and characterized. Objective. Temperature-sensitive middle ear epithelial cell lines are essential for pathophysiologic studies of otitis media. They are useful for studying the pathogen–host interaction, receptor identification, signal transduction, cytokine/mucin production and cellular responses, especially for cell proliferation and differentiation. The purpose of this study was to establish a large T-antigen [simian virus 40 A-gene (SV40)] mutant-immortalized mouse middle ear epithelial cell line for otitis media studies. Material and methods. Primary culture of middle ear epithelial cells was established from the middle ear mucosa of an Immortomouse. The cells were transduced by a temperature-sensitive large T-antigen mutant and cultured for >50 passages. The expression of mRNA transcripts and proteins for epithelial cells was characterized by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. The temperature-sensitive properties of cells cultured at 33°C and 39°C were evaluated using 3H-thymidine incorporation, Trypan blue exclusion and peroxisome proliferator-activated receptor-γ activity. Results. Immortalized middle ear epithelial cells demonstrated a cobblestone-like monolayer culture. The cells expressed mucosal cell markers such as mucins, keratins and collagens. They proliferated at 33°C when the SV40 antigen was active and differentiated at 39°C when the SV40 antigen was inactive.

Quantification of Jκ signal end breaks in developing B cells by blunt-end linker ligation and qPCR

Introduction of a double-strand DNA break at the junction between a rearranging gene segment and its flanking recombination signal sequence (RSS) is the first step of V(D)J recombination. Such DNA breaks can be detected by either Southern blot hybridization or ligation-mediated PCR. While Southern blotting is easily quantifiable, it is often insufficiently sensitive and while LM-PCR is far more sensitive, it is poorly quantifiable. Reported here is a LM-qPCR assay which relies on real-time qPCR to provide an absolute measure of recombinase-mediated, or any other specific, double-strand DNA break in genomic DNA. The efficiency of the initial ligation reaction was found to be relatively low with just 3% of potential targets undergoing linker ligation. Using this assay, approximately 16% of murine bone marrow pre-B cells were determined to contain a dsDNA break adjacent to the Jκ1 gene segment. In addition, the kinetics of JΚκ1 dsDNA breaks in a temperature-sensitive cell line induced to recombine its κ locus was determined.

CAMP-dependent Protein Kinase Regulates Ubiquitin-Proteasome-mediated Degradation and Subcellular Localization of the Nuclear Receptor Coactivator GRIP1

Nuclear receptors and their coactivators are key regulators of numerous physiological functions. GRIP1 (glucocorticoid receptor-interacting protein) is a member of the steroid receptor coactivator family. Here, we show that GRIP1 is regulated by cAMP-dependent protein kinase (PKA) that induces its degradation through the ubiquitin-proteasome pathway. GRIP1 was down-regulated in transiently transfected COS-1 cells after treatment with 8-para-chlorophenylthio-cAMP or forskolin and 3-isobutyl-1-methylxanthine and in adrenocortical Y1 cells after incubation with adrenocorticotropic hormone. Pulse-chase experiments with transiently transfected COS-1 cells demonstrated that the half-life of GRIP1 was markedly reduced in cells overexpressing the PKA catalytic subunit, suggesting that activation of PKA increases the turnover of GRIP1 protein. The proteasome inhibitors MG132 and lactacystin abolished the PKA-mediated degradation of GRIP1. Using ts20 cells, a temperature-sensitive cell line that contains a thermolabile ubiquitin-activating E1 enzyme, it was confirmed that PKA-mediated degradation of GRIP1 is dependent upon the ubiquitin-proteasome pathway. Coimmunoprecipitation studies of COS-1 cells transfected with expression vectors encoding GRIP1 and ubiquitin using anti-GRIP1 and anti-ubiquitin antibodies showed that the ubiquitination of GRIP1 was increased by overexpression of PKA. Finally, we show that PKA regulates the intracellular distribution pattern of green fluorescent protein-GRIP1 and stimulates recruitment of GRIP1 to subnuclear foci that are colocalized with the proteasome. Taken together, these data demonstrate that GRIP1 is ubiquitinated and degraded through activation of the PKA pathway. This may represent a novel regulatory mechanism whereby hormones down-regulate a nuclear receptor coactivator.

Proteolysis of DNA Replication Licensing Factor Cdt1 in S-phase Is Performed Independently of Geminin through Its N-terminal Region

Licensing of replication origins is carefully regulated in a cell cycle to maintain genome integrity. Using an in vivo ubiquitination assay and temperature-sensitive cell lines we demonstrate that Cdt1 in mammalian cells is degraded through ubiquitin-dependent proteolysis in S-phase. siRNA experiments for Geminin indicate that Cdt1 is degraded in the absence of Geminin. The N terminus of Cdt1 is required for its nuclear localization, associates with cyclin A, but is dispensable for the association of Cdt1 with Geminin in cells. This region is responsible for proteolysis of Cdt1 in S-phase. On the other hand, the N terminus-truncated Cdt1 is stable in S-phase, and associates with the licensing inhibitor, Geminin. High level expression of this form of Cdt1 brings about cells having higher DNA content. Proteasome inhibitors stabilize Cdt1 in S-phase, and the protein is detected in the nucleus in a complex with Geminin. This form of Cdt1 associates with chromatin as tightly as that of G1-cells, when no Geminin is detected. Our data show that proteolysis and Geminin binding independently inactivate Cdt1 after the onset of S-phase to prevent re-replication.

Identification and Characterization of the Cytoplasmic Protein TRAF4 as a p53-regulated Proapoptotic Gene

The role of p53 in tumor suppression partly relies on its ability to transcriptionally regulate target genes involved in the initiation of cell cycle arrest or the activation of programmed cell death. In recent years many genes have been identified as p53-regulated genes; however, no single target gene has been shown to be required for the full apoptotic effect. We have identified TRAF4 as a p53-regulated gene in a microarray screen using a Murine 11K Affymetrix GeneChip hybridized with cRNA from the p53 temperature-sensitive cell line, Vm10. TRAF4 is a member the TRAF family of adaptor proteins that mediate cellular signaling by binding to various members of the tumor necrosis family receptor superfamily and interleukin-1/Toll-like receptor super-family. In contrast to its other family members, TRAF4 has not been shown to bind to a member of the tumor necrosis factor receptor superfamily in vivo, nor has it been shown to regulate signaling pathways common to its other family members. Therefore the role of TRAF4 in a signaling pathway has not yet been established and requires further study. TRAF4 is specifically regulated by p53 in response to temperature sensitive p53, overexpression of p53 by use of an adenovirus, and stabilization of p53 in response to DNA damage. The murine TRAF4 promoter contains a functional p53 DNA-binding site approximately 1 kilobase upstream of the initiating methionine. TRAF4 localizes to the cytoplasm and appears to remain in the cytoplasm following DNA damage. Interestingly, the overexpression of TRAF4 induces apoptosis and suppresses colony formation. These data suggest a correlation that the orphan adaptor protein TRAF4 may play a role in p53-mediated proapoptotic signaling in the response to cellular stress.

Microarray Expression Profiling of p53-Dependent Transcriptional Changes in an Immortalized Mouse Embryo Fibroblast Cell Line

The ability of p53 to transcriptionally regulate genes involved with cell cycle progression and apoptosis is critical to its role as a tumor suppressor. Although numerous p53 regulated genes have been identified over the last several years, ablation of any one of these genes cannot account for the full p53-mediated cellular response. Therefore, we performed microarray analysis using two related p53 temperature sensitive cell lines, Val5 and Vm10, to identify novel p53 regulated genes. The Val5 cells undergo p53-mediated cell cycle arrest and the Vm10 cells undergo p53-mediated apoptosis when p53 is in the wild-type conformation. By using these two cell lines, we can compare which genes are regulated by p53 in two different conditions as well as analyze which genes are common to both cell lines. Using the information obtained in the microarray analysis, we confirmed whether a small sub-set of the genes was regulated by p53 using northern blot analysis. By identifying and confirming the regulation of specific genes by p53, we can further characterize biologically why p53 transcriptionally regulates these genes.

Immortalized epithelial cells from human autosomal dominant polycystic kidney cysts.

Autosomal dominant polycystic kidney disease (ADPKD) is the result of mutations in one allele of the PKD1 or PKD2 genes, followed by "second hit" somatic mutations of the other allele in renal tubule cells. Continued proliferation of clonal cells originating from different nephron segments leads to cyst formation. In vitro studies of the mechanisms of cyst formation have been hampered by the scarcity of nephrectomy specimens and the limited life span of cyst-derived cells in primary culture. We describe the development of a series of immortalized epithelial cell lines from over 30 individual renal cysts obtained from 11 patients with ADPKD. The cells were immortalized with either wild-type (WT) or temperature-sensitive (TS) recombinant adeno-simian virus (SV)40 viruses. SV40 DNA integration into the cell genome was verified by PCR analysis. The cells have been passaged over 50 times with no apparent phenotypic change. By light microscopy, the cells appear pleomorphic but mostly polygonal and resemble the primary cultures. Transmission electron microscopy shows polarized epithelia with tight junctions. The SV40 large T antigen was detected by immunocytochemistry and by Western blot analysis at 37 degrees C in the WT cell lines and at 33 degrees C in the TS cell lines. It disappeared in TS cells 72 h following transfer to 39 degrees C. The majority (29) of the cell lines show binding of Dolichos biflorus lectin, suggesting distal tubule origin. Three cell lines show binding of Lotus tetragonolobus lectin or express aminopeptidase N, suggesting proximal tubule origin. Three cell lines were derived from a mixture of cysts and express features of both tubules. The PKD1 and PKD2 mRNA and protein were detected in all cells by RT-PCR and by immunocytochemistry. The majority of the cells tested also express the epidermal growth factor receptor, cystic fibrosis transmembrane conductance regulator, epithelial sodium channel, and renin. These new series of cyst-derived cell lines represent useful and readily available in vitro models for studying the cellular and molecular biology of ADPKD.

Specific ubiquitin-conjugating enzymes promote degradation of specific nuclear receptor coactivators.

Nuclear receptor coactivators (NRCoAs) are nuclear hormone receptor-associated regulatory proteins that interact with members of the nuclear receptor superfamily in the presence of their cognate ligand, enhancing their transcriptional activity. The identification of ubiquitin-proteasome pathway proteins as coactivators provides evidence that ubiquitin-proteasome-mediated protein degradation plays an integral role in eukaryotic gene transcription. It has also been observed that nuclear receptors themselves are ubiquitinated and degraded in a hormone-dependent manner and that ubiquitin-proteasome function is essential for most nuclear receptors to function as transactivators. Here, we show that specific ubiquitin-proteasome pathway enzymes target specific NRCoA proteins in vivo and in vitro. First, using a temperature-sensitive cell line that contains a thermolabile ubiquitin-activating E1 enzyme, we confirmed that NRCoA proteins are targets of the ubiquitin-proteasome pathway. Then using coimmunoprecipitation studies, we also demonstrate that in vivo, NRCoA proteins are ubiquitinated. Finally, we illustrate that in vitro, NRCoA ubiquitination and degradation depend on the ubiquitin-activating enzyme (E1) and on specific ubiquitin-conjugating enzymes (E2) for each of the coactivators.

Conditionally Immortalized Clonal Cell Lines from the Mouse Olfactory Placode Differentiate into Olfactory Receptor Neurons

To test extracellular signals that direct the development of the olfactory system, we have generated clonal temperature-sensitive cell lines that represent distinct cellular lineages derived from the E10 mouse olfactory placode. Two of these lines, OP6 and OP27, express (at the permissive temperature), a transcriptional profile representing intermediate-late developmental stages in the olfactory receptor neuron (ORN) lineage. At the nonpermissive temperature, both OP6 and OP27 cells can be induced by all-trans retinoic acid to differentiate into a population of mature bipolar ORN-like cells. In response to retinoic acid, differentiated OP6 and OP27 down-regulate neuron-specific transcription factors required for early stages of neuronal differentiation, and shift active components of the neurotrophin signaling cascade (Trk receptors) into a kinase inactive state. When morphologically mature, OP6 and OP27 express the mature ORN chemosensory signaling components, olfactory G-protein (Golf), Type III adenylate cyclase (ACIII), OCNC1, and the olfactory marker protein (OMP). OP27 expresses one odorant receptor, OR 27-3. OP6 expresses two very closely related receptors, OR 6–13 and OR 6–8. Voltage-gated sodium and potassium channels resembling those recorded from primary cultures of ORNs can also be recorded from a subset of differentiated OP6 cells.

Effect of wortmannin on the suppression of thermotolerance in tsAF8 cells

Phosphatidylinositol 3 kinases (PI-3 kinases) play a crucial role in the inositol phospholipid signaling pathway. The effect of wortmannin, an inhibitor of PI-3 kinases, on the suppression of thermotolerance development was investigated in the temperature-sensitive cell line tsAF8. When wortmannin was added immediately after the preheating, the thermotolerance was suppressed, with this suppression being dose-dependent from 0.01 to 0.1 microM, but not dose-dependent from 0.1 to 20 microM. Our results suggest that PI-3 kinases might play an important role in thermotolerance development in tsAF8 cells.

Analysis of HCF, the cellular cofactor of VP16, in herpes simplex virus-infected cells.

Herpes simplex virus (HSV) immediate-early (IE) gene expression is initiated via the recruitment of the structural protein VP16 onto specific sites upstream of each IE gene promoter in a multicomponent complex (TRF.C) that also includes the cellular proteins Oct-1 and HCF. In vitro results have shown that HCF binds directly to VP16 and stabilizes TRF.C. Results from transfection assays have also indicated that HCF is involved in the nuclear import of VP16. However, there have been no reports on the role or the fate of HCF during HSV type 1 (HSV-1) infection. Here we show that the intracellular distribution of HCF is dramatically altered during HSV-1 infection and that the protein interacts with and colocalizes with VP16. Moreover, viral protein synthesis and replication were significantly reduced after infection of a BHK-21-derived temperature-sensitive cell line (tsBN67) which contains a mutant HCF unable to associate with VP16 at the nonpermissive temperature. Intracellular distribution of HCF and of newly synthesized VP16 in tsBN67-infected cells was similar to that observed in Vero cells, suggesting that late in infection the trafficking of both proteins was not dependent on their association. We constructed a stable cell line (tsBN67r) in which the temperature-sensitive phenotype was rescued by using an epitope-tagged wild-type HCF. In HSV-1-infected tsBN67r cells at the nonpermissive temperature, direct binding of HCF to VP16 was observed, but virus protein synthesis and replication were not restored to levels observed at the permissive temperature or in wild-type BHK cells. Together these results indicate that the factors involved in compartmentalization of VP16 alter during infection and that late in infection, VP16 and HCF may have additional roles reflected in their colocalization in replication compartments.

Inhibition of thermotolerance observed on tsAF8 cells

The characteristic development of thermotolerance was examined in tsAF8 cells, which were temperature sensitive cell line derived from BHK21. tsAF8 cells lose RNA polymerase II activity and arrest in the G1 phase of the cell cycle at the nonpermissive temperature of 39.5-40.6°C. tsAF8 cells were pre-heated at 45°C for 20 min, incubated at 34 or 39.7°C for different durations, and then heated again at 45°C for 30 min. The cells were trypsinized immediately after the second heating to evaluate cell survival. As a result, thermotolerance was developed rapidly in the cells incubated at 34°C. However, the survival rate of the cells incubated at 39.7°C was almost constant. Thermotolerance of BHK21 cells, which were wild-type cells of tsAF8 cells, developed at 39.7°C. These results suggest that thermotolerance may be inhibited in tsAF8 cells incubated at 39.7°C, and that some metabolism which is inhibited in tsAF8 cells at 39.7°C may be related to thermotolerance development.

Proteasomes – an appetite for mRNA

In eukaryotic cells, mRNAs can have vastly different half lives, and mRNA decay is thus an important regulatory event in gene expression. Rapid degradation of mRNAs is facilitated by the presence of a 3′ AU-rich element (ARE). Members of a family of ARE-binding proteins known as AUF1 proteins have been implicated in the decay process, and there is evidence that mRNA decay can be triggered by translation, but the general mechanism of decay has largely been unclear. This paper now demonstrates that the ubiquitin–proteasome pathway is crucial for mRNA decay1xControl of mRNA decay by heat shock-ubiquitin–proteasome pathway. Laroia, G., Cuesta, R., Brewer, G., and Schneider, R.J. Science. 1999; 284: 499–502Crossref | PubMed | Scopus (318)See all

Impaired induction of heat shock protein implicated in decreased thermotolerance in a temperature-sensitive multinucleated cell line.

Cells of a temperature-sensitive mutant line (tsFT101) derived from a mouse mammary carcinoma cell line (FM3A) become multinucleated at a non-permissive temperature of 39 degrees C because of disturbed cytokinesis. To explore how this relates to thermotolerance, we examined the proliferative activity of, and heat shock protein (HSP) expression in, FM3A and tsFT101 cells cultured at 37 degrees C and 39 degrees C after heat shock pretreatment (15 min exposure at 45 degrees C). FM3A cells developed thermotolerance when cultured at both 37 degrees C and 39 degrees C, but whereas tsFT101 cells developed thermotolerance at 37 degrees C, this was markedly reduced at 39 degrees C. Western blot analysis showed similar degrees of expression of constitutive HSP70 (HSP73) in FM3A and tsFT101 cells after heat shock pretreatment at both 37 degrees C and 39 degrees C. However, expression of inducible HSP70 (HSP72) was reduced in tsFT101 cells at 39 degrees C compared to 37 degrees C and to FM3A cells at both 37 degrees C and 39 degrees C. Heat shock pretreatment activated DNA binding of heat shock transcription factor (HSF) in FM3A cells at 37 degrees C and 39 degrees C, but only at 37 degrees C in tsFT101 cells. These results indicate that (1) multinucleation caused by disturbed cytokinesis increases temperature sensitivity, (2) HSP70 is critical for the development of thermotolerance in both FM3A and tsFT101 cells, and (3) decreased expression of inducible HSP70 parallels deficient development of thermotolerance in tsFT101 cells cultured at a non-permissive temperature.

DAD1 Is Required for the Function and the Structural Integrity of the Oligosaccharyltransferase Complex

Asparagine-linked glycosylation is a highly conserved protein modification reaction that occurs in all eukaryotic organisms. The oligosaccharyltransferase (OST), which has its active site exposed on the luminal face of the endoplasmic reticulum (ER), catalyzes the transfer of preassembled high mannose oligosaccharides onto certain asparagine residues of nascent polypeptides. The mammalian OST complex was initially thought to be composed of three transmembrane proteins, ribophorin I (RI), ribophorin II (RII), and OST48. Most recently, a small integral membrane protein of 12 kDa called DAD1 has been identified as an additional member of the mammalian OST complex. A point mutation in the DAD1 gene is responsible for the temperature-sensitive phenotype of a baby hamster kidney-derived cell line (tsBN7) that undergoes apoptosis at the non-permissive temperature. Furthermore, the mutant protein DAD1 is not detectable in tsBN7 cells 6 h after shifting the cells to the non-permissive temperature. This temperature-sensitive cell line offered unique opportunities to study the effects caused by the loss of one OST subunit on the other three subunits and also on N-linked glycosylation. Western blot analysis of cell lysates showed that after 6 h at the non-permissive temperature, steady-state levels of the ribophorins were reduced by about 50%, and OST48 was barely detectable. On the other hand, steady-state levels of other components of the rough ER, such as the alpha-subunits of the TRAP (translocon-associated membrane protein) and the Sec61 complex, which are components of the translocation apparatus, are not affected by the instability of the OST subunits. Furthermore, N-glycosylation of the ribophorins was seriously affected 6 h after shifting the cells to the non-permissive temperature, and after 12 h they were synthesized only in the non-glycosylated form. As may be expected, this defect in the OST complex at the non-permissive temperature caused also the underglycosylation of a secretory glycoprotein. We concluded that degradation of DAD1 at the non-permissive temperature not only affects the stability of OST48 and the ribophorins but also results in the functional inactivation of the OST complex.