Physiological quiescence in plasma-derived serum: Influence of platelet-derived growth factor on cell growth in culture

A platelet-derived growth factor can be shown to be the principal stimulant of DNA synthesis in whole blood serum for those cells that require serum for maintenance and growth in culture. Cell free plasma-derived serum lacks such platelet-derived material. 3T3 cells and primate arterial smooth muscle cells can be maintained in a quiescent state in culture for as long as six weeks in plasma-derived serum. Such cells can grow logarithmically after exposure to 5% whole blood serum or as little as 100 ng/ml of partially purified platelet factor. The cell cycle of smooth muscle cells has been studied in the quiescent (5% plasma-derived serum) and growing state (5% whole blood serum or 5% plasma-derived serum plus platelet factor). The generation time of smooth muscle cells is 16 to 18 hours as shown by autoradiographic frequency of labelled mitoses. The generation time is the same for cells in the growth fraction in either 5% whole blood serum or 5% plasma-derived serum. Thus, platelet factor acts by recruiting cells into the growth fraction rather than effecting a change in the duration of the cell cycle. Flow microfluorimetry studies on cells growing logarithmically in 5% whole blood serum give the following phase durations: G₁ = 5.6 hours; S = 7.6 hours; and G₂ + M = 3.8 hours. Based on these studies the argument is presented that cells cultured in 5% plasma-derived serum provide a more physiological base for the study of quiescence than do cells in low concentrations of whole blood serum or confluent, density inhibited cells at high (5% or greater) concentrations of whole blood serum. Furthermore, 5% plasma-derived serum represents an appropriate state to examine the perturbation of quiescent cells.     

Effect of B-mercaptoethanol on the viability of IVM/IVF/IVC bovine embryos during long-distance transportation in plastic straws

Experiments were conducted to assess the effect of beta-mercaptoethanol (beta-ME) on the quality and viability of bovine blastocysts derived from in-vitro culture (IVC) of in-vitro matured and fertilized (TVM-IVF) oocytes during their transport between 2 distant places. Follicular oocytes were collected from ovaries obtained at a slaughterhouse and were cultured for 20 to 21 h in modified TCM-199. The IVM oocytes were fertilized in vitro with frozen-thawed spermatozoa. Fertilized oocytes were cultured for 7 d, and embryos that developed to the blastocyst stage were used for the experiments. The blastocysts, packed in straws with transportation medium that consisted of modified TCM-199 with HEPES equilibrated in air and supplemented with 20 % calf serum and 0, 10, 50, 100 or 150 microM beta-ME, were transported at 37 degrees C from Tokyo to Sapporo by air (18.3 h). The quality of blastocysts was assessed and ranked as excellent (A), good (B), fair (C) or poor (D) after transportation. The percentages of blastocysts ranked as A or B were significantly higher (P < 0.05) when the embryos were transported in beta-ME supplemented medium (80 to 100%) than when transported without beta-ME (54 %). Blastocysts ranked as A or B after transportation in medium with or without 150 microM beta-ME were nonsurgically transferred to synchronous recipients; 60 d after embryo transfer, 21/36 and 19/35 cows, respectively, were diagnosed as pregnant by palpation per rectum. These results indicate that beta-ME maintains the quality of bovine blastocysts in plastic straws for several hours without control of CO2 and that the concentration of beta-ME used in this experiment is not detrimental to the blastocysts.     

A model for microcarrier motion inside a horizontally rotating bioreactor for animal cell culture

The motion of microcarriers inside the horizontally rotating bioreactor was simulated in order to obtain some insight as to how particle motion can affect radial mass transfer. Fluid motion was modeled taking into account momentum transfer induced by particle motion. The force balance on the particle included the viscous drag, inertia, gravitational and buoyant forces. The main characteristics of observed particle motion under conditions of low particle concentration were reproduced by the model. Some implications of particle motion to mass transfer are discussed.     

The beta3 chain short arm of laminin-332 (laminin-5) induces matrix assembly and cell adhesion activity of laminin-511 (laminin-10)

The basement membrane (BM) protein laminin-332 (Lm332) (laminin-5) has unique activity and structure as compared with other laminins: it strongly promotes cellular adhesion and migration, and its alpha3, beta3, and gamma2 chains are all truncated in their N-terminal regions (short arms). In the present study, we investigated the biological function of the laminin beta3 chain. When the beta3 chain short arm (beta3SA) was overexpressed in HEK293 cells (beta3SA-HEK), they deposited a large amount of beta3SA and a small amount of laminin-511 (Lm511) (laminin-10) on culture plates. Control HEK293 cells secreted Lm511 but failed to deposit it. The extracellular matrix (ECM) deposited by beta3SA-HEK cells strongly promoted cell attachment and spreading. The beta3SA-HEK ECM did not directly bind Lm511, but it stimulated control HEK293 cells to deposit Lm511 on the culture plates. Although purified beta3SA did not support cell adhesion by itself, it enhanced the cell adhesion activity of Lm511. Experiments with anti-integrin antibodies also suggested that the strong cell adhesion activity of the beta3SA-HEK ECM was derived from the synergistic action of beta3SA and Lm511. It has previously been found that beta3SA binds an unknown cell surface receptor. Taken together, the present study suggests that the short arm of the laminin beta3 chain enhances the matrix assembly of Lm511 and its cell adhesion activity by interacting with its receptor.     

Selective accumulation of hybrid liposomes into adult T-cell leukemia cells along with induction of apoptosis

Markedly inhibitory effects of hybrid liposomes (HL-n) composed of 90 mol % l-α-dimyristoylphosphatidylcholine (DMPC) and 10 mol % polyoxyethylene(n) dodecyl ethers on the growth of adult T-cell leukemia cells were obtained for the first time. It is noteworthy that HL-n could selectively accumulate into the adult T-cell leukemia cells and induce apoptosis via caspase-3 activation.     

Wnt/beta-catenin signaling down-regulates N-acetylglucosaminyltransferase III expression: the implications of two mutually exclusive pathways for regulation

In previous studies, we reported that N-acetylglucosaminyltransferase III (GnT-III) activity and the enzyme product, bisected N-glycans, both were induced in cells cultured under dense conditions in an E-cadherin-dependent manner (Iijima, J., Zhao, Y., Isaji, T., Kameyama, A., Nakaya, S., Wang, X., Ihara, H., Cheng, X., Nakagawa, T., Miyoshi, E., Kondo, A., Narimatsu, H., Taniguchi, N., and Gu, J. (2006) J. Biol. Chem. 281, 13038-13046). Furthermore, we found that α-catenin, a component of the E-cadherin-catenin complex, was also required for this induction (Akama, R., Sato, Y., Kariya, Y., Isaji, T., Fukuda, T., Lu, L., Taniguchi, N., Ozawa, M., and Gu, J. (2008) Proteomics 8, 3221-3228). To further explore the molecular mechanism of this regulation, the roles of β-catenin, an essential molecule in both cadherin-mediated cell adhesion and canonical Wnt signaling, were investigated. Unexpectedly, shRNA knockdown of β-catenin resulted in a dramatic increase in GnT-III expression and its product, the bisected N-glycans, which was confirmed by RT-PCR and GnT-III activity and by E4-PHA lectin blot analysis. The induction of GnT-III expression increased bisecting GlcNAc residues on β1 integrin, which led to down-regulation of integrin-mediated cell adhesion and cell migration. Immunostaining showed that nuclear localization of β-catenin was greatly suppressed; intriguingly, the knockdown of β-catenin in the nuclei was more effective than that in cell-cell contacts in the knockdown cells, which was also confirmed by Western blot analysis. Stimulation of the Wnt signaling pathway by the addition of exogenous Wnt3a or BIO, a GSK-3β inhibitor, consistently and significantly inhibited GnT-III expression and its products. Conversely, the inhibition of β-catenin translocation into the nuclei increased GnT-III activation. Taken together, the results of the present study are the first to clearly demonstrate that GnT-III expression may be precisely regulated by the interplay of E-cadherin-catenin complex-mediated cell-cell adhesion and Wnt/β-catenin signaling, which are both crucial in the process of epithelial-mesenchymal transitions in physiological and pathological conditions.     

Potential roles of N-glycosylation in cell adhesion

The functional units of cell adhesion are typically multiprotein complexes made up of three general classes of proteins; the adhesion receptors, the cell-extracellular matrix (ECM) proteins, and the cytoplasmic plaque/peripheral membrane proteins. The cell adhesion receptors are usually transmembrane glycoproteins (for example E-cadherin and integrin) that mediate binding at the extracellular surface and determine the specificity of cell-cell and cell-ECM recognition. E-cadherin-mediated cell-cell adhesion can be both temporally and spatially regulated during development, and represents a key step in the acquisition of the invasive phenotype for many tumors. On the other hand, integrin-mediated cell-ECM interactions play important roles in cytoskeleton organization and in the transduction of intracellular signals to regulate various processes such as proliferation, differentiation and cell migration. ECM proteins are typically large glycoproteins, including the collagens, fibronectins, laminins, and proteoglycans that assemble into fibrils or other complex macromolecular arrays. The most of these adhesive proteins are glycosylated. Here, we focus mainly on the modification of N-glycans of integrins and laminin-332, and a mutual regulation between cell adhesion and bisected N-glycan expression, to address the important roles of N-glycans in cell adhesion.     

Inhibition of autophagy by chloroquine induces apoptosis in primary effusion lymphoma in vitro and in vivo through induction of endoplasmic reticulum stress

Autophagy plays a crucial role in cancer cell survival and the inhibition of autophagy is attracting attention as an emerging strategy for the treatment of cancer. Chloroquine (CQ) is an anti-malarial drug, and is also known as an inhibitor of autophagy. Recently, it has been found that CQ induces cancer cell death through the inhibition of autophagy; however, the underlying mechanism is not entirely understood. In this study, we identified the role of CQ-induced cancer cell death using Primary Effusion Lymphoma (PEL) cells. We found that a CQ treatment induced caspase-dependent apoptosis in vitro. CQ also suppressed PEL cell growth in a PEL xenograft mouse model. We showed that CQ activated endoplasmic reticulum (ER) stress signal pathways and induced CHOP, which is an inducer of apoptosis. CQ-induced cell death was significantly decreased by salbrinal, an ER stress inhibitor, indicating that CQ-induced apoptosis in PEL cells depended on ER stress. We show here for the first time that the inhibition of autophagy induces ER stress-mediated apoptosis in PEL cells. Thus, the inhibition of autophagy is a novel strategy for cancer chemotherapy.     

Two types of protein kinase C with different functions in cultured rabbit aortic smooth muscle cells

Incubation of cultured rabbit aortic smooth muscle cells (SMC) with phorbol-12, 13-dibutyrate (PDBu) for 48 h caused the down-regulation of protein kinase C (PKC) to the level of 30-40% of that in the control cells. The proliferative and antiproliferative actions of PKC were abolished in parallel with the loss of the down-regulation-sensitive component of PKC, but the inhibitory actions in the whole blood serum (WBS)-induced phospholipase C (PLC) reactions and intracellular Ca2+ mobilization were not affected. Immunoblot analysis with specific monoclonal antibodies against three PKC isozymes (type I, II and III) revealed that only the type III isozyme was detected in rabbit aortic SMC and that this isozyme completely disappeared after the incubation with PDBu. These results indicate that the type III isozyme is responsible for the proliferative and antiproliferative actions and suggest that the unidentified isozyme(s) is involved in the inhibitory actions in the WBS-induced PLC reactions and intracellular Ca2+ mobilization in rabbit aortic SMC.