Cytoskeletal Actin Structure in Osteosarcoma Cells Determines Metastatic Phenotype via Regulating Cell Stiffness,Migration, and Transmigration

Osteosarcoma is the most common primary malignant bone tumor. The cause of death due to osteosarcoma is typically a consequence of metastasis to the lung. Controlling metastasis leads to improved prognosis for osteosarcoma patients. The cell stiffness of several tumor types is involved in metastatic potential; however, it is unclear whether the metastatic potential of osteosarcoma depends on cell stiffness. In this study, we analyzed the cell stiffness of the low metastatic Dunn cell line and its highly metastatic LM8 subline, and compared actin organization, cell proliferation, and metastasis. Actin cytoskeleton, polymerization, stiffness, and other cellular properties were analyzed. The organization of the actin cytoskeleton was evaluated by staining F-actin with Alexa Fluor 488 phalloidin. Cell stiffness was measured using Atomic Force Microscopy (AFM). Cell proliferation, migration, invasion, and adhesion were also evaluated. All experiments were performed using mouse osteosarcoma cell lines cultured in the absence and presence of cytochalasin. In LM8 cells, actin polymerization was strongly suppressed and actin levels were significantly lower than in Dunn cells. Stiffness evaluation revealed that LM8 cells were significantly softer than Dunn. Young’s modulus images showed more rigid fibrillar structures were present in Dunn cells than in LM8 cells. LM8 cells also exhibited a significantly higher proliferation. The migration and invasion potential were also higher in LM8 cells, whereas the adhesion potential was higher in Dunn cells. The administration of cytochalasin resulted in actin filament fragmentation and decreased actin staining intensity and cell stiffness in both LM8 and Dunn cells. Cells with high metastatic potential exhibited lower actin levels and cell stiffness than cells with low metastatic potential. The metastatic phenotype is highly correlated to actin status and cell stiffness in osteosarcoma cells. These results suggest that evaluation of actin dynamics and cell stiffness is an important quantitative diagnostic parameter for predicting metastatic potential. We believe that these parameters represent new reliable quantitative indicators that can facilitate the development of new drugs against metastasis.     

Stabilization of 30 S ribosomal subunits of Bacillus subtilis W168 by spermidine and magnesium ions

An explanation for the fragility of 30 S ribosomal subunits of Bacillus subtilis has been studied. Degradation of 16 S ribosomal RNA, rather than degradation of ribosomal proteins, was found to cause the inactivation of 30 S subunits. Although RNAases were bound specifically to 30 S ribosomal subunits, the RNAases were able to function. Spermidine was found to contribute to the stabilization of 30 S ribosomal subunits by inhibiting the degradation of 16 S ribosomal RNA. A high concentration of Mg²⁺ also stabilized the 30 S ribosomal subunits of Bacillus subtilis. The polypeptide synthetic activity of 30 S ribosomal subunits prepared in the presence of spermidine was at least 4-times greater than that of 30 S ribosomal subunits prepared in the absence of spermidine; this activity was maintained without any loss for 3 months at ?70°C.     

Increase of degree of spermidine stimulation of polypeptide synthesis in the presence of phosphate

The addition of phosphate caused an increase in the degree of spermidine stimulation of polypeptide synthesis in an Escherichia coli and a wheat germ cell-free system. Optimal stimulation of polypeptide synthesis was observed at 20 mm phosphate for both systems, but concentrations of phosphate up to 40 mm had no additional effect. The increase of degree of spermidine stimulation in the presence of phosphate in an E. coli cell-free system occurred at the level of aminoacyl-tRNA binding to ribosomes and not at the level of peptide bond formation, translocation, or aminoacyl-tRNA formation. From the results of studies on RNase A sensitivity of ribosomal subunits and the effect of antibiotics known to act on the 30 S ribosomal subunits, it is suggested that the nature of the 30 S ribosomal subunits is changed by phosphate so that the degree of spermidine stimulation of polypeptide synthesis is increased.     

Signaling flux redistribution at toll-like receptor pathway junctions

Various receptors on cell surface recognize specific extracellular molecules and trigger signal transduction altering gene expression in the nucleus. Gain or loss-of-function mutations of one molecule have shown to affect alternative signaling pathways with a poorly understood mechanism. In Toll-like receptor (TLR) 4 signaling, which branches into MyD88- and TRAM-dependent pathways upon lipopolysaccharide (LPS) stimulation, we investigated the gain or loss-of-function mutations of MyD88. We predict, using a computational model built on the perturbation-response approach and the law of mass conservation, that removal and addition of MyD88 in TLR4 activation, enhances and impairs, respectively, the alternative TRAM-dependent pathway through signaling flux redistribution (SFR) at pathway branches. To verify SFR, we treated MyD88-deficient macrophages with LPS and observed enhancement of TRAM-dependent pathway based on increased IRF3 phosphorylation and induction of Cxcl10 and Ifit2. Furthermore, increasing the amount of MyD88 in cultured cells showed decreased TRAM binding to TLR4. Investigating another TLR4 pathway junction, from TRIF to TRAF6, RIP1 and TBK1, the removal of MyD88-dependent TRAF6 increased expression of TRAM-dependent Cxcl10 and Ifit2. Thus, we demonstrate that SFR is a novel mechanism for enhanced activation of alternative pathways when molecules at pathway junctions are removed. Our data suggest that SFR may enlighten hitherto unexplainable intracellular signaling alterations in genetic diseases where gain or loss-of-function mutations are observed.     

Continuous hemodiafiltration with PMMA Hemofilter in the treatment of patients with septic shock

Septic shock is the most severe form of sepsis. It is widely accepted that cytokines play pivotal roles in the pathophysiology of septic shock. We reported previously that continuous hemodiafiltration (CHDF) using a polymethylmethacrylate (PMMA) membrane hemofilter removed various cytokines from blood continuously and efficiently, mainly by adsorption to membrane matrix of the hemofilter. Furthermore, in April 2000, we introduced to clinical practice a rapid assay system that determines blood levels of IL (interleukin)-6 in approximately 30 min. This enabled us to routinely measure blood IL-6 as an index of cytokine cascade activation in critically ill patients for real-time clinical monitoring of hypercytokinemia. The aim of the present cohort study was to evaluate the clinical efficacy of PMMA-CHDF in septic shock, a typical condition associated with hypercytokinemia. Forty-three patients with septic shock were assessed by monitoring of blood IL-6 level with a rapid assay system and immediate initiation of critical care including PMMA-CHDF for cytokine removal. Following initiation of PMMA-CHDF, early improvement of hemodynamics was noted, as well as an increase in urine output. PMMA-CHDF treatment improved both hypercytokinemia (assessed by measurement of blood IL-6 level) and dysoxia (assessed by measurement of blood lactate level). The present findings suggest that cytokine-oriented critical care using PMMA-CHDF might be an effective strategy for the treatment of septic shock.     

Myristoylation of p39 and p35 is a determinant of cytoplasmic or nuclear localization of active cyclin-dependent kinase 5 complexes

Cdk5 is a member of the cyclin-dependent kinases (Cdks), activated by the neuron-specific activator p39 or p35. The activators also determine the cytoplasmic distribution of active Cdk5, but the mechanism is not yet known. In particular, little is known for p39. p39 and p35 contain localization motifs, such as a second Gly for myristoylation and Lys clusters in the N-terminal p10 region. Using mutant constructs, we investigated the cellular distribution mechanism. We observed that p39 localizes the active Cdk5 complex in the perinuclear region and at the plasma membrane as does p35. We demonstrated the myristoylation of both p39 and p35, and found that it is a major determinant of their membrane association. Plasma membrane targeting depends on the amino acid sequence containing the Lys-cluster in the N-terminal p10 region. In contrast, a non-myristoylated Ala mutant (p39G2A or p35G2A) showed nuclear localization with stronger accumulation of p39G2A than p35G2A. These results indicate that myristoylation regulates the membrane association of p39 as well as p35 and that the Lys cluster controls their trafficking to the plasma membrane. The differential nuclear accumulation of p39 and p35 suggests their segregated functions, p35–Cdk5 in the cytoplasm and p39–Cdk5 in the nucleus.     

An IkappaB-beta COOH terminal region protein is essential for the proliferation of CHO cells under acidic stress

CHO-K1 cells were able to proliferate and maintain pHi homeostasis at pH 6.3. A novel acidic sensitive mutant, AS-5B, which proliferated at pH 7.4 but failed to either proliferate or maintain pHi homeostasis at pH 6.3, was derived from CHO-K1 using a replica method. The acidic-sensitivity of AS-5B was not due to deficiencies in sodium proton exchangers, HCO3- (co)transporters or H+-ATPases. A cDNA clone encoding a COOH terminal region of IkappaB-beta conferred partial acidic-resistance on AS-5B, and the encoded protein was present in CHO-K1, but was nearly absent from AS-5B. Our data demonstrated that the expression of this small protein was essential for the proliferation of CHO cells under acidic stress.     

Massspectrometric analyses of transmembrane proteins in human erythrocyte membrane

It is difficult to understand the functional mechanisms of integral membrane proteins without having protein chemical information on these proteins. Although there have been many attempts to identify functionally important amino acids in membrane proteins, chemically and enzymatically cleaved peptides of integral membrane proteins have been difficult to handle because of their hydrophobic properties. In the present study, we have applied an analytical method to transmembrane proteins combining amino acid sequencing, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, and liquid chromatography with electrospray ionization (LC/ESI) mass spectrometry. We could analyze most (97%) of the tryptic fragments of the transmembrane domains of band 3 as well as other minor membrane proteins. The peptide mapping of the transmembrane domain of band 3 was completed and the peptide mapping information allowed us to identify the fragments containing lysine residues susceptible to 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and to 2,4-dinitrofluorobenzene (DNFB). This method should be applicable to membrane proteins not only in erythrocyte membranes but also in other membranes.