Journal of Cellular Physiology
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Effect of swainsonine, an inhibitor of glycoprotein processing, on cultured mammalian cells Abstract Swainsonine is an indolizidine alkaloid that inhibits glycoprotein processing by inhibiting mannosidase II. Thus, cells grown in the presence of this alkaloid exhibit a decreased amount of complex types of oligosaccharides at their cell surface, and instead have hybrid types of structures. Since this compound could be useful for studying functional roles of glycoproteins, it was important to determine whether it affected the growth of mammalian cells in culture, and whether it was cytotoxic to these cells. At levels of up to 1 μg/ml, swainsonine did not affect the growth rate of Madin‐Darby canine kidney (MDCK) cells, Chinese hamster ovary (CHO), simian virus‐181 (SV‐101), B‐16 melanoma, or intestine 407 cells, as measured by the increase in cell numbers over a 5‐day period. There was also no apparent change in cell size or cell shape in cells grown in the presence of this inhibitor. Swainsonine also did not appear to be cytotoxic, nor to cause alterations in cell morphology, as evidenced by comparison of thin sections of normal and swainsonine‐grown cells in the electron microscope. Since alterations in the oligosaccharide chains of cell surface glycoproteins could greatly affect cell surface properties, we examined the binding of various lectins and bacteria to cells grown in swainsonine as a measure of changes in their cell surface carbohydrates. Thus, when MDCK cells, CHO cells, or B‐16 melanoma cells were grown for several days in the presence of swainsonine (100–500 ng/ml), these cells showed a 50–100% increase in their ability to bind [3 H]concanavalin A, and a substantial decrease in the binding of [3 H]wheat germ agglutinin. These alterations suggested an increase in high‐mannose (or hybrid) types of receptors and a decrease in the complex types. The adhesion of E. coli B‐886, a bacterium that binds to high‐mannose glycoproteins, was also increased 1.5‐to twofold, in cells grown in swainsonine. However, the binding of E. coli SS‐142, another bacterial strain that does not bind to high‐mannose receptors, was not altered by growth in swainsonine. In addition to the decrease in wheat germ agglutinin binding, another indication of a decrease in complex chains was the finding that CHO cells grown in swainsonine were more resistant to the toxic effects of the lectin, ricin. This increased resistance could be measured microscopically by the decrease in the number of cells remaining attached to the plates, or by the inhibition of amino acid incorporation, at various ricin concentrations. The effect of swainsonine on the incorporation of amino acids and sugars into protein was also examined. When MDCK cells were grown overnight in swainsonine (1 μg/ml), or were incubated in the alkaloid for several hours before the start of the experiment, there was no alteration in the incorporation of [3 H]leucine or [3 H]proline into protein. There was, however, a significant inhibition in the incorporation of [3 H]fucose, [3 H]glucosamine, and [3 H]galactose caused by this alkaloid. Fucose incorporation was decreased by about 40%, glucosamine by about 40 or 50%, and galactose by about 50%. In many cases (but not all), the incorporation of mannose was enhanced about 20–30% in cells grown in swainsonine.
Journal of Cellular Physiology - Tập 115 Số 3 - Trang 265-275 - 1983
Mitotic catastrophe and apoptosis induced by docetaxel in hormone‐refractory prostate cancer cells Abstract Studies performed in different experimental and clinical settings have shown that Docetaxel (Doc) is effective in a wide range of tumors and that it exerts its activity through multiple mechanisms of action. However, the sequence of events induced by Doc which leads to cell death is still not fully understood. Moreover, it is not completely clear how Doc induces mitotic catastrophe and whether this process is an end event or followed by apoptosis or necrosis. We investigated the mechanisms by which Doc triggers cell death in hormone‐refractory prostate cancer cells by analyzing cell cycle perturbations, apoptosis‐related marker expression, and morphologic cell alterations. Doc induced a transient increase in G2/M phase followed by the appearance of G0/1 hypo‐ and hyperdiploid cells and increased p21 expression. Time‐ and concentration‐dependent apoptosis was induced in up to 70% of cells, in concomitance with Bcl‐2 phosphorylation, which was followed by caspase‐2 and ‐3 activation. In conclusion, Doc would seem to trigger apoptosis in hormone‐refractory prostate cancer cells via mitotic catastrophe through two forms of mitotic exit, in concomitance with increased p21 expression and caspase‐2 activation. J. Cell. Physiol. 217: 494–501, 2008. © 2008 Wiley‐Liss, Inc.
Journal of Cellular Physiology - Tập 217 Số 2 - Trang 494-501 - 2008
Differential expression of cell surface glycoproteins on various organ‐derived microvascular endothelia and endothelial cell cultures Abstract Glycoproteins expressed on the luminal surfaces of microvascular endothelium derived from various murine organs were analyzed and compared with those expressed by cultured vascular endothelial cells. Cell‐surface vascular proteins were radiolabeled in situ via intracardiac perfusion with lactoperoxidase/Na125 l. Autoradiography confirmed that the radiolabel was restricted to the vessel lumen in most tissues. Controls contained 125 l‐labeled serum proteins to identify adsorbed serum components. Glycoproteins were analyzed by western enzymelinked lectin analysis using detergent extracts of 125 l‐labeled microvessels isolated from different organs. The western transfers were probed with a panel of lectin‐peroxidase conjugates to determine differences in protein glycosylation. The same transfers were also screened for exposed 125 l‐labeled cell‐surface proteins by autoradiography. This dual analysis detected glycoprotein patterns unique for each organ. At least seven major proteins (Mr ∼ 180 K, 130 K, 95 K, 80 K, 75 K, 60 K, 12 K) were common to microvessels derived from each organ; however, certain glycoproteins appeared to be expressed differentially in particular organs. For example, a Mr ∼ 135 K WGA‐binding glycoprotein was detected in brain microvessels, whereas another WGA‐binding glycoprotein of Mr ∼ 40 K was detected only in kidney. In lung microvessels, a Mr ∼ 140 K WGA binding glycoprotein and a Mr ∼ 55 K RCA‐l‐binding galactoprotein were expressed preferentially, and liver microvessels displayed Mr ∼ 220 K protein and a Mr ∼ 135 K PNA‐binding galactoprotein. The cell‐surface‐iodinated protein profiles from in situ labeled microvessels were similar to profiles derived from cultured bovine aortic endothelial cells and several short‐term endothelial cell cultures isolated from different organs. The results from this study suggest that organ‐associated endothelia express glycoprotein fingerprints unique to each organ.
Journal of Cellular Physiology - Tập 136 Số 3 - Trang 398-410 - 1988
Mineral trioxide aggregate enhances the osteogenic capacity of periodontal ligament stem cells via NF‐κB and MAPK signaling pathways Mineral trioxide aggregate (MTA), as a bioactive material, has a widespread application in clinical practice. To date, the effects of MTA on the proliferation and differentiation of human periodontal ligament stem cells (hPDLSCs) remain unclear. hPDLSCs were isolated from human periodontal ligament tissues and cultured with MTA conditioned media. Cell counting kit‐8 (CCK‐8) assay was performed to assess the proliferation capacity of MTA‐treated hPDLSCs. Immunofluorescence assay, alkaline phosphatase (ALP) activity, alizarin red staining, real‐time RT‐PCR, and western blot analyses were used to investigate the odonto/osteogenic capacity of hPDLSCs as well as the involvement of NF‐κB and MAPK pathways. ALP activity assay revealed that 2 mg/ml was the optimal concentration for the induction of hPDLSCs by MTA. The protein expression of DSP, RUNX2, OCN, OSX, OPN, DMP1, ALP, and COL‐I in MTA‐treated hPDLSCs was significantly higher than those in control group (p < 0.01). When hPDLSCs were treated with the inhibitors of NF‐κB and MAPK pathways (U0126, SP600125, SB203580, and BMS345541), the effects of MTA on the differentiation of hPDLSCs were suppressed. Mechanistically, P65 was detected to transfer from cytoplasm to nuclei, as indicated by western blot and immunofluorescence assay. Moreover, MAPK‐related proteins and its downstream transcription factors were also upregulated in MTA‐treated hPDLSCs. Together, mineral trioxide aggregate can promote the odonto/osteogenic capacity of hPDLSCs via activating the NF‐κB and MAPK pathways.
Journal of Cellular Physiology - Tập 233 Số 3 - Trang 2386-2397 - 2018
Tributyltin induces distinct effects on cortical and trabecular bone in female C57Bl/6J mice The retinoid X receptors (RXR), peroxisome proliferator activated receptor gamma (PPARγ), and liver X receptors (LXR) all have been shown to regulate bone homeostasis. Tributyltin (TBT) is an environmental contaminant that is a dual RXRα/β and PPARγ agonist. TBT induces RXR, PPARγ, and LXR‐mediated gene transcription and suppresses osteoblast differentiation in vitro. Bone marrow multipotent mesenchymal stromal cells derived from female C57BL/6J mice were more sensitive to suppression of osteogenesis by TBT than those derived from male mice. In vivo, oral gavage of 12 week old female, C57Bl/6J mice with 10 mg/kg TBT for 10 weeks resulted in femurs with a smaller cross‐sectional area and thinner cortex. Surprisingly, TBT induced significant increases in trabecular thickness, number, and bone volume fraction. TBT treatment did not change the Rankl :Opg RNA ratio in whole bone, and histological analyses showed that osteoclasts in the trabecular space were minimally reduced. In contrast, expression of cardiotrophin‐1, an osteoblastogenic cytokine secreted by osteoclasts, increased. In primary bone marrow macrophage cultures, TBT marginally inhibited the number of osteoclasts that differentiated, in spite of significantly suppressing expression of osteoclast markers Nfatc1 , Acp5 , and Ctsk and resorptive activity. TBT induced expression of RXR‐ and LXR‐dependent genes in whole bone and in vitro osteoclast cultures. However, only an RXR antagonist, but not an LXR antagonist, significantly inhibited TBTs ability to suppress osteoclast differentiation. These results suggest that TBT has distinct effects on cortical versus trabecular bone, likely resulting from independent effects on osteoblast and osteoclast differentiation that are mediated through RXR.
Journal of Cellular Physiology - Tập 233 Số 9 - Trang 7007-7021 - 2018
Activated K‐Ras and INK4a/Arf deficiency promote aggressiveness of pancreatic cancer by induction of EMT consistent with cancer stem cell phenotype Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most frequently diagnosed cancers and the fourth leading cause of cancer‐related death in the United States, suggesting that there is an urgent need to design novel strategies for achieving better treatment outcome of patients diagnosed with PDAC. Our previous study has shown that activation of Notch and NF‐κB play a critical role in the development of PDAC in the compound K‐RasG12D and Ink4a/Arf deficient transgenic mice. However, the exact molecular mechanism by which mutated K‐Ras and Ink4a/Arf deficiency contribute to progression of PDAC remains largely elusive. In the present study, we used multiple methods, such as real‐time RT‐PCR, Western blotting assay, and immunohistochemistry to gain further mechanistic insight. We found that the deletion of Ink4a/Arf in K‐RasG12D expressing mice led to high expression of PDGF‐D signaling pathway in the tumor and tumor‐derived cell line (RInk‐1 cells). Furthermore, PDGF‐D knock‐down in RInk‐1 cells resulted in the inhibition of pancreatosphere formation and down‐regulation of EZH2, CD44, EpCAM, and vimentin. Moreover, we demonstrated that epithelial–mesenchymal transition (EMT) was induced in the compound mice, which is linked with aggressiveness of PDAC. In addition, we demonstrated that tumors from compound transgenic mice have higher expression of cancer stem cell (CSC) markers. These results suggest that the acquisition of EMT phenotype and induction of CSC characteristics could be linked with the aggressiveness of PDAC mediated in part through the activation of PDGF‐D, signaling. J. Cell. Physiol. 228: 556–562, 2013. © 2012 Wiley Periodicals, Inc.
Journal of Cellular Physiology - Tập 228 Số 3 - Trang 556-562 - 2013
Regulation of fibrogenic/fibrolytic genes by platelet‐derived growth factor C, a novel growth factor, in human dermal fibroblasts Abstract Platelet‐derived growth factor (PDGF) is a potent mitogenic and chemotactic cytokine, and PDGF‐C is a novel growth factor belonging to the PDGF family. In this study, to determine whether this growth factor can contribute to fibrosis and tissue remodeling, we examined the effect of PDGF‐CC on the expression of fibrogenic/fibrolytic genes such as type I collagen, fibronectin (FN), matrix metalloproteinases (MMPs), and their inhibitors (TIMPs) in normal human dermal fibroblasts in vitro. PDGF elevated the levels of MMP‐1 or TIMP‐1 protein as well as mRNA, whereas this cytokine had no influence on the expression of type I collagen, FN, or TIMP‐2. PDGF‐CC also increased the levels of MMP‐1 catalytic activity in the cultured media and mRNA expression, which was paralleled that on the levels of promoter activation. Additionally, PDGF‐CC induced the mitogenic and migratory activity of human dermal fibroblasts in a dose‐dependent manner. On the other hand, we also determined the specificity of the inhibitory effect of monoclonal antibodies against PDGF‐CC generated by immunizing balb/c mice with recombinant human PDGF‐CC. This antibody could inhibit the regulatory effects of MMP‐1 or TIMP‐1 synthesis as well as the mitogenic effects on human dermal fibroblasts induced by PDGF‐CC, whereas this antibody did not affect those induced by other PDGF forms such as PDGF‐AA, ‐AB, or ‐BB. These results suggest that this cytokine plays a role in the tissue remodeling. © 2004 Wiley‐Liss, Inc.
Journal of Cellular Physiology - Tập 202 Số 2 - Trang 510-517 - 2005
Transport by the (Na<sup>+</sup>, K<sup>+</sup>) ATPase: Modulation by differentiation inducers and inhibition of protein synthesis in the MDCK kidney epithelial cell line Abstract MDCK kidney epithelial cell cultures exposed to the differentiation inducer hexamethylene bisacetamid (HMBA) for 24 hours exhibited a 50% decrease in transport activity per (Na+ , K+ )‐ATPase molecule (turnover number) but an unchanged number of pump sites (Kennedy and Lever, 1984). Inhibition of protein synthesis by either 10 μM cycloheximide or 2 μM emetine blocked the inhibitory effects of HMBA on Na+ /K+ pump efficiency assessed by measurements of [3 H]‐ouabain binding to intact cells, (Na+ , K+ ) ATPase activity of detergent‐activated cell extracts, and ouabain‐sensitive Rb+ uptake. In the absence of inducer treatment, inhibition of protein synthesis increased Na+ /K+ pump turnover number by twofold while maintaining Na+ /K+ pump activity per cell at a constant level. Intracellular Na+ levels were decreased after cycloheximide treatment; therefore, pump stimulation was not due to substrate effects. Furthermore, cycloheximide effects of Rb+ uptake could be dissociated from effects on tight junctions. These observations suggest that the transport activity of the (Na+ , K+ ) ATPase is tightly regulated by factors dependent on protein synthesis.
Journal of Cellular Physiology - Tập 123 Số 3 - Trang 410-416 - 1985
Activation of cell membrane potassium conductance by mercury in cultured renal epitheloid (MDCK) cells Abstract To elucidate mechanisms of mercury toxicity, the cell membrane potential has been determined continuously in cultured kidney (MDCK)‐cells during reversible application of mercury ions to extracellular perfusate. Exposure of the cells to 1μM mercury ions is followed by rapid, sustained, and slowly reversible hyperpolarization of the cell membrane, increase of cell membrane potassium selectivity, and decrease of cell membrane resistance. Thus, mercury ions enhance the potassium conductance of the cell membrane. Half maximal hyperpolarizing effect is elicited by ≈0.2 μM. Higher concentrations of mercury ions (> 10 μM) eventually depolarize the cell membrane. At extracellular calcium activity reduced to less than 0.1 μM, 1 μM mercury ions still leads to a sustained hyperpolarization and increase of potassium selectivity of the cell membrane. As evident from fluorescence measurements, 10 μM, but not 1 μM mercury ions leads to a rapid increase of intracellular calcium activity. Pretreatment of the cells with either pertussis toxin or cholera toxin does not blunt the hyperpolarizing effect of mercury ions. In conclusion, mercury ions activate the potassium conductance by a mechanism independent of increase of intracellular calcium activity and of cholera toxin‐or pertussis toxin‐sensitive G‐proteins. This activation of potassium conductance may account for early effects of mercury intoxication, such as kaliuresis.
Journal of Cellular Physiology - Tập 146 Số 1 - Trang 25-33 - 1991
Cellular mechanisms of ATP‐induced hyperpolarization in renal epitheloid MDCK‐cells Abstract Previous studies have shown that ATP enhances intracellular calcium concentration and activates potassium channels in Madin Darby canine kidney (MDCK)‐cells, thus leading to hyperpolarization of the cell membrane. The present study has been performed to elucidate the intracellular mechanisms involved. To this end, the effects of ATP on the potential difference across the cell membrane (PD), on formation of inositol phosphates, and on intracellular calcium concentration (Cai) have been analyzed in cells without or with pretreatment with pertussis toxin or 12‐O‐tetradecanoyl phorbol 13‐acetate diester (TPA). In untreated cells, ATP leads to a sustained hyperpolarization and an increase of inositol 1,4,5‐tris‐phosphate (IP3), inositol 1,3,4,5‐tetrakisphosphate (IP4), and Cai. In the absence of extracellular calcium, the effect of ATP on PD and Cai is only transient. In cells pretreated with pertussis toxin, the effect of ATP on inositol trisphosphate is almost abolished, but ATP still leads to an increase of PD and Cai, which is sustained in the presence, and transient in the absence, of extracellular calcium. In cells pretreated with TPA, the effect of ATP on inositol trisphosphate is reduced and the effect on Cai blunted; but ATP still leads to a hyperpolarization of the cell membrane, which is sustained in the presence, and transient in the absence, of extracellular calcium. The observations indicate that ATP activates phospholipase C by a phorbol ester and pertussis toxin sensitive mechanism. In addition, ATP enhances Cai by pertussis toxin insensitive mechanisms allowing recruitment of calcium from both, extracellular fluid and intracellular stores. Calcium then activates the potassium channels and thus leads to the hyperpolarization of the cell membrane.
Journal of Cellular Physiology - Tập 147 Số 1 - Trang 68-75 - 1991
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