BBA - Molecular Cell Research (v.1763, #9)

Biphasic electric current stimulates proliferation and induces VEGF production in osteoblasts by In Sook Kim; Jong Keun Song; Yu Lian Zhang; Tae Hyung Lee; Tae Hyung Cho; Yun Mi Song; Do Kyun Kim; Sung June Kim; Soon Jung Hwang (907-916).
This study investigated biphasic electric current (BEC) functions as a new type of electrical stimulation to induce rat calvarial osteoblasts to proliferate, differentiate and synthesize cytokines. The culture system was designed so that biphasic current flowed between upper and lower gold plates. BEC helps to minimize the net charge accumulation during cell exposure to the electrical stimulation. Osteoblasts were exposed to electrical stimulation of 1.5 μA/cm2 at 3000 Hz, and the effect of BEC was assessed in the interrupted mode (6 h daily) and in the continuous mode (24 h daily), depending on the interval of stimulation. Whereas proliferation increased by 31% after stimulation in the continuous mode for 2 days, it was unaffected in the interrupted mode. The transcriptional expression of osteogenesis-related genes such as alkaline phosphatase (ALP), osteopontin, and type I collagen was unchanged 4 days after stimulation in both modes, while cbfa1 was decreased under the same conditions. There was no detectable change in mRNA expression of growth factors (BMP-2, -4, IGF-2 and TGF-β1) that promote osteoblast differentiation. However, real-time RT-PCR and ELISA demonstrated that vascular endothelial growth factor (VEGF) was markedly up-regulated by BEC. Induction of VEGF by BEC was not hypoxia driven. In conclusion, the present in vitro study demonstrates that BEC increases cell proliferation and induces the production of VEGF. The BEC was more effective with continuous stimulation than with interrupted stimulation. To confirm whether BEC can enhance osteogenesis, further in vivo studies are needed.
Keywords: Biphasic electric current (BEC); VEGF; Osteoblast differentiation; Proliferation; Bone formation;

Increased stability of P21 WAF1/CIP1 mRNA is required for ROS/ERK-dependent pancreatic adenocarcinoma cell growth inhibition by pyrrolidine dithiocarbamate by Massimo Donadelli; Elisa Dalla Pozza; Chiara Costanzo; Maria Teresa Scupoli; Paolo Piacentini; Aldo Scarpa; Marta Palmieri (917-926).
We present evidence that pyrrolidine dithiocarbamate (PDTC) inhibits growth of p53-negative pancreatic adenocarcinoma cell lines via cell cycle arrest in the S-phase, while it has no effect on primary fibroblast proliferation. Growth inhibition of cancer cells is dependent on ROS and ERK1/2 induction as indicated by a significantly reduced PDTC-associated growth inhibition by the free radical scavenger N-acetyl-l-cysteine (NAC) or the MEK/ERK1/2 inhibitor (PD98059). Moreover, ERK1/2 induction is dependent on ROS production as demonstrated by a complete removal of PDTC-mediated ERK1/2 phosphorylation by NAC. p21WAF1/CIP1 activation has a central role in growth inhibition by PDTC, as revealed by P21 WAF1/CIP1 silencing experiments with antisense oligonucleotide, and occurs via increased mRNA stability largely mediated by ROS/ERK induction. Conversely, PDTC does not affect P21 WAF1/CIP1 gene expression in primary fibroblasts, although it is able to activate p53 and the p53-regulated antioxidant SESN2. These results suggest that the resistance of fibroblasts to the cytotoxic action of PDTC may be related to the up-regulation of p53-dependent antioxidant genes. Finally, in vivo studies on PaCa44 cells subcutaneously xenografted in nude mice show that treatment with 100 or 200 mg/kg PDTC reduces of 30% or 60% the tumour volume, respectively, and does not cause any apparent form of toxicity.
Keywords: Pancreatic adenocarcinoma; Pyrrolidine dithiocarbamate; Oxidative stress; ERK1/2; p21WAF1/CIP1; p53;

Advanced glycation end products (AGEs) that arise from the reaction of sugars with protein side chains and the terminal amino group are supposed to be involved in the pathogenesis of several diseases and therefore the effects of AGEs on cells are the objective of numerous investigations. The effects of AGEs on cells are commonly assumed to be transduced via the receptor for AGEs (RAGE) but there are also other receptors known to interact with AGEs and they are likely to be involved in signal transduction. The primary cellular effect of AGEs on cultured cells was found to be the formation of reactive oxygen species (ROS). For the present study one murine and three human cell lines were used. The effects of a set of different highly modified AGEs and AGE-like compounds derived from the incubation of different modifiers with BSA were tested for their effects on these cells. Almost all AGEs tested induced the production of reactive oxygen species (ROS) in the different cell lines although the intensity of the detected signals varied considerably between the cell lines and are strongly dependent on the AGE used for cell activation. The most highly modified BSA-species were shown to inhibit cell growth in all cell lines, whereas a moderately modified glucose derived BSA-AGE and BSA-GAred did not show any inhibitory effect on cell growth even when a high ROS formation was detected.
Keywords: Advanced glycation end product; AGEs; Reactive oxygen species; ROS; Growth inhibition;

Cytoplasmic Ca2+ signals and cellular death by apoptosis in myocardiac H9c2 cells by Antonio Lax; Fernando Soler; Francisco Fernández-Belda (937-947).
The incubation of H9c2 cells with 10 μM thapsigargin (TG) was associated with the appearance of a two-component cytoplasmic Ca2+ peak. Experiments performed in a Ca2+-free medium indicated that both components came from intracellular sources. The first component of the signal corresponded to the discharge of the sarco-endoplasmic reticulum (SER) Ca2+ store. The appearance of the second component was prevented by cell preincubation with cyclosporin A (CsA) and gave rise to a clear and permanent depolarization of the mitochondrial inner membrane. These features were indication of a mitochondrial origin. The observed release of mitochondrial Ca2+ was related with opening of the permeability transition pore (PTP). The two-component cytoplasmic Ca2+ peak, i.e., treatment with 10 μM TG, as compared with the first component alone, i.e., treatment with 3 μM TG, was associated with a faster process of cellular death. In both cases, chromatin fragmentation and condensation at the nuclear periphery were observed. Other prominent apoptotic events such as loss of DNA content and cleavage of poly(ADP-ribose) polymerase (PARP) were also dependent on TG concentration and occurred in different time windows. PTP opening induced by 10 μM TG was responsible for the faster apoptotic death.
Keywords: Ca2+ signal; Thapsigargin; Sarco-endoplasmic reticulum; Permeability transition pore; Apoptosis; Cardiac cell line;

A kinase activity that phosphorylated myelin basic protein in vitro was detected in RalA and RalB immunoprecipitates from human platelets. Protein–protein interaction studies using recombinant GST-RalA, GST-RalB and GST-cH-Ras confirmed that the kinase specifically associates with the Ral GTPase. The Ral Interacting Protein 1 (RIP1), a GTPase Activating Protein (GAP) for Cdc42 and Rac1, was found to be the preferred substrate for the Ral Interacting Kinase (RIK). Phosphoamino acid analysis demonstrated that RIK phosphorylated serine residue in RIP1. The Ral–RIK interaction was not dependent on the guanine nucleotide status of Ral. RIK was detected in a variety of rat tissues with testis containing the highest and skeletal muscle the lowest activity. In-gel kinase renaturation assay using RIP1 as the substrate demonstrated that the kinase activity was associated with polypeptides of molecular mass of ∼36–40 kDa and was detected in most rat tissues with a prominent 38 kDa band in testis and a 40 kDa band in brain. Human platelets contained a single band of ∼36 kDa. RIK was distinct from MAPKs, CDKs, cyclic AMP dependent protein kinase and Ca2+/calmodulin dependent kinases. To demonstrate in vivo interaction, the endogenous Ral–RIK complex was isolated using a calmodulin affinity column. The Ral–RIK complex co-eluted from this column upon washing with a 13 residue peptide that encompasses the calmodulin-binding domain in RalA. The data suggest that RIK is a serine specific kinase that phosphorylates RIP1 and is constitutively associated with Ral. The current study provides additional support for a link between Ral and the Cdc42/Rac1 signalling pathways in the cell.
Keywords: Ral; GTPase; Serine Kinase; RIP1; Platelet; Testis; Phosphorylation;

Interplay between PI3K/Akt and MAPK signaling pathways in DNA-damaging drug-induced apoptosis by Eung-Ryoung Lee; Jang-Yong Kim; Yong-Jin Kang; Jae-Yeon Ahn; Jung-Hyun Kim; Bong-Woo Kim; Hye-Yeon Choi; Mi-Young Jeong; Ssang-Goo Cho (958-968).
In order to elucidate the role of the mitogen-activated protein kinases, including JNK, p38 MAPK and ERK, as well as the survival-associated PI3K/Akt signaling pathway, in the response to chemotherapy, we have conducted a comparative study regarding the effects of doxorubicin on these pathways. Doxorubicin was determined to elicit the apoptosis of NIH3T3 cells in a dose-dependent manner. Prior to cell death, both Akt and p38 MAPK were transiently activated, and subsequently inactivated almost wholly, whereas ERK and JNK evidenced sustained activations in response to the drug treatment. The inhibition of PI3K/Akt and p38 MAPK both accelerated and enhanced doxorubicin-induced apoptosis and ERK inhibition apparently exerted negative effect on apoptosis. The modulation of PI3K/Akt activation by treatment of LY294002 or expression of Akt mutants such as Akt-DN or Myr-Akt exerted a significant effect on the activation of ERK1/2. We also observed that PI3K/Akt and sustained ERK activation were associated intimately with the etoposide-induced apoptosis. Taken together, our results clearly suggest that the differential regulation of the PI3K/Akt, ERK1/2, and p38 MAPK signaling pathways are crucial in the context of DNA-damaging drug-induced apoptosis, and this has compelled us to propose that the sustained activation of ERK1/2 pathway may be generally involved in the apoptosis induced by anticancer DNA-damaging drugs, including doxorubicin and etoposide.
Keywords: DNA-damaging drug; Doxorubicin; Etoposide; Akt; ERK; Apoptosis;

We evaluated the role of reactive oxygen species (ROS) for the contraction induced increase in expression of PGC-1α, HKII and UCP3 mRNA. Rat skeletal muscle cells were subjected to acute or repeated electrostimulation in the presence and absence of antioxidants. Contraction of muscle cells lead to an increased H2O2 formation, as measured by oxidation of H2HFF. Acute contraction of the muscle cells lead to a transient increase in PGC-1α and UCP3 mRNA by 172 and 65%, respectively (p  < 0.05), whereas this increase was absent in the presence of antioxidants. Repeated contraction sessions induced a sustained elevation in PGC-1α and UCP3 mRNA and a transient increase in HKII (p  < 0.05) and this effect was not present with treatment of cells with either an antioxidant cocktail or with GPX + GSH. Incubation of cells for 10 days with ROS produced by xanthine oxidase/xanthine increased the level of PGC-1α, HKII and UCP3 mRNA by 175, 58 and 115%, respectively (p  < 0.05). A 10-day incubation of cells with antioxidants was found to have no effect on the basal mRNA content (p  > 0.05). The present data demonstrate that contraction of skeletal muscle cells leads to an enhanced formation of ROS and an elevation in PGC-1α, UCP3 and HKII mRNA content which is abolished in the presence of antioxidants, suggesting that ROS are of importance for the contraction induced increase in expression of these genes in skeletal muscle.
Keywords: Antioxidant; Reactive oxygen species; Gene expression; Rat skeletal muscle cell; Contraction;

Phosphoproteomic profiling of human SH-SY5Y neuroblastoma cells during response to 6-hydroxydopamine-induced oxidative stress by Megumi Nakamura; Masaki Yamada; Takako Ohsawa; Hiraku Morisawa; Tsutomu Nishine; Osamu Nishimura; Tosifusa Toda (977-989).
Dopaminergic neurons are known to be vulnerable to age-related neuronal disorders due to reactive oxygen species (ROS) generated during dopamine metabolism. However, it remains unclear what kinds of proteins are involved in the response to oxidative stress. We examined changes in whole proteins and phosphoproteins in the human dopaminergic neuroblastoma cell line SH-SY5Y under oxidative stress induced by the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). Proteins of SH-SY5Y cells at various stages of oxidative stress were separated by two-dimensional gel electrophoresis for comparative analysis. Increase in glutathione-S-transferase pi was detected on SYPRO Ruby-stained gels by computer-aided image analysis. Stress-induced alterations in phosphoproteins were detected by Pro-Q Diamond staining. Elongation factor 2, lamin A/C, T-complex protein 1, and heterogeneous nuclear ribonucleoprotein H3 were identified by MALDI-TOF mass spectrometry as stress-responsive elements.
Keywords: Oxidative stress; SH-SY5Y cells; Phosphoproteome; 6-hydroxydopamine;