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Biomedical subjects

Ryuji Higashikubo

Publications and source records attributed to Ryuji Higashikubo.

14 recordsLinked to original sources
PubMed2005

Combined inhibition of extracellular signal-regulated kinases and HSP90 sensitizes human colon carcinoma cells to ionizing radiation.

Indomethacin, a common nonsteroidal anti-inflammatory drug, has been shown to enhance radiation-mediated cell-killing effect through the activation of p38 mitogen-activated protein kinase (MAPK). We found that indomethacin strongly reduced the basal level of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in HT-29 human colon carcinoma cells. The inhibition of ERK1/2 by indomethacin was only observed in cells with high basal activities of ERK1/2 such as HT-29 cells, but not in cells with low basal activities, such as HeLa. Cell cycle analysis of HT-29 cells exposed with indomethacin showed a partial G1/S arrest and slow DNA synthesis. However, the treatment with NS398, a specific COX-1/2 inhibitor, failed to show any effect on cell cycle, indicating that the inhibition of COX-1/2 is not responsible for cell cycle arrest. Since U0126, a specific inhibitor for MEK1/2, also induced a partial G1/S arrest, the G1/S arrest induced by indomethacin is, at least in part, caused by the inhibition of ERK1/2. Cell proliferation of HT-29 was inhibited by the treatment of U0126 but not in HeLa cells, and the treatment of HT-29 cells with U0126 enhanced radiation sensitivity possibly due to the accumulation of cells in G1 phase. We found that 17-allylamino-17-demethoxygeldanamycin, a geldanamycin delivative, radiosensitized HT-29 cells at a relatively low dose of irradiation, and indomethacin and U0126 further enhanced this effect. Therefore, tumor cells with elevated ERK1/2 activity can be effectively sensitized to radiation treatment by a combinational inhibition of HSP90 and MAPK activity.

Carcinoma↗
PubMed2005

Retinamide-induced apoptosis in glioblastomas is associated with down-regulation of Bcl-xL and Bcl-2 proteins.

Glioblastomas are among the most difficult neoplasms to treat with continued poor prognosis for long-term survival. Glioblastomas have developed effective mechanisms to resist chemotherapy including levels anti-apoptotic proteins, Bcl-xL and Bcl-2. Chemotherapy agents that promote down-regulation of Bcl-xL and Bcl-2 may enhance sensitivity to chemotherapy in glioblastomas. The ability of the synthetic retinoid N-(4-hydroxyphenyl) retinamide to modulate these anti-apoptotic proteins and to enhance apoptosis and chemotherapy was examined in glioblastoma cells. Expression of Bcl-2 family member proteins Bcl-xL and Bcl-2 were assessed in glioblastomas from three cell lines including U87, U251, and U138. Cells were treated with either retinamide alone or in combination with the chemotherapy agent, BCNU. The incidence of apoptosis was determined with flow cytometry analysis (FACS). Based on Western blots the levels of Bcl-2 and Bcl-xL were decreased in glioblastoma cells after treatment with retinamide. Retinamide treatment resulted in increased ratios of deamidated verses transamidated levels of Bcl-xL in U87 cells. BCNU chemotherapy combined with retinamide markedly down-regulated levels of both Bcl-xL and Bcl-2 proteins in glioblastoma and enhanced the incidence of apoptosis in U87 cells. These studies demonstrate that modulation of levels of the anti-apoptotic proteins, Bcl-xL and Bcl-2, may enhance the sensitivity of glioblastoma toward chemotherapy.

Antineoplastic Agents↗
PubMed2005

Tissue transglutaminase 2 inhibition promotes cell death and chemosensitivity in glioblastomas.

Tissue transglutaminase 2 belongs to a family of transglutaminase proteins that confers mechanical resistance from proteolysis and stabilizes proteins. Transglutaminase 2 promotes transamidation between glutamine and lysine residues with the formation of covalent linkages between proteins. Transglutaminase 2 also interacts and forms complexes with proteins important in extracellular matrix organization and cellular adhesion. We have identified the novel finding that treatment of glioblastoma cells with transglutaminase 2 inhibitors promotes cell death and enhances sensitivity to chemotherapy. Treatment with either the competitive transglutaminase 2 inhibitor, monodansylcadaverine, or with highly specific small-molecule transglutaminase 2 inhibitors, KCA075 or KCC009, results in induction of apoptosis in glioblastoma cells. Treatment with these transglutaminase 2 inhibitors resulted in markedly decreased levels of the prosurvival protein, phosphorylated Akt, and its downstream targets. These changes promote a proapoptotic profile with altered levels of multiple intracellular proteins that determine cell survival. These changes include decreased levels of the antiapoptotic proteins, survivin, phosphorylated Bad, and phosphorylated glycogen synthetase kinase 3beta (GSK-3beta), and increased levels of the proapoptotic BH3-only protein, Bim. In vivo studies with s.c. murine DBT glioblastoma tumors treated with transglutaminase 2 inhibitors combined with the chemotherapeutic agent, N-N'-bis (2-chloroethyl)-N-nitrosourea (BCNU), decreased tumor size based on weight by 50% compared with those treated with BCNU alone. Groups treated with transglutaminase 2 inhibitors showed an increased incidence of apoptosis determined with deoxynucleotidyl transferase-mediated biotin nick-end labeling staining. These studies identify inhibition of transglutaminase 2 as a potential target to enhance cell death and chemosensitivity in glioblastomas.

Animals↗
PubMed2004

Mitochondrial O2*- and H2O2 mediate glucose deprivation-induced stress in human cancer cells.

The hypothesis that glucose deprivation-induced cytotoxicity in transformed human cells is mediated by mitochondrial O2*- and H2O2 was first tested by exposing glucose-deprived SV40-transformed human fibroblasts (GM00637G) to electron transport chain blockers (ETCBs) known to increase mitochondrial O2*- and H2O2 production (antimycin A (AntA), myxothiazol (Myx), or rotenone (Rot)). Glucose deprivation (2-8 h) in the presence of ETCBs enhanced parameters indicative of oxidative stress (i.e. GSSG and steady-state levels of oxygen-centered radicals) as well as cytotoxicity. Glucose deprivation in the presence of AntA also significantly enhanced cytotoxicity and parameters indicative of oxidative stress in several different human cancer cell lines (PC-3, DU145, MDA-MB231, and HT-29). In addition, human osteosarcoma cells lacking functional mitochondrial electron transport chains (rho0) were resistant to glucose deprivation-induced cytotoxicity and oxidative stress in the presence of AntA. In the absence of ETCBs, aminotriazole-mediated inactivation of catalase in PC-3 cells demonstrated increases in intracellular steady-state levels of H2O2 during glucose deprivation. Finally, in the absence of ETCBs, overexpression of manganese containing superoxide dismutase and/or mitochondrial targeted catalase using adenoviral vectors significantly protected PC-3 cells from toxicity and oxidative stress induced by glucose deprivation with expression of both enzymes providing greater protection than was seen with either alone. Overall, these findings strongly support the hypothesis that mitochondrial O2*- and H2O2 significantly contribute to glucose deprivation-induced cytotoxicity and metabolic oxidative stress in human cancer cells.

Adenosine Triphosphate↗
PubMed2004

HSP70 and genomic stability.

The 70 kDa heat shock proteins (HSP70s) were initially identified by their elevated expression following hyperthermic cell stress, however, these highly conserved proteins also protect critical cellular functions from a wider range of important environmental and physiological stresses. At least one result of HSP70 expression is inhibition of stress induced caspase activation as well as downstream events in the apoptotic cell death pathway. HSP70 have been reported upregulated in tumor cells, selective inhibition of such proteins might be valuable approach to treat cancer. A recent study revealed that cells with inactivated HSP70 displayed telomere instability and high frequency of spontaneous chromosomal aberrations, indicating a possible role for HSP70 proteins in the maintenance of genomic stability.

Animals↗
PubMed2004

A comparative analysis of alphaA- and alphaB-crystallin expression during the cell cycle in primary mouse lens epithelial cultures.

AlphaA- and alphaB-crystallins are small heat shock proteins and molecular chaperones that prevent non-specific aggregation of denaturing proteins. Previous work in our laboratory has shown that lens epithelial cells derived from alphaA-/- mice exhibit slower growth, whereas alphaB-/- lens epithelial cells hyperproliferate at a higher rate in culture [Andley et al., J. Biol. Chem. 273 (1998) 31252; FASEB J. 15 (2001) 221]. Although both have been implicated in apoptosis and cell proliferation, direct analysis of their expression during the cell cycle has not been investigated. This study was undertaken to define the expression levels of alphaA and alphaB-crystallins during the cell cycle. Primary lens epithelial cell cultures derived from wild type mice were synchronized by serum starvation, and pulsed with bromodeoxyuridine (BrdU) at different times after re-stimulation with serum. Dual parameter flow cytometric studies with BrdU and propidium iodide (PI)-labeled cells were performed. Cells entered S phase 14 hr after serum re-stimulation. The duration of the S phase was 6 hr, and the total cell cycle transit time was between 24-27 hr. Enhanced expression of cyclin A, a protein essential for DNA synthesis was used as an additional marker to define the initiation of the S phase. Immunoblotting analysis demonstrated that the expression of alphaA and alphaB-crystallin was up to 10-fold higher in cells synchronized in G0 phase than in G1 phase. The levels of the proteins increased three-fold again as the cells entered the S phase and progressed to mitosis, but did not rise to the levels observed in G0 phase. This increase in expression of alphaA-crystallin resulted in part from enhanced synthesis during the S phase, as shown by an increase in [35S]methionine-labeling and immunoprecipitation of the radiolabeled alphaA-crystallin. The results were further confirmed by flow cytometric analysis using DNA content and alphaA-crystallin expression. The increase in alphaB-crystallin in S phase was paralleled by an increase in gene expression as shown by real-time RT-PCR analysis. These results demonstrate for the first time that in lens epithelial cells, alphaA and alphaB-crystallin levels are modulated during the cell cycle. Since the absence of alphaA and alphaB- crystallin in lens epithelial cells has been associated with disturbance of the tubulin cytoskeleton during mitosis, and with increased cell death or genomic instability, our results indicating that the alphaA- and alphaB-crystallin expression increases prior to mitosis are significant. The differential expression of these crystallins in the cell cycle may be important for optimal lens epithelial growth and lens transparency.

Animals↗
PubMed2004

Cell kinetic status of mouse lens epithelial cells lacking alphaA- and alphaB-crystallin.

alphaA- and alphaB-crystallins are small heat shock proteins and molecular chaperones that are known to prevent non-specific aggregation of denaturing proteins. Recent work indicates that alphaA-/- lens epithelial cells grow at a slower rate than wild-type cells, and cultured alphaB-/- cells demonstrate increased hyperproliferation and genomic instability, suggesting that these proteins may exert a direct effect on the cell cycle kinetics, and influence cell proliferation. However, the cell cycle parameters of alphaA/alphaBKO (double knockout) cells have not been analyzed. Here we investigate the cell cycle kinetics of synchronized mouse lens epithelial cultures derived from wild-type and alphaA/alphaB double knockout (alphaA/alphaBKO) mice using BrdU labeling of proliferating cells, and flow cytometric analysis. We also provide data on the changing pattern of expression of HSP25, a small heat shock protein in alphaA/alphaBKO and wild-type cells during the cell cycle. Using serum starvation to synchronize cells in the quiescent G0 phase, and restimulation with serum followed by BrdU labeling and flow cytometry, the data indicated that as compared to wild-type cells, a <50% smaller fraction of the alphaA/alphaBKO cells entered the DNA synthetic S phase of the cell cycle. Furthermore, there was a delay in cell cycle transit through S phase in alphaA/alphaBKO cells, suggesting that although capable of entering S phase, the alphaA/alphaBKO cells are blocked in G1 phase, and are delayed in their cell cycle progression. Immunoblot analysis with antibodies to the small heat shock protein HSP25 indicated that although HSP25 increased in G1 phase of wild-type cells, and remained elevated on further progression through the cell cycle, HSP25 accumulation was delayed to S phase in alphaA/alphaBKO cells. These data can be interpreted to indicate that mouse lens epithelial cell progression through the cell cycle is significantly affected by expression of alphaA and alphaB-crystallin.

Animals↗
PubMed2004

Lovastatin-induced up-regulation of the BH3-only protein, Bim, and cell death in glioblastoma cells.

The mechanism of lovastatin-induced cell death was examined in three established human glioblastoma cell lines; U87, U251, and U138. Changes in potential modifiers of apoptosis, including Bcl-2 family proteins and MAP kinase targets after such lovastatin treatment, were evaluated. Lovastatin (5 microm) treatment causes extensive cell death in two of the cell lines, U87 and U251; but only minimal in a third, U138. Lovastatin-induced death occurs in correlation with significantly increased levels of the BH3-only protein, Bim. The up-regulation of Bim levels was directly associated with an increased incidence of apoptosis. Lovastatin treatment in U87 cells results in activation of targets of three major mitogen-activating protein kinase cascades including Erk1/2, JNK and p38. Changes in levels of Bim, as well as increase phosphorylation of Erk1/2, c-jun, and p38 are all prevented by co-incubation of lovastatin and the isoprenylation metabolite, geranylgeranyl pyrophosphate. Inhibition of the MAP kinase pathways failed to block the increased expression of Bim expression or cell death. Further elucidation of the mechanisms of lovastatin-induced up-regulation of Bim and apoptosis in glioblastoma cells are important in determining a potential role for lovastatin as a chemotherapy agent.

Antineoplastic Agents↗
PubMed2004

Measurement of DNA damage and apoptosis in Molt-4 cells after in vitro exposure to radiofrequency radiation.

To determine whether exposure to radiofrequency (RF) radiation can induce DNA damage or apoptosis, Molt-4 T lymphoblastoid cells were exposed with RF fields at frequencies and modulations of the type used by wireless communication devices. Four types of frequency/modulation forms were studied: 847.74 MHz code-division multiple-access (CDMA), 835.62 MHz frequency-division multiple-access (FDMA), 813.56 MHz iDEN(R) (iDEN), and 836.55 MHz time-division multiple-access (TDMA). Exponentially growing cells were exposed to RF radiation for periods up to 24 h using a radial transmission line (RTL) exposure system. The specific absorption rates used were 3.2 W/kg for CDMA and FDMA, 2.4 or 24 mW/kg for iDEN, and 2.6 or 26 mW/kg for TDMA. The temperature in the RTLs was maintained at 37 degrees C +/- 0.3 degrees C. DNA damage was measured using the single-cell gel electrophoresis assay. The annexin V affinity assay was used to detect apoptosis. No statistically significant difference in the level of DNA damage or apoptosis was observed between sham-treated cells and cells exposed to RF radiation for any frequency, modulation or exposure time. Our results show that exposure of Molt-4 cells to CDMA, FDMA, iDEN or TDMA modulated RF radiation does not induce alterations in level of DNA damage or induce apoptosis.

Apoptosis↗
PubMed2004

Evaluation of parameters of oxidative stress after in vitro exposure to FMCW- and CDMA-modulated radiofrequency radiation fields.

The goal of this study was to determine whether radiofrequency (RF) radiation is capable of inducing oxidative stress or affecting the response to oxidative stress in cultured mammalian cells. The two types of RF radiation investigated were frequency-modulated continuous-wave with a carrier frequency of 835.62 MHz (FMCW) and code division multiple access centered on 847.74 MHz (CDMA). To evaluate the effect of RF radiation on oxidative stress, J774.16 mouse macrophage cells were stimulated with gamma-interferon (IFN) and bacterial lipopolysaccharide (LPS) prior to exposure. Cell cultures were exposed for 20-22 h to a specific absorption rate of 0.8 W/kg at a temperature of 37.0 +/- 0.3 degrees C. Oxidative stress was evaluated by measuring oxidant levels, antioxidant levels, oxidative damage and nitric oxide production. Oxidation of thiols was measured by monitoring the accumulation of glutathione disulfide (GSSG). Cellular antioxidant defenses were evaluated by measuring superoxide dismutase activity (CuZnSOD and MnSOD) as well as catalase and glutathione peroxidase activity. The trypan blue dye exclusion assay was used to measure any changes in viability. The results of these studies indicated that FMCW- and CDMA-modulated RF radiation did not alter parameters indicative of oxidative stress in J774.16 cells. FMCW- and CDMA-modulated fields did not alter the level of intracellular oxidants, accumulation of GSSG or induction of antioxidant defenses in IFN/LPS-stimulated cells. Consistent with the lack of an effect on oxidative stress parameters, no change in toxicity was observed in J774.16 cells after either optimal (with or without inhibitors of nitric oxide synthase) or suboptimal stimulation.

Analysis of Variance↗
PubMed2003

Redox regulation of the G1 to S phase transition in the mouse embryo fibroblast cell cycle.

The hypothesis that intracellular oxidation/reduction (redox) reactions regulate the G(0)-G(1) to S-phase transition in the mouse embryonic fibroblast cell cycle was investigated. Intracellular redox state was modulated with a thiol-antioxidant, N-acetyl-L-cysteine (NAC), and cell cycle progression was measured using BrdUrd pulse-chase and flow cytometric analysis. Treatment with NAC for 12 h resulted in an approximately 6-fold increase in intracellular low-molecular-weight thiols and a decrease in the MFI of an oxidation-sensitive probe, dihydrofluorescein diacetate, indicating a shift in the intracellular redox state toward a more reducing environment. NAC-induced alterations in redox state caused selective delays in progression from G(0)-G(1) to S phase in serum-starved cells that were serum stimulated to reenter the cell cycle as well as to inhibit progression from G(1) to S phase in asynchronous cultures with no significant alterations in S phase, and G(2)+M transits. NAC treatment also showed a 70% decrease in cyclin D1 protein levels and a 3-4-fold increase in p27 protein levels, which correlated with decreased retinoblastoma protein phosphorylation. Cells released from the NAC treatment showed a transient increase in dihydrofluorescein fluorescence and oxidized glutathione content between 0 and 8 h after release, indicating a shift in intracellular redox state to a more oxidizing environment. These changes in redox state were followed by an increase in cyclin D1, a decrease in p27, retinoblastoma protein hyperphosphorylation and subsequent entry into S phase by 8-12 h after the removal of NAC. These results support the hypothesis that a redox cycle within the mammalian cell cycle might provide a mechanistic link between the metabolic processes early in G(1) and the activation of G(1)-regulatory proteins in preparation for the entry of cells into S phase.

Acetylcysteine↗
PubMed2002

A novel p53-inducible apoptogenic gene, PRG3, encodes a homologue of the apoptosis-inducing factor (AIF).

The p53 tumor suppressor protein induces cell cycle arrest or apoptosis in response to cellular stresses. We have identified PRG3 (p53-responsive gene 3), which is induced specifically under p53-dependent apoptotic conditions in human colon cancer cells, and encodes a novel polypeptide of 373 amino acids with a predicted molecular mass of 40.5 kDa. PRG3 has significant homology to bacterial oxidoreductases and the apoptosis-inducing factor, AIF, and the gene was assigned to chromosome 10q21.3-q22.1. Expression of PRG3 was induced by the activation of endogenous p53 and it contains a p53-responsive element. Unlike AIF, PRG3 localizes in the cytoplasm and its ectopic expression induces apoptosis. An amino-terminal deletion mutant of PRG3 that lacks a putative oxidoreductase activity retains its apoptotic activity, suggesting that the oxidoreductase activity is dispensable for the apoptotic function of PRG3. The PRG3 gene is thus a novel p53 target gene in a p53-dependent apoptosis pathway.

Adenosine Diphosphate↗
PubMed2002

Evidence that protein disulfide isomerase (PDI) is involved in DNA-nuclear matrix anchoring.

DNA-nuclear matrix (NM) anchoring plays a critical role in the organization of DNA within the nucleus and in functional access to DNA for transcription, replication, and DNA repair. The cellular response to oxidative stress involves both gene expression and DNA repair. We, therefore, determined if changes in the oxidative-reductive environment can affect DNA-NM anchoring. The present study used two approaches to study the effect of the reducing agent DTT on DNA-NM anchoring. First, the relative stringency of the DNA-NM attachment was determined by measuring the ability of NM attached DNA loops to undergo supercoiling changes. Second, the effects of DTT on the association of nuclear proteins with DNA were determined by cisplatin crosslinking. When nucleoids (nuclear matrices with attached DNA loops) were prepared from HeLa cells with 1 mM dithiothreitol (DTT), supercoiled DNA loops unwound more efficiently compared with control in the presence of increasing propidium iodide (PI) concentrations. In addition, the rewinding of DNA supercoils in nucleoids treated with DTT was inhibited. Both effects on DNA supercoiling ability were reversed by diamide suggesting that they are dependent on the oxidation state of the protein thiols. When DTT treated nucleoids were isolated from gamma-irradiated cells, the inhibition of DNA supercoil rewinding was equal to the sum of the inhibition due to DTT and gamma-rays alone. Nucleoids isolated from heat-shocked cells with DTT, showed no inhibition of DNA rewinding, except a small inhibition at high PI concentrations. Nuclear DNA in DTT-treated nuclei was digested faster by DNase I than in untreated nuclei. These results suggest that DTT is altering DNA-NM anchoring by affecting the protein component(s) of the anchoring complex. Extracting NM with increasing concentrations of DTT did not solubilize any protein to a significant extent until measurable NM disintegration occurred. Therefore, we determined if 1 mM DTT affected the ability of 1 mM cisplatin to crosslink proteins to DNA. Isolated nuclei were treated with 1 mM DTT for 30 min or left untreated prior to crosslinking with 1 mM cisplatin for 2 h at 4 degrees C. The ability of capsulation to crosslink DNA to proteins per se, did not appear to be affected by 1 mM DTT because relative amounts of at least four proteins, 69, 60, 40, and 35 kDa, were crosslinked to DNA to the same extent in DTT-treated and untreated nuclei. However, protein disulfide isomerase (PDI) crosslinked to DNA in untreated nuclei, but did not crosslink DNA in nuclei that were treated with 1 mM DTT; 1 mM DTT did not affect the intranuclear localization of PDI. Thus, DTT appears to alter the conformation of PDI, as suggested by the DTT-induced change in DNA association, but not its NM association. These results also imply that DNA-NM anchoring involves the redox state of protein sulfhydryl groups.

Cisplatin↗