Biomedical subjects

T Tsuruo

Publications and source records attributed to T Tsuruo.

At least 73 records · Page 4Linked to original sources

PubMed1999

Imaging of caspase-3 activation in HeLa cells stimulated with etoposide using a novel fluorescent probe.

Microscopic visualization of intracellular enzyme activity can provide information about the physiological role of the enzyme. Caspases are cysteine proteases that have critical roles in the execution of apoptosis. General fluorometric substrates of caspase-3, such as DEVD-MCA, are unsuitable for imaging because they are excited at short wavelength, so we designed and synthesized novel fluorescent probes that are excited at suitable wavelengths for detecting caspase-3 activity in living cells. Using one of these probes, we succeeded in microscopic visualization of caspase-3-like activity within HeLa cells treated with etoposide. The caspase-3-like activity was increased in the cytosol at first, then expanded to the whole cell.

Apoptosis↗

PubMed1999

Effect of cyclosporin A or tacrolimus on the function of blood-brain barrier cells.

Recently, it has been reported that continuous treatment with cyclosporin A or tacrolimus induces encephalopathy in transplant patients. The mechanism of immunosuppressant-induced encephalopathy is unclear. We investigated the cytotoxicity to brain capillary endothelial cells and the effect of these two drugs on P-glycoprotein function using mouse brain capillary endothelial (MBEC4) cells. The transcellular transport of [3H]sucrose was significantly increased and the cellular viability, based on 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and trypan blue exclusion test, was decreased by cyclosporin A (approximately 50% at 5 microM; P<0.005), while tacrolimus showed a much smaller effect. These findings indicate that the toxicity of cyclosporin A was greater than that of tacrolimus. The uptake of [3H]vincristine, a substrate of P-glycoprotein, was increased by these two drugs. The expression of P-glycoprotein in MBEC4 cells was reduced, but there was no effect on mdr1b mRNA levels. The decrease in the expression of P-glycoprotein may be due to the inhibition of the turnover of P-glycoprotein, which involves translation. In conclusion, the direct cytotoxic effect on the brain capillary endothelial cells and the inhibition of P-glycoprotein may be partly involved in the occurrence of immunosuppressant-induced encephalopathy.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed1999

Glucose starvation and hypoxia induce nuclear accumulation of proteasome in cancer cells.

Solid tumors commonly contain regions with glucose-starved and hypoxic conditions. Tumor cells under the adverse conditions can survive through the stress response, such as cell cycle arrest. In this study, we found that the stress conditions stimulated nuclear accumulation of proteasomes, large multicatalytic protease complexes, in human colon cancer HT-29 cells. The nuclear proteasome levels both in amount and in activity were increased approximately 4 and 2 times by glucose starvation and hypoxia, respectively. No changes were detected in the total expression levels of proteasome. The nuclear proteasome accumulation was also observed in ovarian cancer A2780 cells under glucose starvation, suggesting that this response was regardless of the origin of cancer cells. Our results indicate that the nuclear proteasome distribution is enhanced by glucose starvation and hypoxia, and suggest that the proteolysis by proteasome in the nucleus may play roles in the stress response of solid tumor cells.

Cell Cycle↗

PubMed1999

Caspase-mediated cleavage of cytoskeletal actin plays a positive role in the process of morphological apoptosis.

Tumors result from the imbalance between cell growth and apoptosis. One of the characteristic changes in cancers is the abnormality in cytoskeleton, which suggests some roles of cytoskeletal proteins in tumorigenesis or the maintenance of tumor cells. Previously we showed that cytoskeletal actin is the substrate of caspases, the proteases responsible for apoptosis, while the role of actin cleavage in apoptosis remained unknown. To examine the cleavage of actin in vivo, we extensively performed immunoblot analysis using actin fragment-specific antibody. Here, we showed that, in some solid tumor cells, induction of apoptosis was accompanied by caspase-dependent actin-cleavage to 15 and 31 kDa fragments in vivo. To elucidate the role of actin-cleavage further, we introduced actin cleaved-fragments. We found that ectopic expression of an actin 15 kDa fragment induces morphological changes resembling those of apoptotic cells. The expression of the actin fragment induced a dramatic change of cellular actin localization, as visualized by enhanced green fluorescent protein (EGFP)-tagged actin, while the actin fragment expression did not cause caspase activation nor the cleavage of a marker substrate protein, poly (ADP-ribose) polymerase. These results indicate that actin cleavage could play a positive role in the morphological changes of apoptosis downstream of caspase activation.

Actins↗

PubMed1999

A synthetic inhibitor of histone deacetylase, MS-27-275, with marked in vivo antitumor activity against human tumors.

Synthetic benzamide derivatives were investigated for their ability to inhibit histone deacetylase (HDA). In this study, one of the most active benzamide derivatives, MS-27-275, was examined with regard to its biological properties and antitumor efficacy. MS-27-275 inhibited partially purified human HDA and caused hyperacetylation of nuclear histones in various tumor cell lines. It behaved in a manner similar to other HDA inhibitors, such as sodium butyrate and trichostatin A; MS-27-275 induced p21(WAF1/CIP1) and gelsolin and changed the cell cycle distribution, decrease of S-phase cells, and increase of G1-phase cells. The in vitro sensitivity spectrum of MS-27-275 against various human tumor cell lines showed a pattern different than that of a commonly used antitumor agent, 5-fluorouracil, and, of interest, the accumulation of p21(WAF1/CIP1) tended to be faster and greater in the cell lines sensitive to MS-27-275. MS-27-275 administered orally strongly inhibited the growth in seven of eight tumor lines implanted into nude mice, although most of these did not respond to 5-fluorouracil. A structurally analogous compound to MS-27-275 without HDA-inhibiting activity showed neither the biological effects in cell culture nor the in vivo therapeutic efficacy. These results suggest that MS-27-275 acts as an antitumor agent through HDA inhibition and may provide a novel chemotherapeutic strategy for cancers insensitive to traditional antitumor agents.

Animals↗

PubMed1999

Retroviral expression of a mutant (Gly-533) human DNA topoisomerase I cDNA confers a dominant form of camptothecin resistance.

In previous studies, we isolated a mutant DNA topoisomerase I cDNA from a camptothecin (CPT)-resistant human T-lymphoblastic leukemia cell line, CPT-K5, and demonstrated that an amino acid change from Asp to Gly at residue 533 is responsible for the CPT resistance of the enzyme. In the present study, we have constructed a bicistronic retroviral vector, Ha-TM1-IRES-neo, that carries the mutant (Gly-533) TOP1 cDNA (TM1) and a neomycin-resistance gene to examine the effect of mutant DNA topoisomerase I (topo I) expression on CPT resistance of cells. HeLa S3 cells were transduced with Ha-TM1-IRES-neo, and the transduced cells were selected with G418. Two independently isolated populations of the G418-resistant cells and 2 clones showed 1.7- to 1.8-fold higher resistance to CPT than the control cells. Integration and expression of the exogenous TOP1 were confirmed by genomic and RT-PCR analyses. The topo I enzyme (mixture of mutant and wild-type) expressed in the transduced cells showed 3-fold resistance to CPT in cleavable-complex-formation assay and DNA-relaxation assay. Mutant topo I activity in the transduced cells was as much as 10% that of the endogenous enzyme. Our results clearly show that expression of Gly-533 topo I confers a dominant form of CPT resistance in cells expressing wild-type topo I. The mutant TOP1 could be used for the protection of normal bone marrow cells of cancer patients from the severe hematotoxicity of CPT-derivative anti-tumor agents.

Anti-Bacterial Agents↗

PubMed1999

Monocyte chemoattractant protein-1 gene modification of multidrug-resistant human lung cancer enhances antimetastatic effect of therapy with anti-P-glycoprotein antibody in SCID mice.

Distant metastases and multidrug resistance are critical problems in the therapy of human small cell lung cancer (SCLC). In this study, we investigated whether transduction of the monocyte chemoattractant protein-1 (MCP-1) gene into multidrug-resistant (MDR) human lung cancer cells affected the formation of metastases or their inhibition by the anti-P-glycoprotein (P-gp) monoclonal antibody (MAb) MRK16. MDR human SCLC (H69/VP) cells were transduced with the human MCP-1 gene inserted into the expression vector BCMGSNeo. MCP-1 gene transduction had no effect on drug sensitivity, the expression of surface antigens or the in vitro proliferation of H69/VP cells. Using the metastatic model of NK cell-depleted SCID mice, H69/VP cells transduced with the MCP-1 gene were inoculated intravenously (i.v.) and formed metastatic colonies in the liver, kidneys and lymph nodes, similar to those formed by parent or mock-transduced cells. However, systemic treatment of the mice with MRK16 reduced the metastases of H69/VP cells in the liver, kidneys and lymph nodes, and was significantly more effective in inhibiting the metastases of MCP-1 producing H69/VP than those of mock-transduced cells. MCP-1 gene transduction significantly prolonged the survival of tumor-bearing mice treated with MRK16. Our findings suggest that local production of MCP-1 in the tumor site increases the anti-P-gp antibody-dependent cell-mediated cytotoxicity, and the MCP-1 gene-induced modification of MDR human SCLC cells thereby enhances the antimetastatic effect of therapy with anti-P-gp antibody. Thus, the accumulation of effector cells in the tumor site is a very important factor in the therapy using the anti-P-gp antibody.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Direct interaction between a quinoline derivative, MS-209, and multidrug resistance protein (MRP) in human gastric cancer cells.

MS-209 is a novel quinoline derivative reversing P-glycoprotein-mediated multidrug resistance (MDR). We investigated the interaction between MS-209 and multidrug resistance protein (MRP) in MRP-overexpressing human gastric cancer cells. We measured [3H]leukotriene C4 uptake into the membrane vesicles of the cells and intracellular calcein and [3H]vincristine accumulation with or without MS-209. In multi-drug-resistant MKN45R0.8 cells selected by doxorubicin, MS-209 dose dependently reduced MRP-mediated [3H]leukotriene C4 uptake and increased calcein accumulation. In both resistant and unselected cell lines expressing the MRP gene, MS-209 increased [3H]vincristine accumulation in proportion with the level of MRP mRNA expression and enhanced the cytotoxicity of etoposide, doxorubicin, and vincristine. The reversal effects correlated with the level of MRP mRNA expression in these cells. Our results indicate that MS-209 effectively reverses intrinsic and acquired MRP-mediated MDR of gastric cancer cells by interacting directly with MRP.

ATP-Binding Cassette Transporters↗

PubMed1999

Activation of c-Abl tyrosine kinase requires caspase activation and is not involved in JNK/SAPK activation during apoptosis of human monocytic leukemia U937 cells.

Genotoxic stress triggers the activation of several sensor molecules, such as p53, JNK1/SAPK and c-Abl, and occasionally promotes the cells to apoptosis. We previously reported that JNK1/SAPK regulates genotoxic stress-induced apoptosis in p53-negative U937 cells by activating caspases. c-Abl is expected to act upstream of JNK1/SAPK activation upon treatment with genotoxic stressors, but its involvement in apoptosis development is still unclear. We herein investigated the kinase activities of c-Abl and JNK1/SAPK during apoptosis elicited by genotoxic anticancer drugs and tumor necrosis factor (TNF) in U937 cells and their apoptosis-resistant variant UK711 cells. We found that the activation of JNK1/SAPK and c-Abl correlated well with apoptosis development in these cell lines. Unexpectedly, however, the JNK1/SAPK activation preceded the c-Abl activation. Moreover, the caspase inhibitor Z-Asp suppressed c-Abl activation and the onset of apoptosis but not the JNK1/SAPK activation. Interestingly, c-Abl tyrosine kinase inhibition by CGP 57148 reduced apoptosis without interfering with JNK1/SAPK activation. These results indicate that c-Abl acts not upstream of JNK1/ SAPK but downstream of caspases during the development of p53-independent apoptosis and is possibly involved in accelerating execution of the cell death pathway.

Apoptosis↗

PubMed1999

Modulation of multidrug resistance in tumor cells by taxinine derivatives.

Among a series of taxinine (1) and its designed derivatives (2-33), two taxoids (29 and 33) increased cellular accumulation of vincristine in multidrug-resistant tumor cells more potently than verapamil, while the activities of eight taxoids (11, 14-16, 22, and 30-32) were comparable with that of verapamil. These results reveal that some taxinine derivatives are good modifiers of multidrug resistance in tumor cells.

Animals↗

PubMed1999

Caspase-mediated cleavage of p21Waf1/Cip1 converts cancer cells from growth arrest to undergoing apoptosis.

The cyclin-dependent kinase inhibitor p21waf1/Cip1 is a downstream effector of the p53-dependent cell growth arrest. We report herein that p21 was cleaved by caspase-3/CPP32 at the site of DHVD112L during the DNA damage-induced apoptosis of cancer cells. The cleaved p21 fragment could no more arrest the cells in G1 phase nor suppress the cells undergoing apoptosis because it failed to bind to the proliferating cell nuclear antigen (PCNA) and lost its capability to localize in the nucleus. Thus, caspase-3-mediated cleavage and inactivation of p21 protein may convert cancer cells from growth arrest to undergoing apoptosis, leading to the acceleration of chemotherapy-induced apoptotic process in cancer cells.

Apoptosis↗

PubMed1999

Constitutive activation of the 41-/43-kDa mitogen-activated protein kinase signaling pathway in human tumors.

The 41-kDa and 43-kDa mitogen-activated protein (MAP) kinases play a pivotal role in the mitogenic signal transduction pathway and are essential components of the MAP kinase cascade, which includes MAP kinase kinase (MEK) and Raf-1. As aberrant activation of signal transducing molecules such as Ras and Raf-1 has been linked with cancer, we examined whether constitutive activation of the 41-/43-kDa MAP kinases is associated with the neoplastic phenotype of 138 tumor cell lines and 102 primary tumors derived from various human organs. Constitutive activation of the MAP kinases was observed in 50 tumor cell lines (36.2%) in a rather tissue-specific manner: cell lines derived from pancreas, colon, lung, ovary and kidney showed especially high frequencies with a high degree of MAP kinase activation, while those derived from brain, esophagus, stomach, liver and of hematopoietic origin showed low frequencies with a limited degree of MAP kinase activation. We also detected constitutive activation of the 41-/43-kDa MAP kinases in a relatively large number of primary human tumors derived from kidney, colon and lung tissues but not from liver tissue. Many tumor cells, in which point mutations of ras genes were detected, showed constitutive activation of MAP kinases, however, there were also many exceptions to this observation. In contrast, the activation of the 41-/43-kDa MAP kinases was accompanied by the activation of Raf-1 in the majority of tumor cells and was completely associated with the activation of MEK and p90rsk in all the tumor cells examined. These results suggest that the constitutive activation of 41-/43-kDa MAP kinases in tumor cells is not due to the disorder of MAP kinases themselves, but is due to the disorder of Raf-1, Ras, or some other signaling molecules upstream of Ras.

Amino Acid Sequence↗

PubMed1999

Basic fibroblast growth factor induces cyclooxygenase-2 expression in endothelial cells derived from bone.

Although histological studies have suggested that endothelial cells in bone (BDECs) are associated with some osteolytic bone diseases, it is still unclear how BDECs contribute to bone remodeling. Here we examined the response of BDECs to basic fibroblast growth factor (bFGF, FGF-2) using primary and cloned murine BDECs isolated from the femurs of BALB/c mice. Treatment of primary and cloned BDECs with bFGF induced cyclooxygenase-2 (COX-2) mRNA and protein expression. Furthermore, bFGF promotes the production of prostaglandin E2 (PGE2), which is known to be a potent stimulator of bone resorption and to induce osteoclast formation. Because the secretion of PGE2 was suppressed by COX-2 specific inhibitor NS-398 and by COX-2 antisense oligodeoxynucleotides, bFGF promotes the synthesis of PGE2 in a COX-2-dependent manner. Therefore, endothelial cells in bone are associated with bone remodeling by controlling COX-2 expression and consequently PGE2 production.

Animals↗

PubMed1999

ASK1 mediates apoptotic cell death induced by genotoxic stress.

ASKI mediates apoptotic cell death induced by genotoxic stress Genotoxic stress-induced apoptosis is mediated by caspase family proteases as triggered by other stimuli. In this study, we found that the DNA-damaging agent cisplatin (cDDP) activated MAP kinase kinase kinase ASK1 and subsequent downstream subgroups of MAP kinase kinase, SEK1 (or MKK4) and MKK3/MKK6, which in turn activated c-Jun N-terminal kinase 1/stress-activated protein kinase (JNK1/SAPK) and p38 MAP kinase prior to caspase family protease activation and the onset of apoptosis in human ovarian carcinoma (OVCAR-3) and human kidney (293T) cells. As reported previously, benzyloxy carbonyl-Asp-CH2OC(O)-2, 6-dichlorobenzene (Z-Asp), a preferential inhibitor of caspase family proteases, blocked the apoptosis of OVCAR-3 cells induced by the genotoxic stress cDDP. Z-Asp, however, did not inhibit ASKI activation and the subsequent kinase cascades. Overexpression of kinase-negative ASK1 (K709R), which inhibited ASK1 activation and the downstream MKK3-p38 and MKK4-JNK1 pathways, also suppressed the caspase protease activation and apoptosis induced by cDDP. These results indicate that the ASK1 pathway is involved in genotoxic stress-induced apoptosis and mediates apoptosis at a step upstream of caspase protease activation.

Apoptosis↗

PubMed1999

Interleukin-1 alpha induced cyclooxygenase-2 expression in bone-derived endothelial cells.

Histological studies have suggested that vascular endothelial cells in bone are members of a complex network that regulates bone development and remodeling by producing soluble factors or by mediating cell-cell adhesion. To clarify the role of bone-derived endothelial cell lines (BDECs) in bone remodeling, we established several clones of BDECs from the femurs of BALB/c mice after transformation with the SV40 virus. Then we examined the response of these clones to interleukin-1alpha (IL-1alpha). IL-1alpha is known to induce bone resorption in part by increasing the expression of cyclooxygenase-2 (COX-2) that is associated with the production of PGE2 in osteoblast-lineage cells. Treating the primary and established BDECs with IL-1alpha induced COX-2 mRNA expression. A transcriptional activation assay revealed that the treatment with IL-1alpha increased COX-2 promoter activity in a dose-dependent manner, and IL-1alpha promoted COX-2 protein expression in BDECs. Treatment with IL-1alpha promoted PGE2 production from BDECs in a dose-dependent manner. These results indicate that IL-1alpha stimulates PGE2 synthesis largely by inducing BDECs to express COX-2. Because PGE2 stimulates bone resorption, these vascular endothelial cells, as well as osteoblast cells, play important roles in bone remodeling.

Animals↗

PubMed1999

Glucose-regulated stresses cause degradation of DNA topoisomerase IIalpha by inducing nuclear proteasome during G1 cell cycle arrest in cancer cells.

The glucose-regulated stress response of cancer cells leads to a decreased expression of DNA topoisomerase IIalpha (topo IIalpha) and a cell cycle arrest at the G1 phase. In this study, we found that the topo IIalpha decrease occurred specifically during the G1 arrest in human colon adenocarcinoma HT-29 cells. The intracelluar level of topo IIalpha in HT-29 cells was relatively constant regardless of cell cycle position in the exponentially growing state, determined using a centrifugal elutriation technique and synchronizing the cells with a mitotic inhibitor nocodazole. Interestingly, when the cell cycle was arrested in the M phase by nocodazole, the topo IIalpha level remained high even in stressed cells. After the stressed cells were released from the M phase, topo IIalpha steeply decreased along with cell cycle progression followed by the next G1 arrest. This decrease in nuclear topo IIalpha protein was completely inhibited by selective inhibitors for proteasome. Furthermore, we found that proteasome activity was elevated three to fourfold in the nuclear extract of stressed cells over unstressed cells. Accordingly, there were increased amounts of nuclear proteasome subunits, although total intracellular content of the subunits did not change in stressed cells. These findings indicate that the expression of topo IIalpha in stressed cells is downregulated at the G1 phase by proteasome-mediated degradation and that the proteolysis of topo IIalpha can be facilitated by the nuclear accumulation of proteasome.

Antigens, Neoplasm↗

PubMed1999

Contribution of P-glycoprotein to bunitrolol efflux across blood-brain barrier.

In this study, we investigated the mechanism of the blood-brain barrier (BBB) transport of bunitrolol (BTL), as a model of beta-blocker, in vivo and in vitro. In order to define the contribution of P-glycoprotein (P-gp) to the active efflux of BTL from brain to blood, we examined the in vivo brain distribution of BTL in mdr1a(-/-) mice with a disrupted mdr1a gene. After intravenous administration of BTL to mdr1a(-/-) mice, the brain concentration and Kp value of BTL were significantly increased as compared with those in mdr1a(+/+) mice. Next, the contribution of the mdr1a P-gp to in vitro uptake of BTL was compared in LV500 cells and L cells (mouse mdr1a-expressing cells and host cells, respectively). The intracellular accumulations of [3H]vinblastine and BTL by LV500 cells were lower than those by L cells, but were significantly increased by verapamil, a P-gp inhibitor. Furthermore, the BTL uptake by KB-VJ300 cells, which express human P-gp, was also significantly lower than that by KB host cells, and was increased by verapamil. The steady-state uptake of BTL by LLC-GA5-COL300 cells, expressing human P-gp, was significantly increased in the presence of 20 microM cyclosporin A (another P-gp inhibitor), which had no effect in the LLC-PK1 host cells. On the other hand, the steady-state intracellular accumulation of BTL by MBEC4 cells, which express mdr1b P-gp instead of mdr1a P-gp, was not significantly changed in the presence of verapamil. This finding suggested that BTL is not a good substrate for mdr1b P-gp. In conclusion, our results suggest that BTL is transported from brain to blood by mdr1a P-gp in mice and by MDR1 in humans, and this presumably accounts for the low brain distribution of BTL.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Cellular sensitization to cisplatin and carboplatin with decreased removal of platinum-DNA adduct by glucose-regulated stress.

PURPOSE: Stress conditions, such as glucose starvation and hypoxia, that induce glucose-regulated proteins (GRPs) in cells, are seen in most solid tumors. These conditions have been shown to cause cellular resistance to multiple anticancer drugs, such as etoposide, doxorubicin, and camptothecin. We examined the effect of the GRP-inducing conditions on cellular sensitivity to cisplatin and carboplatin, which are widely used drugs against solid tumors. METHODS: We generated the GRP-inducing culture conditions by exposing cells to 2-deoxyglucose (2DG), calcium ionophore A23187 and tunicamycin, and examined cellular sensitivity to cisplatin and carboplatin under these conditions. We next measured platinum accumulation and DNA-bound platinum in 2DG-stressed cells after cisplatin exposure. RESULTS: The GRP-inducing stress conditions led to cellular sensitization to cisplatin and carboplatin. This sensitization was reversible, as the cellular sensitivity returned to normal levels 12 h after removal of 2DG. Platinum accumulation and DNA-bound platinum that were found immediately after exposure to cisplatin for 1 h were slightly increased in 2DG-stressed cells as compared with nonstressed cells. After a drug-free recovery incubation of 8 h, the DNA-bound platinum in the nonstressed cells was reduced by 33% while the amount in the 2DG-stressed cells was sustained at the initial levels. CONCLUSIONS: These results indicated that the decreased removal of platinum-DNA adducts was associated with increased sensitivity to cisplatin and carboplatin in the stressed cells. The sensitization of cancer cells under the GRP-inducing stress conditions would explain, in part, the clinical potency of platinum drugs against solid tumors.

Antineoplastic Agents↗