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Biomedical subjects

N Kubota

Publications and source records attributed to N Kubota.

At least 109 records · Page 6Linked to original sources
PubMed1994

Characterization of an etoposide-resistant human ovarian cancer cell line.

Etoposide (VP-16) is one of the most important anticancer agents available and is used in many chemotherapeutic regimens. To characterize resistance to this drug, we established a VP-16-resistant human ovarian cancer cell line, SKOV3/VP, by continuous stepwise exposure of SKOV3 cells to VP-16. The degree of resistance to VP-16 of SKOV3/VP was about 25 times that of the parent cell line (SKOV3), and SKOV3/VP showed cross-resistance to teniposide, adriamycin, CPT-11, and vincristine. The accumulation of [3H]-VP-16 observed in SKOV3/VP cells was about half that seen in SKOV3 cells, and the accumulation of Adriamycin by this resistant cell line was also lower than that of its parent. Overexpression of neither the multidrug resistance gene mdr-1, the multidrug-resistance-associated protein (mrp) gene, nor P-glycoprotein was detected using reverse transcriptase-polymerase chain reaction analysis and flow cytometry with MRK-16, a monoclonal antibody against P-glycoprotein. The topoisomerase II activity of nuclear extracts from SKOV3/VP cells was lower than that from the parental cells, as was the amount of DNA topoisomerase II, demonstrated by immunoblotting. These results suggest that the mechanism responsible for the multidrug resistance of this cell line may be attributable to changes on its DNA topoisomerase II and to its reduced accumulation of the drugs as compared with the parental line SKOV3.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Characterization of a taxol-resistant human small-cell lung cancer cell line.

Taxol is a novel anticancer agent with activity against a broad range of tumors. It has a unique ability to stabilize polymerized tubulin into microtubule bundles within the cell. We have established a taxol-resistant human small-cell lung cancer cell line (H69/Txl) by exposing H69 cells to stepwise increases in taxol concentration. The resistance of H69/Txl cells to taxol was 4.7-fold that of the original H69 cells: the IC50 values for H69 and H69/Txl were 113.7 +/- 56.54 nM and 538.7 +/- 214.7 nM by the tetrazolium dye assay, respectively. Removal of the drug from the medium resulted in a 38% decrease in the growth rate of H69/Txl as compared with that in the presence of 30 nM taxol, suggesting that the growth of H69/Txl was partially dependent on taxol. H69/Txl showed higher sensitivity to vinca alkaloids such as vindesine, vincristine and vinblastine than the parental H69. There was no significant difference in intracellular [3H]taxol content between H69 and H69/Txl cells. No MDR-1 mRNA was detected in H69/Txl by the reverse transcription polymerase chain reaction. There was no significant difference of total and polymerized tubulin content between H69 and H69/Txl cells. Altered mobility of one of the alpha-tubulin isoforms in H69/Txl was revealed by using isoelectric focusing and Western blotting with anti-alpha-tubulin antibody. In H69, two alpha-tubulin isoforms were observed, whereas three were evident in H69/Txl, two of them comigrating with the isoforms of H69 and the other being more acidic. We observed the increased acetylation of alpha-tubulin in H69/Txl cells as compared with that in H69 cells. The acetylation of alpha-tubulin may be responsible for the taxol resistance and/or taxol-dependent growth of H69/Txl.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

A novel antitumor antibiotic, KW-2189 is activated by carboxyl esterase and induces DNA strand breaks in human small cell lung cancer cells.

KW-2189 has been selected as a lead compound for clinical trial among duocarmycin derivatives with structural similarity to CC-1065, a cyclopropylpyrroloindole. The purpose of this study was to examine the DNA-binding potency and the mechanisms of cytotoxicity of KW-2189. In order to analyze DNA-binding activity of KW-2189, plasmid pBR322 was treated with KW-2189 with or without pretreatment with carboxyl esterase, which we demonstrated to be an activating enzyme, and the products were examined by agarose gel electrophoresis and restriction enzyme analysis. Cytotoxic activity was examined by exposing a human small cell lung cancer cell line, NCI-H69 to KW-2189 with or without carboxyl esterase. Alkaline elution was performed to examine whether KW-2189 induces DNA strand breaks. DNA treated with KW-2189 and carboxyl esterase migrated faster than KW-2189-treated DNA, which migrated at the same rate as untreated DNA. In addition DNA treated with esterase-activated KW-2189 was protected from digestion by some restriction enzymes. KW-2189 showed concentration- and time-dependent growth inhibitory effect with IC50 values (drug concentration required for 50% growth inhibition) of 58 nM (96 h) to 1900 nM (1 h) in H69 cells. The IC50 values of 4-h exposure of H69 to KW-2189 with 0, 26, 130, 650 mU/ml carboxyl esterase were 460, 120, 30, and 7 nM, respectively. Time-dependent enhancement of cytotoxicity by carboxyl esterase was also observed. KW-2189 induced DNA strand breaks in H69 cells in a concentration-dependent manner around the IC50 value. We conclude that 1) KW-2189 is activated by carboxyl esterase to its active form(s), 2) activated KW-2189 has a stronger DNA-binding activity and cytotoxicity than KW-2189, 3) DNA cleavage is one of the major mechanisms of KW-2189-mediated cytotoxicity.

Antibiotics, Antineoplastic↗
PubMed1994

Immunohistochemical localization of estrogen receptors in the submandibular gland tumors of female rats.

The localization of estrogen receptors (EsR) in the tumor tissues of submandibular glands was examined in female rats, using the indirect immunoperoxidase method in combination with the in situ hybridization technique. Tumors were experimentally produced by 9,10-dimethyl-1,2-benzanthracene (DMBA), and the tumor tissues were fixed with formalin or paraformaldehyde and then embedded in paraffin. In the DMBA-induced submandibular gland tumors, immunoreactivity to EsR-peroxidase conjugate was found in nuclei of the tumor cells which occupied the peripheral rim of the tumor cell nests. In contrast, the reactivity in the normal submandibular glands without tumor was mostly confined to nuclei of the duct cells. When EsR mRNA expression was analyzed in the tumor tissue by in situ hybridization with a cDNA probe, its distribution was identical with that of immunoreactivity to EsR. These data suggest that the ductal cells of the submandibular gland are responsive to ovarial steroids, and that estrogens may play an important role in the maintenance of growth of the submandibular gland tumors.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1994

Turbidimetric latex agglutination inhibition immunoassay for primidone.

We have developed a quick, highly sensitive immunoassay method for drugs by latex agglutination inhibition. An antiserum against primidone (PRM) was obtained by immunizing rabbits with PRM-bovine serum albumin conjugate. PRM-rabbit serum albumin conjugate sensitized latex was agglutinated with diluted antiserum, and the agglutination was inhibited by free PRM quantitatively. Turbidity of the agglutination suspension was measured by spectrophotometry as absorbance. Larger latex gave higher sensitivity than the smaller, because its agglutination was inhibited more intensely by free PRM. The assay values of this method were correlated well with those obtained by an enzyme immunoassay method.

Animals↗
PubMed1994

[Observation of the eyelid structure with opening and closing of the eye in magnetic resonance imaging].

To confirm the changes in the upper eyelid structure during normal movements such as opening and closing we observed the changes with magnetic resonance imaging and obtained the following results. (1) In the anterior lobe of the upper eyelid, the lower part moves more than the upper part. (2) The tarsus slides toward posterior and upper direction when the eye opens. (3) The orbital septum and the orbital fat move posteriorly and thicken when the eye opens. (4) When the eye opens Whitnall's ligament moves posteriorly with a little change of the acting vector direction of the levator muscle. (5) Aponeurosis of the levator muscle clearly separates from Müller's muscle when the eye opens. (6) We suspected that there is some suspending tissue of Whitnall's ligament that consists of a membranous or mesh-like structure in the orbital fat above the levator muscle.

Adult↗
PubMed1994

The effects of X-ray energy and an iodine-based contrast agent on chromosome aberrations.

A study was undertaken to evaluate the effect of combining irradiation with X rays of various energies and an iodine-based contrast agent on the induction of chromosome aberrations in the peripheral lymphocytes of blood samples taken from healthy young donors. Although no enhancement of the effect of radiation was induced when blood samples with the iodine-based contrast agent were given 35 kV X irradiation, an 80 kV X-ray exposure induced an enhanced level of chromosome aberrations, and at 250 kV X irradiation, an enhancement of the frequencies of chromosome aberrations was seen in blood samples with the iodine-based contrast agent, especially when a Lucite phantom was employed in studies to increase the scattered rays. It was thus shown by microdosimetric analysis that X irradiation combined with an iodine-based contrast agent causes an enhancement of the absorbed radiation dose, which is dependent on the X-ray energies employed. This phenomenon may have clinical use in the radiotherapeutic management of tumors, although further extensive studies of tumor vascularity must be pursued before this can be applied clinically.

Adult↗
PubMed1994

[Changes in liver tissue amino acids during hemorrhagic shock].

Changes in amino acids in liver tissue during hemorrhagic shock were examined in eight mature mongrel dogs using the microdialysis method. The mean blood pressure was maintained at 50 mmHg by exsanguination, and the values of liver tissue amino acids after 30 minutes showed significant decreases of aspartate, glutamate, histidine, glutamine, glycine and taurine. The decrease of taurine was especially marked. The values dropped further 30 minutes after returning the blood into circulation with some exceptions. These results suggest the possibility that hemorrhagic shock causes reduction in uptake of amino acids by the liver or consumption of amino acids in the liver, and that bile acid metabolism appears to be disturbed by the marked drop in the taurine level. It is necessary to give careful consideration to the possible existence of reduced amino acid metabolism in the liver in the treatment of hemorrhagic shock.

Amino Acids↗
PubMed1994

[A pregnant woman with convulsion treated with diazepam which was reversed with flumazenil just prior to cesarean section].

A 37 year-old female in 36th week gestation with convulsion treated with intravenous diazepam underwent an emergency cesarean section. Just prior to induction of anesthesia flumazenil was administrated intravenously to reverse the effects of diazepam in both mother and fetus. Cesarean section was successful under general anesthesia (N2O-pentazocine). The neonate's Apgar score was 6 points at 1 minute and 7 points at 5 minutes after delivery. We conclude that intravenous administration of flumazenil just prior to Cesarean section is effective in a patient with convulsion treated with diazepam.

Adult↗
PubMed1993

Cis-diamminedichloroplatinum(II) inhibits p34cdc2 protein kinase in human lung-cancer cells.

cis-Diamminedichloroplatinum(II) (CDDP) induced G2-phase arrest in PC-9 human cancer cells. To elucidate how CDDP acts on cell-cycle regulation, we analyzed the effect of CDDP on cell-cycle regulators such as p34cdc2 protein kinase. p34cdc2 protein kinase activity was maximum in G2 phase and decreased after G2/M transition in synchronized PC-9 human lung cancer cells. Evidence for a phosphorylated p34cdc2 protein kinase complexed with cyclin B was obtained from cells in G2 phase and the p34cdc2 protein kinase appeared to be dephosphorylated at M phase. After exposure to CDDP in G1 phase, PC-9 cells were arrested in G2 phase. The activation of p34cdc2 protein kinase was inhibited by CDDP. Cyclin A and wee-I kinase were not affected by the exposure to CDDP. Cyclin B was degraded in M phase in PC-9 cells. Exposure to CDDP did not affect the degradation of cyclin B. Our data suggest that the effect of CDDP on cell-cycle phase might be regulated by the dephosphorylation of p34cdc2 protein kinase. To determine whether the p34cdc2 protein kinase is a primary target for CDDP, we examined the direct effect of CDDP on tyrosine dephosphorylation of p34cdc2 protein kinase in cellular extracts. Cell lysates from synchronized PC-9 in G2 phase were immunoprecipitated with p13-Sepharose beads. In vitro dephosphorylation of phosphotyrosine of p34cdc2 protein kinase was observed after exposure to okadaic acid in a concentration-dependent manner. The dephosphorylation of p34cdc2 protein kinase by okadaic acid was inhibited by CDDP. We hypothesize that inhibition of p34cdc2 dephorphorylation by CDDP is important for its growth-inhibiting properties.

CDC2 Protein Kinase↗
PubMed1993

Apoptosis of lung cancer cells caused by some anti-cancer agents (MMC, CPT-11, ADM) is inhibited by bcl-2.

To determine whether the apoptotic cell death induced by anti-cancer agents could be inhibited by bcl-2, we established a bcl-2-transfected human small cell lung cancer cell line, SBC-3/Bcl2. SBC-3/Bcl2 showed higher resistance to ADM, CPT-11 and MMC compared with the parental line SBC-3, with relative resistance values of 3.4, 7.6 and 5.7, respectively. However, there was no difference in sensitivity to CDDP, VP-16, ACNU, MTX and taxol between SBC-3 and SBC-3/Bcl2. Agarose gel electrophoresis showed typical DNA fragmentation of SBC-3 following treatment with CPT-11 or MMC, in a concentration-dependent manner. In contrast, the same concentration of the drugs did not induce DNA fragmentation in SBC-3/Bcl2. Treatment with CDDP resulted in the same degree of DNA fragmentation in SBC-3 and SBC-3/Bcl2. These studies indicate that bcl-2 can modulate the cytotoxicity of some anti-cancer agents by inhibiting the process of apoptosis.

Animals↗
PubMed1993

Hyperthermic enhancement of cell killing by five platinum complexes in human malignant melanoma cells grown as monolayer cultures and multicellular spheroids.

The cytotoxic properties of hyperthermia combined with cis-diammine-dichroloplatinum(II) (CDDP), and recently developed platinum complexes, (Glycolato-O-O')diammineplatinum(II) (254-S), cis-1-1-cyclobutane-dicarboxylate-(R)-2-methyl-1-4-butanediammine platinum(II) (NK-121), cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) (CBDCA), and (-)-R-[2-(aminomethyl)pyrrolodine](1,1- cyclobutanedicarboxylato)-platinum(II)monohydrate (DWA-2114R) were studied in vitro in monolayer cultures and multicellular spheroids of HMV-I human malignant melanoma cells. Hyperthermia at 44 degrees C for 30 min was applied during the latter part of 1 hr drug exposure. Cell survival was compared after drug treatments in cells exposed or not exposed to heat. Cytotoxicity was assessed by clonogenic assays. In exponentially growing monolayer cultures, marked hyperthermic sensitization was observed by each of the five platinum complexes studied. The dose modifying factors obtained were almost the same in these drugs. Unlike monolayer cells, the spheroids were appreciably different with regard to hyperthermic sensitization among platinum complexes. The order of the magnitude was as follows: CDDP, DWA-2114R, 254-S, CBDCA, and NK-121. In the low dose region, however, 254-S was the most thermally sensitized. These results suggest that the microenvironment factor within spheroids may significantly affect the cytotoxicity of platinum complexes combined with hyperthermia. On the basis of these findings using spheroids, CDDP, DWA2114R, and 254-S appear to be promising platinum complexes for use with hyperthermia clinically as far as hyperthermic sensitization is concerned.

Antineoplastic Agents↗
PubMed1993

Characteristics of neutron beam generated by 500 MeV proton beam.

A neutron irradiation facility was constructed at PARMS, University of Tsukuba to produce an ultrahigh energy neutron beam with a depth dose distribution superior to an x-ray beam generated by a modern linac. This neutron beam was produced from the reaction on a thick uranium target struck by a 500 MeV proton beam from the booster synchrotron of the High Energy Physics Laboratory. The percentage depth dose of this neutron beam was nearly equivalent to that of x-rays around 20 MV and the dose rate was 15 cGy per minute. The relative biological effectiveness (RBE) of this neutron beam has been estimated using the cell inactivation effect and the HMV-I cell line. The survival curve of cells after neutron irradiation has a shoulder with n and Dq of 8 and 2.3 Gy, respectively. The RBE value at the 10(-2) survival level for the present neutron beam as compared with 137Cs gamma rays was 1.24. The results suggest that the biological effects of ultrahigh energy neutrons are not large enough to be useful, although the depth dose distribution of neutrons can be superior to that of high energy linac x-rays.

Cell Survival↗
PubMed1993

The effect of sevoflurane on rat liver mitochondrial respiration.

The effect of sevoflurane on rat liver mitochondrial respiration has been investigated with a Clark type oxygen electrode at 25 degrees C, pH 7.4. The higher susceptibility of NADH-linked substrate (glutamate) oxidation (with respect to succinate oxidation) to the damages by sevoflurane has been confirmed.

Journal Article↗
PubMed1993

Novel water-soluble derivatives of docosahexaenoic acid increase diacylglycerol production mediated by phosphatidylcholine-specific phospholipase C.

The effect of ascorbic acid 6-docosahexaenoate (DHA-VC) on the phospholipase-C-mediated hydrolysis of phosphatidylcholine was investigated. In human non-small cell lung cancer cells (PC-14) exposed to DHA-VC for 24 hr, a dose-dependent increase in phosphatidylcholine-specific phospholipase C (PC-PLC) activity was seen. PC-PLC activity in whole-cell homogenate of PC-14 cells was increased about 2.5-fold by 2 hr of treatment with DHA-VC (20 micrograms/ml). Treatment with DHA-VC also augmented PC-PLC activity in the crude membrane extract. On the other hand, DHA-VC inhibited the activity of phospholipase A2 (ID50 = 800 micrograms/ml). Another water-soluble analog, choline docosahexaenoate, also stimulated PC-PLC activity. To explore the effect of DHA-VC on phosphatidylcholine turnover, we analyzed phospholipids labeled with [14C] choline or [3H]myristate by thin-layer chromatography, and found that the amount of [14C]- and [3H]-labeled phosphatidylcholine was constant in the presence of DHA-VC. These results suggest that phosphatidylcholine turnover was not influenced by DHA-VC. DHA-VC treatment increased protein kinase C activity of the cells in the late phase (120 min), suggesting that DHA-VC-induced diacylglycerol production mediated by PC-PLC causes protein kinase C activation. Considering that significant inhibition of DNA synthesis occurred 12 hr after 2 hr of treatment with DHA-VC (20 micrograms/ml), DHA-VC-induced PC-PLC activation seems to be an early event in DHA-VC-induced cytotoxicity, which suggests that the effects of DHA-VC on signal transduction pathways may play an important role in the cytotoxicity of DHA-VC.

Ascorbic Acid↗
PubMed1993

[The effects of sevoflurane anesthesia with hypothermia on ischemic brain cellular respiration].

We examined the effects of sevoflurane anesthesia with hypothermia on ischemic brain cellular respiration using Wistar strain rats. Ischemia was induced by decapitation. L/P in brain increased during no anesthesia-normothermia-ischemia (1), then gradually, but not fully recovered during 2.5% sevoflurane anesthesia-normothermia-ischemia (2) and during 2.5% sevoflurane anesthesia-hypothermia-ischemia (3). Energy charge decreased during (1) and slight recovery was observed during (3). Respiratory control ratio in brain mitochondria decreased during (1) and recovery during (2) and (3) was slight. No recovery was observed in ADP/O. These findings indicate that during sevoflurane anesthesia with hypothermia, ischemic brain energy metabolism has an effect not on the mitochondrial energy producing processes but on the consuming system.

Anesthesia, Inhalation↗
PubMed1993

[The development of occlusion amblyopia following atropine therapy for strabismic amblyopia].

For the treatment of strabismic amblyopia the authors have used atropine in the sound eye under usual optical correction as soon as central fixation of the amblyopic eye is secured by means of proceeding occlusion therapy. Although there may be little effect of occlusion in atropine therapy, six children under five years of age developed amblyopia in their sound eyes. In every case, the first sign of developing amblyopia was the decrease of fixation ability in the sound eye in a binocular fixation test prior to the reduction of visual acuity. On the other hand, fixation ability of the originally amblyopic eye was reinforced more and more not only during atropinization but after its finish until deep amblyopia developed in the sound eye. In three cases, eccentric fixation occurred at 7, 9, and 17 weeks after atropinization. These facts suggest that occlusion amblyopia following atropine therapy is not the result of deprivation but is due to the reversal of the eye used for fixation. Strabismic amblyopia may have the tendency toward an alternation between fixation abilities of the two eyes and such a binocular sensory anomaly may cause strabismic amblyopia itself.

Amblyopia↗