Biomedical subjects

K Nishio

Publications and source records attributed to K Nishio.

At least 163 records · Page 9Linked to original sources

PubMed1995

[Blood gas changes in Duchenne type muscular dystrophy].

We studied changes in blood gas data from 32 patients with Duchenne type muscular dystrophy (DMD), who were followed for more than 5 years. Regression lines for each blood gas variable were obtained by the least square regression method, from 276 observations. The mean change per year was calculated by averaging the individual slopes. All blood gas variables (PaO2, PaCO2, pH, and [HCO3-]) correlated significantly with age the regression lines were: PaO2 = 95.6-0.45x Age, PaCO2 = 34.7 + 0.70x Age, pH = 7.397-0.0023x Age, [HCO3-] = 20.7 + 0.28x Age. The rates of change were -0.74 Torr/year for PaO2, 1.07 Torr/year for PaCO2, -0.0028 per year for pH and 0.55 mEq/l/year for [HCO3-]. The slope of each regression line was within the average individual yearly change +/- one standard deviation. AaDO2 did not change significantly with age, but chronic respiratory acidosis worsened because of alveolar hypoventilation. As an index of the severity of DMD, PaCO2 was better than PaO2, because the former was more strongly correlated with age and the values of PaCO2 deviated less from the the regression line.

Adolescent↗

PubMed1995

Cell cycle-dependent localization of tissue inhibitor of metalloproteinases-1 immunoreactivity in cultured human gingival fibroblasts.

Localization of the tissue inhibitor of metalloproteinases-1 (TIMP-1) was investigated in cultured human gingival fibroblasts by immunohistochemistry. TIMP-1 immunostaining was observed in the cytoplasm of a majority of cells, and in the nucleus of some cells. Depletion of fetal calf serum (FCS) from the culture medium reduced the density of immunoreactive TIMP-1 in the nucleus more remarkably than in the cytoplasm, and the following FCS-induced cell growth was accompanied by a recovery of immuno-reactive TIMP-1 in both the nucleus and the cytoplasm. The frequency of the nuclear TIMP-1-positive cells changed in line with that of PCNA-positive cells and was always much higher than that of S-phase cells, which were estimated by a pulse labelled 5-Bromo-2' deoxyuridine (BrdU). These results suggest a localization of TIMP-1 or a related substance in the nucleus of proliferating human fibroblasts, and its depletion from the nucleus due to an arrest of cell growth.

Cell Cycle↗

PubMed1995

Cross-sectional transmission electron microscopy of ZnO tetrapod-like particles.

ZnO tetrapod-like particles having legs as long as several tens of micrometers have been observed by cross-sectional electron microscopy. The specimen is prepared by a new method where a photo-resist suspension of the particles fills up holes of an electron microscopy grid and is solidified for the subsequent ion-milling. Electron diffraction and high-resolution electron microscopy of a section of a ZnO tetrapod reveal the orientation relationship among its four legs, which enables us to discuss on the growth mechanism of these particles.

Microscopy, Electron↗

PubMed1994

Effect of suramin on p34cdc2 kinase in vitro and in extracts from human H69 cells: evidence for a double mechanism of action.

We examined the effect of suramin, an anticancer agent and a functional analog of naturally occuring glycosaminoglycans, on p34cdc2 kinase. We find that suramin strongly inhibits the catalytic activity of purified p34cdc2 kinase (IC50 approximately 4 microM), whereas it only weakly inhibits the p13-agarose precipitated kinase activity from nuclear and cytoplasmic extracts of the asynchronous H69 human small cell lung cancer cells. We also find that the tyrosine phosphorylation of p34cdc2 kinase in the nuclear extract is increased about twice when the extracts are preincubated with 50 microM of suramin prior to the p13-agarose precipitation. We propose that this increase might result from the inhibitory effect of suramin towards p34cdc2-specific tyrosine phosphatases. These results suggest both a direct and an indirect effect of suramin on p34cdc2 kinase. We also find that heparin is a potent inhibitor of purified cdc2 kinase (IC50 approximately 3.5 micrograms/ml). Therefore, glycosaminoglycans might be physiological regulators of p34cdc2 kinase in vivo.

Antineoplastic Agents↗

PubMed1994

Establishment of a human small-cell lung-cancer subline resistant to okadaic acid.

Okadaic acid (OA), a specific protein phosphatase inhibitor, has various biological functions. To elucidate the mechanism of OA resistance, we have established a small-cell lung-cancer subline (H69/OA100) resistant to the growth-inhibitory effect of OA; this was done by using the parental cell line (H69) and increasing the concentration of OA. H69/OA100 was about 8 times more resistant to OA than H69. Intracellular retention of the fluorescent OA derivative in H69/OA100 was the same as that in H69. The catalytic activity of protein phosphatase from H69/OA100 was significantly reduced compared with that from H69. The protein phosphatase from H69/OA100 was 3.6 times more resistant to OA than that from H69. We examined the effect of OA on the activity of the immunoprecipitated protein phosphatase type I (PPI) and type 2A (PP2A) from the 2 cell lines. The PPI and PP2A from H69/OA100 showed more resistance to OA than those from H69. We next examined the effect of OA on the cell cycle of H69 and H69/OA100. In H69, G2/M block was observed at an OA concentration of 30 ng/ml whereas in H69/OA100, no G2/M block was observed at concentrations up to 100 ng/ml OA. We finally evaluated the amount of p34cdc2 kinase expression and the phosphorylation status of p34cdc2. There was no difference in p34cdc2 expression between H69 and H69/OA100 at several concentrations of OA. However, dephosphorylation of p34cdc2 was observed at 30 ng/ml OA in H69, but not in H69/OA100 up to 100 ng/ml OA. These data suggest that the resistance to OA and the resistance of the cell-cycle block to OA in H69/OA100 might be due to alteration of protein phosphatase activity.

Base Sequence↗

PubMed1994

Reversal of multidrug resistance by tyrosine-kinase inhibitors in a non-P-glycoprotein-mediated multidrug-resistant cell line.

We have already established a human leukemia sub-line resistant to the growth-inhibitory effect of TPA (12-O-tetradecanoylphorbol 13-acetate) (K562/TPA) derived from K562. K562/TPA was found to be a non-P-glycoprotein-mediated multidrug-resistant cell line, in which intracellular drug accumulation was not reduced. In K562/TPA, adriamycin (ADM) was distributed mainly in the cytoplasm and was scarcely observed in the nucleus. We determined the relative levels of multidrug-resistance-associated protein (MRP), which was recently identified as the novel transporter. The relative levels of MRP in K562/TPA were the same as in K562. Although the catalytic activity of K562/TPA topoisomerase II was about half that of the parental cells, resistance to other drugs could not be explained by topoisomerase-II activity. To elucidate the mechanism of drug resistance in K562/TPA, we tried to find chemicals that would reverse the drug resistance. Tyrosine-kinase inhibitors enhanced the cytotoxicity of anti-neoplastic drugs against K562/TPA. Therefore we examined the modification of nuclear ADM accumulation in K562/TPA by one of these tyrosine-kinase inhibitors, genistein. Although the amount of ADM was decreased in the nuclei of K562/TPA cells, it was significantly increased after incubation in the presence of genistein. The formation of DNA single-strand breaks by ADM, etoposide, and ACNU was significantly lower in K562/TPA than in K562, but was significantly increased in the presence of genistein. These results suggest that genistein could overcome drug resistance by enhancing the accumulation of drug into the nuclear fraction of K562/TPA.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Ouabain-resistant non-small-cell lung-cancer cell line shows collateral sensitivity to cis-diamminedichloroplatinum(II) (CDDP).

We have reported that the cellular uptake of cis-diamminedichloroplatinum(II) (CDDP) was inhibited by an Na+,K(+)-adenosine triphosphatase (ATPase) inhibitor, ouabain, in a human non-small-cell lung-cancer cell line, PC-14, but not in its CDDP-resistant cell line, PC-14/CDDP. [3H]Ouabain binding of PC-14/CDDP was about 50% lower than that of PC-14. Accordingly, we speculated that a decrease in Na+,K(+)-ATPase activity in PC-14/CDDP might contribute to the decrease in cellular CDDP accumulation. To clarify the relationship between the activity or expression of Na+,K(+)-ATPase and cellular CDDP accumulation, we established an ouabain-resistant non-small-cell lung-cancer cell line (PC-14/OB300), which showed 1.9-fold resistance to the cytotoxicity of ouabain. Interestingly, this cell line was 4.2-fold more sensitive to CDDP than PC-14. The accumulation of CDDP in PC-14/OB300 was increased to 2.7-fold that in PC-14. This elevation of CDDP accumulation was not considered to be caused by increased passive diffusion, because the accumulation of CDDP in PC-14/OB300 was also inhibited by ouabain compared to PC-14. As one of the indices of Na+,K(+)-ATPase activity, we determined cellular 86Rb+ influx rates. The 86Rb+ influx rate was 1.5-fold higher in PC-14/OB300 and fell to 0.7-fold in PC-14/CDDP compared with PC-14. The mRNA expression of Na+,K(+)-ATPase was increased in PC-14/OB300 and decreased in PC-14/CDDP. There was no difference in cellular [3H]ouabain binding between PC-14/OB300 and PC-14. It is possible that Na+,K(+)-ATPase of PC-14/OB300 has a different affinity for ouabain from that of PC-14. Our results suggest that the enzyme activity or the level of expression of Na+,K(+)-ATPase may contribute to the cellular uptake of CDDP and determine the sensitivity to CDDP.

Adenocarcinoma↗

PubMed1994

7-Ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy camptothecin: mechanism of resistance and clinical trials.

The camptothecin derivative 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxy camptothecin (CPT-11) has attracted the attention of clinicians because of its high antitumor activity against refractory solid cancers. We established two CPT-11-resistant cell lines, a non-small-cell lung-cancer cell line (PC-7/CPT-11) from the parental PC-7 line and an ovarian cancer cell line (HAC-2/CPT-11) from the parental HAC-2 line. The mechanisms of resistance to CPT-11 in PC-7/CPT-11 cells were reduced conversion of CPT-11 to its active metabolite SN-38 and point mutation topoisomerase I. Those in HAC-2/CPT-11 cells were reduction of topoisomerase I activity and decreased sensitivity of topoisomerase to topoisomerase I inhibitors. No point mutation of the topoisomerase was observed in HAC-2/CPT-11 cells. We conducted two phase I trials using CPT-11 in combination with other anticancer agents. One was a phase I trial of CPT-11 and cisplatin given with a fixed dose of vindesine to patients with advanced non-small-cell lung-cancer and the other was a phase I study on a topoisomerase-targeting combination of CPT-11 and etoposide (VP-16) in patients with various malignant solid tumors. The results of the first trial indicated that the recommended dose of CPT-11 for phase II studies was 80 mg/m2 combined with 3 mg/m2 vindesine on days 1 and 8 and 60 mg/m2 cisplatin on day 1. In the second trial, the recommended dose of CPT-11/VP-16 given with recombinant granulocyte colony-stimulating factor (on days 4-17) was found to be 60/60 mg/m2. In both trials, diarrhea and granulocytopenia were considered to be dose-limiting toxicities.

Amino Acid Sequence↗

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

Apoptosis induced by etoposide in small-cell lung cancer cell lines.

The DNA fragmentation, a parameter of apoptosis, in non-small (NSCLC) and small (SCLC) cell lung cancer cell lines (N231 and PC-9) was evaluated. The DNA fragmentation in SCLC lines, but not in NSCLC lines, was observed in overgrown cells without exposure to anticancer drugs. In etoposide (VP-16)-treated N231 but not PC-9 cells, DNA fragmentation continued to increase up to 42 h, and the increase was dependent on the concentration of VP-16. The endonuclease activity of VP-16-treated N231, but not PC-9, cells required both Ca2+ and Mg2+ for full activity. It was elevated in a time- and concentration-dependent manner. As this activity was not affected by addition of cycloheximide, the activation of the endonuclease activity without protein synthesis may be involved in VP-16-induced cytotoxicity in N231.

Apoptosis↗

PubMed1994

In vitro and in vivo growth of B16F10 melanoma cells transfected with interleukin-4 cDNA and gene therapy with the transfectant.

In an attempt to develop the most effective cytokine gene therapy, we transfected mouse interleukin(IL)-2, mouse IL-4, and human IL-6 cDNAs into mouse melanoma cells, B16F10. Transfection with IL-4 cDNA decreased the tumorigenicity of B16F10 most strongly. We investigated whether gene therapy with IL-4-transfected B16F10 cells was possible. Flow-cytometric analysis showed that major histocompatibility complex class I and II expression in B16F10 and IL-4-cDNA-transfected B16F10 (B16F10-IL4) cells did not differ. Doubling times of B16F10 and B16F10-IL4 were 20.1 and 21.1 h respectively. The growth of B16F10 cells was retarded if C57BL/6 mice were inoculated with B16F10-IL4 at the contralateral sides. When 5 x 10(5) B16F10 cells were transplanted subcutaneously into the flanks of C57BL/6 mice, they all developed a tumor mass, whereas no tumor masses formed in those transplanted with B16F10-IL4 cells within 60 days. No nude, severe combined immunodeficient or beige mice were able to reject parental B16F10 or B16F10-IL4 cells, although, B16F10-IL4 tumor growth in all these immunodeficient mice was slower than that of B16F10. Therefore, we concluded that T and natural killer cells are necessary for rejection of B16F10-IL4 tumor cells.

Animals↗

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

In vitro and in vivo effects of cisplatin and etoposide in combination on small cell lung cancer cell lines.

The effects of cisplatin (CDDP) and etoposide (ETP) in combination were evaluated in vitro and in vivo using small cell lung cancer cell lines. The combination effects in vitro were investigated using isobologram analysis. Used together, CDDP and ETP showed a synergistic effect against cell growth on only 1 cell line (SBC-3), additive effects on 6 (SBC-2, SBC-5, Lu130, Lu134AH, Lu135T and H69) and an antagonistic effect on 1 (SBC-1). In the in vivo experiment, nude mice were inoculated with SBC-1, SBC-3 and SBC-5 cells. Two or 5 mg/kg CDDP and 10 or 30 mg/kg ETP were administered intraperitoneally alone and simultaneously in combination to nude mice. The in vivo effects of the combination were determined by comparing the observed growth ratio in mice treated with the combination with the expected value of this ratio calculated based on the assumption that the effects of the drugs were simply additive. According to this definition, synergistic effects were observed against all 3 tumors. Thus, the in vivo and in vitro effects differed. The toxicity of the combination therapy, which was analyzed by estimating the body weight change of mice, was no higher than that of CDDP or ETP alone. These results suggest that the excellent clinical effects of CDDP and ETP combination therapy may be attributable not to drug interaction at the cellular level but to the feasibility of combined use of them at full doses without overlapping side effects.

Animals↗

PubMed1994

Genistein, a tyrosine kinase inhibitor, decreased the affinity of p56lck to beta-chain of interleukin-2 receptor in human natural killer (NK)-rich cells and decreased NK-mediated cytotoxicity.

Intracellular signal transduction has been reported to be triggered by phosphorylation of interleukin-2 (IL-2) receptor by IL-2. In order to clarify the effect of tyrosine phosphorylation of IL-2 receptors on cell-mediated cytotoxicity by natural killer (NK) cells, we studied the effect of a tyrosine kinase inhibitor, genistein, on the lethal effects of NK-rich cells for K562 cells. Exposure of NK-rich cells to IL-2 (100 U/ml) for 3 days increased their cytotoxicities against K562 cells. The effect was reduced in the presence of 10 micrograms/ml of genistein. Samples immunoprecipitated by anti-IL-2R beta antibodies were prepared from NK-rich fractions with or without exposure to IL-2 and/or genistein. Coprecipitated proteins with 75, 65, and 56 kDa were detected with an antiphosphotyrosine antibody. The amount of phosphorylated tyrosine residues of 56-kDa protein, which was predominantly detected in NK-rich cells, was remarkably increased by IL-2 treatment. The enhanced phosphorylation of 56-kDa protein was reduced by the presence of genistein. These results suggested that IL-2 increased tyrosine phosphorylation and the affinity to IL-2R beta of the 56-kDa proteins in NK-rich cells. Immunoprecipitated samples by anti-IL-2R beta were reblotted with anti-p56lck antibody and revealed that the 56-kDa protein was identified to be p56lck. The increase of coprecipitated p56lck with anti-IL-2R beta antibody by the treatment with IL-2 suggested that the affinity of p56lck to IL-2R beta was increased by IL-2 in NK-rich cells. The amount of coprecipitated p56lck with IL-2R beta was reduced in the sample exposed to genistein. The affinity of p56lck to IL-2R beta was considered to be regulated by IL-2-induced tyrosine phosphorylation. Our results demonstrated a potential role for tyrosine kinase, p56lck, in the signaling events that regulate the cytotoxicity by NK-rich cells.

Cytotoxicity, Immunologic↗

PubMed1994

Gene therapy for Lewis lung carcinoma with tumor necrosis factor and interleukin 2 cDNAs co-transfected subline.

Gene therapy with cytokine cDNA will provide a new tool for cancer treatment. We have already reported that immunization with interleukin-2 (IL2) cDNA transfected Lewis lung carcinoma (LLC) cells induced anti-tumor immunity, which, however, was not strong enough to eradicate an established tumor. In an attempt to develop more effective gene therapy methods, we have used tumor cells co-transfected with IL-2 and tumor necrosis factor (TNF) cDNAs. These cDNAs were introduced into pBMG-Neo and pcDV-X819 vectors, respectively, and then co-transfected into LLC cells. The co-transfectants were selected by incubating them in a medium containing G418 followed by the limiting dilution method twice to obtain IL2 and TNF cDNA co-transfected LLC (LLC-TNF-IL2) cells. When 5 x 10(5)/ml LLC-TNF-IL2 cells were incubated for 48 h, they secreted 7.56 U/ml TNF and 527.0 U/ml IL2 into the culture supernatant. When C57BL/6 mice were transplanted with 1 x 10(6) LLC-TNF-IL2 cells, all the tumors were rejected. The growth of transplanted LLC, but not B16F10 melanoma cells, was retarded in mice inoculated with LLC-TNF-IL2 on their contralateral sides, which suggests specific immunity was induced. The immunization effect by the co-transfectant was superior to that of the IL2- and TNF-transfectants alone.

Animals↗

PubMed1994

[Effect of cisplatin on cell cycle regulators].

An anticancer agent, cisplatin (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 kinase activity was maximum in G2 phase and decreased after G2/M transition in synchronized PC-9 cells. Evidence for a phosphorylated p34cdc2 complexed with cyclin B was obtained from cells in G2 phase and the p34cdc2 appeared to be dephosphorylated at M phase. After exposure to CDDP in G1 phase, PC-9 cells were arrested at G2 phase. The activation of p34cdc2 was inhibited by CDDP. Cyclin A, B and wee-1 kinase were not affected by the exposure to CDDP. Our data suggested that the effect of CDDP on cell-cycle phase might be regulated by the dephosphorylation of p34cdc2. We hypothesize that inhibition of p34cdc2 dephosphorylation by CDDP is important for its growth-inhibiting properties.

CDC2 Protein Kinase↗

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↗