Mutations in the Plk gene lead to instability of Plk protein in human tumour cell lines.

It has been established that mutations in Drosophila Polo cause abnormalities in mitosis. In human cells, maximal Plk activity is reached in the M phase of the cell cycle, and the function of Plk is therefore considered to be required for mitotic cellular events such as spindle formation, chromosome segregation and cytokinesis. Microinjection of anti-Plk antibody into living cells has been found to induce a mitotic abnormality that contributes to the generation of aneuploidy, and this is an important finding in relation to tumour development. Indeed, previous studies have shown that the level of expression of a mitotic checkpoint gene, hsMAD2, is reduced and that another checkpoint gene, BUB1, is mutated in certain human cancer cells.

Cytological criteria of endometrial lesions with emphasis on stromal and epithelial cell clusters: result of 8 years of experience with intrauterine sampling.

OBJECTIVE: There are a number of unresolved issues in endometrial cytology. They include the significance of nuclear atypia for the diagnosis of grade1 adenocarcinoma (G1AC) and atypical endometrial hyperplasia (AEH), cytological criteria of endometrial hyperplasia without atypia, and recognition of stromal cell cluster (SC) and its distinction from epithelial cell cluster (EC). METHODS: We examined nuclear atypia, SC and EC in typical cases of five categories: normal endometrium (NEM), simple endometrial hyperplasia without atypia (SEH), complex endometrial hyperplasia without atypia (CEH), G1AC and grade2 adenocarcinoma (G2AC). We classified EC into four types: simple EC (SPEC), large regular EC (LREC), large irregular EC (LIEC) and small irregular EC (SIEC). Based on the results, we developed criteria of endometrial cytology and have evaluated 13 639 cases over 8 years. RESULTS: Nuclear atypia was significantly more frequent in G2AC than in any of the other four categories (P < 0.001). SC was significantly more frequent in NEM and SEH than in the other three categories (P < 0.001). G1AC and G2AC showed significantly higher frequency of LIEC than the other three categories (P < 0.001). CEH exhibited significantly higher frequency of LREC than the four categories (P < 0.001). The sensitivity and the specificity was 88.8% and 99.0% respectively. CONCLUSIONS: We could diagnose G1AC, G2AC and CEH with high accuracy using the established criteria mainly based on SC and EC. We think that the criteria may facilitate an effective screening and an objective interpretation of endometrial samples.

Involvement of hydrogen peroxide production in erbstatin-induced apoptosis in human small cell lung carcinoma cells.

Tyrosine kinase inhibitor, erbstatin, induced morphological apoptosis and DNA fragmentation in human small cell lung carcinoma (SCLC) cells. Erbstatin-induced apoptosis was inhibited by antioxidants, whereas erbstatin-inhibited tyrosine phosphorylation was not affected by them. Erbstatin was shown by means of flow cytometry to induce hydrogen peroxide generation. Furthermore, hydrogen peroxide induced morphological apoptosis and DNA fragmentation in the SCLC cells. We also demonstrated that erbstatin-induced hydrogen peroxide production and DNA fragmentation were partially suppressed by inhibition of protein synthesis. Thus, erbstatin-induced apoptosis would be due to hydrogen peroxide generation via newly synthesized protein.

Bcl-2-independent induction of apoptosis by neuropeptide receptor antagonist in human small cell lung carcinoma cells.

The broad-spectrum antagonist of neuropeptide receptor, [D-Arg1, D-Phe5, D-Trp7,9, Leu11]substance P, induced apoptosis selectively in human small cell lung carcinoma (SCLC) cells, which express gastrin-releasing peptide receptor, but not in other types of tumor cells as well as normal cells. The addition of gastrin-releasing peptide or bombesin and the inhibitor of caspase-3 suppressed [D-Arg1, D-Phe5, D-Trp7,9, Leu11]substance P-induced apoptosis. Moreover, [D-Arg1, D-Phe5, D-Trp7,9, Leu11]substance P-induced apoptosis was not suppressed by Bcl-2 over-expression. Thus, blockage of gastrin-releasing peptide receptor-mediated signaling may provide a novel therapeutic option in SCLC which has become resistant to conventional chemotherapeutic agents.

[ELISA for type-specific group B streptococcal antibody and its clinical significance].

In order to investigate the neonatal infection of group B streptococci (GBS), vaginal and anal cultures, and measurement of type-specific antibodies to GBS were carried out on 461 pregnant women. Levels of antibody to GBS were measured with ELISA plates coated with type-specific antigen of GBS. These plates were furnished by Toyo Jozo Co., Ltd. The results were as follows: 1) Antibodies to type Ia, Ib, II and III were detected in 41.9, 34.7, 31.7 and 40.1% of subjects, respectively. 2) GBS was isolated in 78 (16.9%) of subjects. 3) Antibody levels against GBS in the sera of colonized mothers and cord blood of their infants were well correlated. 4) Among the colonized mothers, 4 out of 19 (Ia), 9 out of 18 (Ib) 5 out of 8 (II) and 5 out of 17 (III) showed low levels of antibody. 5) Those who had low levels of antibody were administered antibiotic delivery, and there was no case of crisis in both treatment (antibody levels were negative) and non-treatment (antibody levels were positive) groups.

[Type-specific antibody to type Ia, Ib, II and III group B Streptococcus in maternal and neonatal sera measured by ELISA].

In order to determine the type-specific antibody to group B streptococcus (GBA) type Ia, Ib, II and III, the ELISA system was established in Research laboratories, Toyo Jozo Co., Ltd. We assayed type-specific antibody by this ELISA system in both maternal and neonatal (or cord) sera. The cut off levels were determined by the antibody levels of maternal and neonatal sera of 26 infected cases and 90 GBS carriers, that type Ia, Ib, II were 0.20 and type III was 0.15. Prevalence of type-specific antibody levels were studied in 356 maternal sera (14 affected cases, 100 GBS carriers and 242 non carriers). Antibody levels were positive in 47.2% of maternal sera to type Ia, 34.0% to type Ib, 46.9% to type II and 45.5% to type III. Antibody levels to type Ia, Ib, II and III were positive, respectively, in 100%, 88.2%, 25.0% and 42.9% of the sera of carrier mothers whose infants were not affected. Antibody levels in 50 pair sera of maternal and cord blood were well correlated.

[Clinical studies on cefoxitin in the field of obstetrics and gynecology].

Clinical studies were done on cefoxitin (CFX), the first cephamycin antibiotic, in the field of obstetrics and gynecology and following results were obtained. CFX was administrated by intravenous drip infusion for average 6.9 days at a daily dose of 2--6 g to 58 patients; 15 with intrauterine infections, 5 with intrapelvic infections, 11 with adnexitis, 3 with mastitis, 5 with urinary tract infections, 2 with other infections and 17 for prophylaxis of postoperative infections. The clinical results were excellent in 8 cases, good in 39 cases, fair in 4 cases, poor in 6 cases and unknown in 1 case, with the efficacy of 82.5%. Total bacteriological effective rate was 88.9%. As to side effects, eruption was observed only in 1 case. From the results of the present study, the usefulness of CFX was demonstrated in the field of obstetrics and gynecology.

[Mechanisms of multiple myeloma cell growth].

The mechanism of human multiple myeloma cell growth was studied utilizing eleven myeloma cell lines established in vitro or in vivo (Scid mouse). Four bone marrow derived cell lines grew dependently on IL-6 or bone marrow stromal cells. Seven extramedullary lesion derived cell lines grew spontaneously and additively proliferated in response to IL-6. All cell lines expressed the IL-6 receptor (IL-6R) and IL-6RmRNA, but none expressed IL-6mRNA. No IL-6 activity was detected in the myeloma cell culture supernatant. Both the anti-IL-6 antibody and anti-IL-6R antibody neutralized IL-6-induced proliferation, but did not inhibit spontaneous proliferation of extramedullary lesion derived cell lines. While establishing cell lines, it was found that the proliferating fraction was primarily included in a fraction which was non-adherent to stromal cells and composed of undifferentiated plasmablasts. Undifferentiated plasmablasts proliferated in response to IL-6, in contrast to the adherent, mature form of myeloma cells which did not proliferate in response to IL-6. Innoculation of myeloma cells into Scid mice induced subcutaneous tumor formation. These tumors were composed of undifferentiated plasmablasts, which also proliferated in response to IL-6. These results imply that the growth of bone marrow derived myeloma cell lines are dependent on the IL-6 paracrine mechanism and that the growth of extramedullary lesion derived cell lines primarily autonomous and additively dependent on the IL-6 paracrine mechanism.

Polo-like kinase 1 phosphorylates and regulates Bcl-x(L) during pironetin-induced apoptosis.

Bcl-x(L), an anti-apoptotic Bcl-2 family member protein, contributes to the resistance against chemotherapies such as tubulin-binder treatment in many human tumors. Although Bcl-x(L) is phosphorylated after tubulin-binder treatment, the role of the phosphorylation and its responsible kinase(s) are poorly understood. Here, we identified Plk1 (polo-like kinase 1) as a Bcl-x(L) kinase. Same location of Bcl-x(L) and Plk1 was revealed by immunocytochemical analyses at M-phase in situ. Plk1 phosphorylates Bcl-x(L) in vitro, and we identified Plk1 phosphorylation sites in Bcl-x(L). When all of these phosphorylation sites were substituted to alanines, the anti-apoptotic activity of the Bcl-x(L) mutant against the apoptosis induced by pironetin, but not against ultraviolet-induced apoptosis, was increased. These observations suggest that Plk1 is a regulator of Bcl-x(L) phosphorylation and controls the anti-apoptotic activity of Bcl-x(L) during pironetin-induced apoptosis.

Bcl-2 inhibits calcineurin-mediated Fas ligand expression in antitumor drug-treated baby hamster kidney cells.

It is well known that human leukemia cells, such as HL-60 and U937 are sensitive to antitumor drugs, but human normal lung fibroblasts, such as WI-38 cells are resistant to the drugs. However, the mechanisms of the different responses to apoptosis in these cell lines remain unclear. We report here that an increase of Fas and Fas ligand (FasL) expression was required for antitumor drug-induced apoptosis in WI-38 and baby hamster kidney (BHK) cells, but not in HL-60 cells. Then, we used BHK cells transfected with the bcl-2 gene to investigate the involvement of complex formation of Bcl-2 and calcineurin. Calcineurin was imported to the nucleus in response to the drug treatment. Overexpression of Bcl-2 and cyclosporin A treatment inhibited the nuclear import and FasL expression, and as a result, both inhibited apoptosis. Although a caspase inhibitor, z-Asp-CH2-DCB, suppressed the drug-induced apoptosis, it failed to inhibit the drug-induced expression of Fas and FasL. These findings suggest that initially the Fas / FasL system is activated by calcineurin-dependent transcription followed by activation of the downstream caspase cascade resulting in antitumor drug-induced apoptosis in BHK cells, but not in HL-60 cells. Furthermore, Bcl-2 inhibits the nuclear import of calcineurin and suppresses calcineurin-mediated FasL expression during antitumor drug-induced apoptosis.

Requirement of caspase-3(-like) protease-mediated hydrogen peroxide production for apoptosis induced by various anticancer drugs.

Caspase-3(-like) proteases play important roles in controlling mammalian apoptosis. However, the downstream events from the caspase-3(-like) protease activation to death of cells are still unclear. Previously, we reported that hydrogen peroxide (H2O2) was generated by the activation of caspase-3(-like) proteases in the process of tyrosine kinase inhibitor-induced apoptosis in human small cell lung carcinoma Ms-1 cells. In the present study, we examined whether generation of H2O2 is a critical event for the apoptotic pathway downstream of caspase-3(-like) protease activation by various anticancer drugs. Anticancer drugs such as camptothecin, vinblastine, inostamycin, and adriamycin induced activation of caspase-3(-like) proteases and apoptosis. Generation of H2O2 was commonly detected after treatment with each of the four anticancer drugs, and scavenging of H2O2 caused cells to fail to undergo apoptosis. Moreover, anticancer drug-induced H2O2 production was inhibited not only by an inhibitor of caspase-3(-like) proteases but also by diphenyleneiodonium chloride, an inhibitor of flavonoid-containing enzymes such as NADPH oxidase. However, activation of caspase-3(-like) proteases was not inhibited by diphenyleneiodonium chloride. These findings suggest that activation of caspase-3(-like) proteases by various anticancer drugs causes generation of H2O2 presumably through the activation of NADPH oxidase, thereby inducing apoptosis. Therefore, H2O2 may function as a common mediator for apoptosis induced by various anticancer drugs.

Caspase-3 activation is not responsible for vinblastine-induced Bcl-2 phosphorylation and G2/M arrest in human small cell lung carcinoma Ms-1 cells.

Vinblastine arrests cells in the G2/M phase of the cell cycle and subsequently induces cell death by apoptosis. We found that treatment of cells with vinblastine induced phosphorylation of Bcl-2, resulting in the dissociation of Bcl-2 and Bax. Moreover, vinblastine-induced apoptosis was suppressed by an inhibitor of caspase-3, Ac-DEVD-CHO; and a 17-kDa active fragment of caspase-3 was detected following vinblastine treatment, suggesting that caspase-3 is involved in vinblastine-induced apoptosis. However, Ac-DEVD-CHO affected neither vinblastine-induced Bcl-2 phosphorylation nor vinblastine-induced G2/M arrest. Vinblastine caused G2/M arrest prior to apoptosis, whereas vinblastine-induced apoptosis was not dependent on the duration of the G2/M phase. Thus, vinblastine-induced apoptosis might be mediated by the phosphorylation of Bcl-2, resulting in Bcl-2 inactivation, and by subsequent activation of caspase-3.

Potentiation of paclitaxel cytotoxicity by inostamycin in human small cell lung carcinoma, Ms-1 cells.

In the present study, we found that inostamycin increased the ability of paclitaxel to induce apoptosis in Ms-1 cells. A considerably higher concentration of paclitaxel was required for the induction of apoptosis in Ms-1 cells than in other cell lines tested. Treatment of Ms-1 cells with inostamycin, an inhibitor of phoshatidylinositol (PI) synthesis, reduced the dosage of paclitaxel required to induce cell death by apoptosis. This effect of inostamycin is specific to Ms-1 cells, and inostamycin did not increase the cytotoxicity of other antitumor drugs such as adriamycin, vinblastine, methotrexate, cisplatin, etoposide, or camptothecin in Ms-1 cells. Addition of inostamycin to paclitaxel-treated cells caused a significant increase in the sub G1 peak, representing apoptosis, which was accompanied by a decrease in the G2/M peak seen in paclitaxel-treated Ms-1 cells, without affecting paclitaxel-inhibited tubulin depolymerization. Moreover, paclitaxel did not enhance inostamycin-inhibited PI synthesis. The expression levels of Bcl-2, Bax, and Bcl-XL were not changed following the co-treatment with inostamycin plus paclitaxel, whereas the activated form of caspase-3 was markedly increased. Thus, inostamycin is a chemosensitizer of paclitaxel in small cell lung carcinoma Ms-1 cells.