Challenge to improve Japan's deplorable HPV vaccination rate by local government actions.

AIM: In Japan, in 2013, following reports of several alleged adverse reactions in young girls following vaccination, the previously successful national human papillomavirus infection (HPV) vaccination program collapsed rapidly. In the 8 years since vaccination rates have hovered near zero. In October of 2020, in an attempt to mitigate this lingering disaster, the Japanese Ministry of Health, Labor, and Welfare (MHLW) agency finally revised its HPV vaccination informational leaflet that was designed to be distributed by local governments nationwide. Prior to this revision, Toyonaka City, in Japan's Osaka province, had already begun sending out their own unique leaflet to girls in the targeted 6th-10th grades. As a preview of how MHLW's revised leaflet might eventually succeed, we have studied the HPV vaccination results from Toyonaka City's experiment. METHOD: This study was a population-based analysis that compared the monthly rates of new vaccinations in girls of a targeted grade school age group. We looked at rates before and after the leaflets were sent by Toyonaka City's Division of Health Promotion and Senior Services. RESULTS: The vaccination rates between April 2020 and March 2021 were improved across all grades; 1.2% in 6th grade (p = 0.000185), 2.5% in 7th grade (p < 0.0001), 3.5% in 8th grade (p < 0.0001), 6.8% in 9th grade (p < 0.0001), and a remarkable 16.5% in 10th grade (p < 0.0001). CONCLUSION: When a local government sends an HPV informational leaflet targeted at young girls, it can significantly improve their HPV vaccination rates.

Measurement of endometrial thickness in premenopausal women in office gynecology.

Purpose: To define the median endometrial thickness (ET) in office gynecology is thought to be important for clinical practice. However, there are few reports about ET that have included the general female population on a large scale. The median ET was determined prospectively in premenopausal women who attended office gynecology for cervical cancer screening. Methods: In total, 849 women were enrolled. The median ET was determined by using transvaginal ultrasound and the relationships between the ET and various clinical factors were analyzed. Results: The participants' median age was 38.5 years. The median ET was 8.6 mm (90% and 95% quantiles: 13.8 and 15.8 mm). The ET was not related to their age, symptoms, obstetric history, geographical location, or risk factors for endometrial cancer. In the women with a menstrual cycle length of 28-30 days, the ET was 7 mm on days 1-6, but it increased from 5.4 mm immediately after menstruation (day 7 or 8) to 9.2 mm on days 13-14. Subsequently, the ET increased further to 11.1 mm on day 18. Conclusion: In all the women, the upper limit of the ET was 13.8 mm and 15.8 mm in the 90% and 95% quantile, respectively, in office gynecology.

Guidelines for office gynecology in Japan: Japan Society of Obstetrics and Gynecology and Japan Association of Obstetricians and Gynecologists 2011 edition.

Gynecology in the office setting is developing worldwide. Clinical guidelines for office gynecology were first published by the Japan Society of Obstetrics and Gynecology and the Japan Association of Obstetricians and Gynecologists in 2011. These guidelines include a total of 72 clinical questions covering four areas (Infectious disease, Malignancies and benign tumors, Endocrinology and infertility, and Healthcare for women). These clinical questions were followed by several answers, backgrounds, explanations and references covering common problems and questions encountered in office gynecology. Each answer with a recommendation level of A, B or C has been prepared based principally on evidence or consensus among Japanese gynecologists.These guidelines would promote a better understanding of the current standard care practices for gynecologic outpatients in Japan.

Involvement of RelA-associated inhibitor in regulation of trophoblast differentiation via interaction with transcriptional factor specificity protein-1.

Glucose transporter-1 (GLUT1), one of the key functional indicators of placental differentiation, has an important role in placental glucose transport. We previously showed that the protein levels of GLUT1 and nuclear transcription factor specificity protein-1 (Sp1) in rat choriocarcinoma cells (Rcho-1 cells) decreased during the differentiation of these cells to giant cells. We also showed that Sp1 was involved in the regulation of GLUT1 gene expression during this process. RelA-associated inhibitor (RAI) is an inhibitor of nuclear factor-kappaB that was identified by a yeast two-hybrid screen and is preferably expressed in human placenta and heart. RAI was also found to interact with Sp1 and exert an inhibitory effect against the DNA-binding activity of Sp1. We first show here that RAI mRNA expression increased as gestation proceeded and that RAI was localized mainly in the syncytiotrophoblast throughout pregnancy. The chloramphenicol acetyltransferase activity assay in Rcho-1 cells revealed that cotransfection of RAI expression vector resulted in decreased activity of the rat GLUT1 promoter but not in that of a mutated rat GLUT1 promoter lacking the Sp1 binding site. Furthermore, the protein level of RAI increased during differentiation. In addition, transfection of RAI expression vector promoted the morphological differentiation of Rcho-1 cells, and RAI knockdown using RAI-specific small interfering RNA reveals inhibitory effects on the morphological differentiation, as assessed by photomicroscopy. Taken together, these findings suggest that RAI may be involved in the regulation of trophoblast differentiation via interaction with Sp1.

Peritoneal fluids from patients with certain gynecologic tumor contain elevated levels of bioactive lysophospholipase D activity.

Levels of lysophosphatidic acid (LPA), an important phospholipid mediator, in serum and ascitic fluid from ovarian cancer patients were shown to be higher than those from healthy women and from patients with other type of cancer, respectively. Although LPA in human serum seems mainly to be generated by lysophospholipase D (lysoPLD), the source and pathway for LPA in the ascitic fluid remain still obscure. In this study, we examined whether lysoPLD activity producing bioactive LPA in human peritoneal fluid was significantly elevated under pathological statuses. Lysophospholipase D activity in human peritoneal fluids was measured by quantifying choline released from exogenous lysophosphatidylcholine on their incubation at 37 degrees C. We also compared the activity of lysoPLD in sera from patients with different gynecologic diseases. We found relatively high lysoPLD activity in peritoneal fluids from patients with ovarian cancer, dermoid cyst or mucinous cystadenoma, whereas there were no significant differences in the serum lysoPLD activity among clinical groups and healthy subjects. The lysoPLD in the peritoneal fluid was found to have similar substrate specificity and metal ion requirement to those of serum lysoPLD, that has been identified as autotaxin, a tumor cell-motility stimulating protein. Our results suggest that increased lysoPLD activity in peritoneal fluid from patients with certain gynecologic tumors might be relevant to its potential of tumor progression.

Peripheral quantitative computed tomography (pQCT) is useful for monitoring bone mineral density of the patients who receive hormone replacement therapy.

OBJECTIVE: A forearm fracture (Colles' fracture) is often the first sign of osteoporosis and should alert the patient and physician to the possibility of underlying skeletal fragility. Therefore, the establishment of a more accurate and reliable method for the measurement of bone mineral density (BMD) at the distal radius would be beneficial for the patients who suffer from osteoporosis. The objective of the present study was to evaluate the usefulness of peripheral quantitative computed tomography (pQCT) to assess the change of BMD at the distal radius in early postmenopausal women who receive hormone replacement therapy (HRT). METHODS: Twenty healthy early postmenopausal women who were diagnosed as osteoporosis or osteopenia were randomized to either HRT or placebo treatment. We analyzed BMD of the distal radius by pQCT, lumbar spine by dual-energy X-ray absorptiometry (DXA) and the biochemical markers of bone turn over (osteocalcin, deoxypyridinoline) every 6 months. RESULTS: The placebo group showed a significant decrease from the baseline in the trabecular BMD of the radius at 12 months (7.4+/-2.5%) (p<0.05), whereas the HRT group showed a slight increase (0.7+/-2.2%). The changes in the trabecular BMD of the radius between the HRT and placebo groups were statistically different at 12 months (p<0.05). On the other hand, in the cortical BMD of the radius, no significant differences were seen between the changes of bone densities in the HRT and control groups after 1 year of treatment. pQCT could detect a significant loss of BMD of the radius in early postmenopausal women after 1 year and HRT prevented its loss. CONCLUSION: Our preliminary clinical trial showed that pQCT might be useful for the early detection of bone loss in early postmenopausal women and for the monitoring BMD of the patients who receive HRT.

Alendronate inhibits intraperitoneal dissemination in in vivo ovarian cancer model.

Ovarian cancer is characterized by diffuse peritoneal carcinomatosis and often by large volumes of ascites. We previously reported that alendronate, a nitrogen-containing bisphosphonate, inhibited ovarian cancer cell migration by attenuating the activation of Rho through inhibiting the mevalonate pathway. However, questions remain about the ability of alendronate to inhibit the invasiveness of cancer cells to the adherent tissues and the growth of disseminated ovarian cancer in vivo. We established an in vivo ovarian cancer model with i.p. carcinomatosis in athymic immunodeficient mice. In the prevention model, in which alendronate administration started from the day after tumor inoculation, alendronate prevented the stromal invasion, reduced the tumor burden, and inhibited ascites accumulation. Histologic observation revealed that alendronate treatment decreased the stromal invasion of the i.p. tumor while inhibiting the metalloproteinase-2 activity in ascites. This antitumor effect might result from the inhibition of cancer cell migration and proteolytic activity. In the treatment model, in which alendronate was given from 10 days after tumor inoculation when macroscopic tumors are already implanted in the peritoneum, the antitumor effect was weaker but still significant. Furthermore, alendronate administration decreased the serum CA-125 levels of mice bearing disseminated ovarian cancer compared with those of nontreated mice. The potent effects of alendronate in reducing stromal invasion, tumor burden, and ascites suggest that it will be of value in regimens for treatment of women with ovarian cancer.

Inhibition of phosphatidylinositol 3-kinase increases efficacy of cisplatin in in vivo ovarian cancer models.

The phosphatidylinositol 3-kinase (PI3K)/Akt cascade has an important role in the resistance of ovarian cancer cells to cisplatin in vitro; however, there have been no reports about whether blocking the PI3K/Akt cascade enhances the sensitivity to cisplatin in vivo. We investigated whether inhibition of PI3K increased the efficacy of cisplatin in an in vivo ovarian cancer model. Blocking the PI3K/Akt cascade with a PI3K inhibitor (wortmannin) increased the efficacy of cisplatin-induced inhibition of intraabdominal dissemination and production of ascites in athymic nude mice inoculated ip with the Caov-3 human ovarian cancer cell line. In addition, wortmannin increased the efficacy of cisplatin-induced apoptosis in tumors cells. There were no detectable side effects in mice treated with wortmannin. Moreover, the antitumor effect of cisplatin detected in mice inoculated with Caov-3 cells stably transfected with empty vector was significantly attenuated, compared with mice inoculated with Caov-3 cells stably transfected with a dominant-negative Akt, K179M-Akt. We confirmed that wortmannin blocked Akt phosphorylation and the downstream targets of the PI3K/Akt cascade, such as BAD (Bcl-2-associated death protein) and nuclear factor-kappaB in vivo by immunohistochemical staining and Western blotting. In accordance with the previously reported in vitro results, these in vivo results support the idea that combination therapy with cisplatin and a PI3K inhibitor would increase the therapeutic efficacy of cisplatin.

Mechanisms of platinum drug resistance.

Platinum-based drugs are among the most active anticancer agents available and are used widely for the treatment of a variety of human solid tumors. Although patients show high response rates to platinum drugs, most patients develop resistance to these drugs during treatment. Because the acquisition of resistance is a major obstacle to the clinical use of platinum drugs, the processes by which cells develop such resistance are of great interest and efforts have been made to overcome this problem. Both mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) cascades are involved in resistance to these drugs, and clinical trials of some small-molecule inhibitors of the MAPK and PI3K-Akt cascades to overcome resistance to platinum drugs are ongoing.

Estrogen and raloxifene inhibit the monocytic chemoattractant protein-1-induced migration of human monocytic cells via nongenomic estrogen receptor alpha.

OBJECTIVE: To investigate the effects of estradiol (E2) and raloxifene on the migration of human monocytic THP-1 cells to endothelium. DESIGN: A prospective comparative study. THP-1 cells, a human acute monocytic leukemia cell line, were used for the study. Migration assays were performed using transwell inserts. THP-1 cells were exposed to E2 or raloxifene in the presence of monocytic chemoattractant protein-1 (MCP-1), a major chemoattractant for monocytes. The cells were transfected with small interfering RNA (siRNA) against estrogen receptor (ER) alpha and ERbeta for gene silencing. ER expression was evaluated by Western blot analysis. RESULTS: MCP-1 induced the migration of the cells for 90 minutes. The addition of E2 or raloxifene significantly inhibited the MCP-1-induced migration for 90 minutes. Preincubation of THP-1 cells with an ER antagonist, ICI 182780, significantly attenuated the inhibitory effects of E2 and raloxifene. Whereas transfection with siRNA of ERalpha significantly attenuated the inhibition by E2 of MCP-1-induced monocyte migration, transfection with control siRNA or siRNA of ERbeta had no effect on the rapid inhibitory action of E2. Moreover, preincubation of THP-1 cells with a transcriptional inhibitor, actinomycin D, had no effect on the rapid inhibitory action of E2. CONCLUSIONS: Our findings suggest that both E2 and raloxifene inhibited the MCP-1-induced monocyte migration through nongenomic ERalpha. This result may explain one of the antiatherosclerotic effects of E2 and raloxifene on vasculature.

Alendronate inhibits lysophosphatidic acid-induced migration of human ovarian cancer cells by attenuating the activation of rho.

Alendronate, a nitrogen-containing bisphosphonate, is a potent inhibitor of bone resorption used for the treatment and prevention of osteoporosis. Recent findings suggest that alendronate and other nitrogen-containing bisphosphonates inhibit the mevalonate pathway and thereby inhibit the synthesis of products derived from this metabolite. This, in turn, prevents the prenylation of a number of small GTPases, which regulate cell growth, motility, and invasion. We studied the effect of alendronate on in vitro migration of human ovarian cancer cells. Lysophosphatidic acid (LPA) induced a dose-dependent increase of migration of cancer cells by promoting Rho/Rho-associated kinase signaling. The induction of cancer cell migration by LPA was inhibited by the addition of alendronate in a dose-dependent manner. Treatment of ovarian cancer cells with alendronate resulted in inactivation of Rho, changes of cell morphology, loss of stress fiber formation, and focal adhesion assembly, and the suppression of phosphorylation of myosin light chain and tyrosine phosphorylation of focal adhesion proteins, which are essential processes for cell migration. The effects of alendronate on cancer cells were prevented by the addition of geranylgeranyol, which is the metabolic intermediate of the mevalonate pathway. These results suggest that alendronate inhibits Rho activation by preventing geranylgeranylation, which results in inhibition of LPA-induced migration of human ovarian cancer cells.

Induction of hTERT expression and phosphorylation by estrogen via Akt cascade in human ovarian cancer cell lines.

We examined the mechanism by which estrogen regulates telomerase activity in Caov-3 human ovarian cancer cell lines, which express ER, to determine whether the regulation affects the expression and/or phosphorylation of the telomerase catalytic subunit (hTERT). 17beta-Estradiol (E(2)) induced telomerase activity and hTERT expression. Transient expression assays using luciferase reporter plasmids containing various fragments of hTERT promoter showed that the estrogen-responsive element appeared to be partially responsible for the E(2)-induced activation of the hTERT promoter. Either pretreatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, or transfection with a dominant-negative Akt attenuated the E(2)-induced activation of the hTERT promoter. In addition, estrogen induced the phosphorylation of IkappaB inhibitor protein via the Akt cascade, and cotransfection with a dominant-negative subunit of NFkappaB attenuated the response of the ERE-deleted hTERT promoter to E(2). Moreover, E(2) induced the phosphorylation of hTERT, the association of 14-3-3 protein and NFkappaB with hTERT, and nuclear accumulation of hTERT in an Akt-dependent manner. These results indicate that E(2) induces telomerase activity not only by transcriptional regulation of hTERT via an ERE-dependent mechanism and a PI3K/Akt/NFkappaB cascade, but also by post-transcriptional regulation via Akt-dependent phosphorylation of hTERT. Thus, the phosphorylation of Akt is a key event in the induction of telomerase activity by E(2) in human ovarian cancer cells.

Up-regulation of c-met protooncogene product expression through hypoxia-inducible factor-1alpha is involved in trophoblast invasion under low-oxygen tension.

During early pregnancy, the invasion of trophoblast cells into the decidua of the uterus is one of the essential steps in appropriate placentation. In this period, trophoblast cells are exposed to a relatively low-oxygen environment. The c-met protooncogene product (Met), which is a high-affinity receptor for hepatocyte growth factor, plays an important role in controlling the invasion of many types of cells. The present study was designed to investigate the effect of low-oxygen tension on Met expression and the invasiveness of trophoblast cells isolated from early-stage human placenta and trophoblast-derived BeWo cells and JEG-3 cells. RT-PCR and immunoblot analyses demonstrated that low-oxygen tension (1% O2) stimulated the expression of Met mRNA and protein, respectively. Hepatocyte growth factor production in the cells was not affected by oxygen tension. Transient transfection of BeWo cells with a hypoxia-inducible factor (HIF)-1alpha expression vector to induce exogenous expression of HIF-1alpha significantly increased the level of Met mRNA and protein, compared with transfection of a control vector. To examine whether this up-regulation of Met was directly induced by HIF-1alpha, we performed the chromatin immunoprecipitation assay, which revealed that HIF-1alpha binds to the promoter region of the Met gene under low-oxygen tension. JEG-3 cells cultured under 1% O2 showed a more invasive character than those cultured under 20% O2, whereas inhibition of Met expression by small interfering RNAs prevented the low-oxygen, tension-induced invasiveness. These results suggest that the induction of Met expression by low-oxygen tension may play an important role in the physiology of early pregnancy by promoting the invasion of trophoblast cells into the decidua of the uterus.

Hypoxia up-regulates hypoxia-inducible factor-1alpha expression through RhoA activation in trophoblast cells.

During early pregnancy, trophoblast cells are exposed to relatively low-oxygen tension. Recently, the Rho GTPase family has been shown to play a key role in hypoxia-inducible factor-1 (HIF-1) alpha induction in renal cell carcinoma. The present study was designed to investigate the effect of low-oxygen conditions on RhoA expression in trophoblast cells isolated from early stages of human placenta and in trophoblast-derived BeWo cells and JAR cells. Immunoblot and RT-PCR analyses showed that low-oxygen conditions (1% O(2) or 250 mum CoCl(2)) stimulated expression of RhoA protein and mRNA. Pull-down assays demonstrated that these low-oxygen conditions increased RhoA activity. Preincubation of BeWo cells with Clostridium botulinum C3 exoenzyme, a specific inhibitor of Rho, inhibited hypoxia-induced HIF-1alpha expression. Under 1% O(2) or 250 mum CoCl(2), BeWo cells, transfected with a dominant-negative RhoA, exhibited decreased levels of HIF-1alpha protein and mRNA compared with the control vector transfectants. BeWo cells expressing constitutively active RhoA showed enhanced protein levels of not only HIF-1alpha but also vascular endothelial growth factor (VEGF) and glucose transporter 1, which are target gene products of HIF-1alpha. These findings suggest that up-regulation of RhoA induced by low-oxygen conditions may play an important role in regulation of HIF-1alpha expression in trophoblast cells.

Inhibition of inhibitor of nuclear factor-kappaB phosphorylation increases the efficacy of paclitaxel in in vitro and in vivo ovarian cancer models.

We investigated whether inhibition of nuclear factor-kappaB (NFkappaB) increases the efficacy of paclitaxel in in vitro and in vivo ovarian cancer models. Treatment of paclitaxel-sensitive Caov-3 cells with paclitaxel transiently activated the phosphorylation of Akt, the phosphorylation of IkappaB kinase (IKK), and the phosphorylation of inhibitor of NFkappaB (IkappaBalpha). Paclitaxel also caused a transient increase in NFkappaB activity, followed by a decrease in NFkappaB activity. We show an association between Akt and IKK and show that the phosphorylation of IKK induced by paclitaxel is blocked by treatment with a phosphatidylinositol 3-kinase inhibitor (wortmannin or LY294002). Furthermore, interference of the Akt signaling cascade inhibits the transient induction of IkappaBalpha phosphorylation and NFkappaB activity by paclitaxel. Inhibition of NFkappaB activity by treatment with an IkappaBalpha phosphorylation inhibitor (BAY 11-7085) attenuated both basal and transient induction of IkappaBalpha phosphorylation by paclitaxel. Treatment with BAY 11-7085 also enhanced the inhibition of NFkappaB activity by paclitaxel for up to 24 hours. In addition, treatment with BAY 11-7085 decreased the viability of cells treated with paclitaxel. Moreover, treatment with BAY 11-7085 increased the efficacy of paclitaxel-induced inhibition of intraabdominal dissemination and production of ascites in athymic nude mice inoculated intraperitoneally with Caov-3 cells. These results suggest that paclitaxel transiently induces NFkappaB activity via the phosphatidylinositol 3-kinase/Akt cascade and that combination therapy with paclitaxel and an NFkappaB inhibitor would increase the therapeutic efficacy of paclitaxel.

Gestational change of K+ channel opener effect is correlated with the expression of uterine KATP channel subunits.

OBJECTIVE: We analyzed the gestational changes of pharmacological activity and molecular levels of KATP channels in rat myometrium. STUDY DESIGN: Using rat myometrium, the effects of K+ channel openers (KCOs) were examined in an isometric tension study of oxytocin-induced contraction. We also examined the effects of KCOs on the intracellular Ca2+ levels of cultured myometrial cells. The expression of myometrial KATP channels was assessed by RT-PCR and Northern blot analysis. RESULTS: The effect of KCOs were altered during pregnancy, with a significant increase of their potency at day 18 of pregnancy followed by a decline towards the non-pregnant level at the day of delivery. KCOs suppressed the Ca2+ influx across the cell membrane. The mRNAs encoding each component of myometrial KATP channels, Kir6.1 and SUR2B, exhibited gestational stage-dependent alterations similar to those of the effects of KCOs. CONCLUSION: These findings suggest that KCOs inhibit uterine myometrial contraction more effectively during pregnancy than in the non-pregnant state due to gestation-enhanced expression of KATP channels, implying that KCOs might be useful for preventing premature delivery.

Expression of nectin-2 in mouse granulosa cells.

OBJECTIVE: The development and maturation of the ovarian follicles are characterized by structural changes that require components involved in cell-cell adhesion systems. Recently a novel group of cell adhesion molecules named nectins has been identified. The present study examined expression and cell-specific localization of nectins during mouse follicular development. STUDY DESIGN: Expression of nectins in mouse ovary was investigated by immnuoblot analysis and immunohistochemistry. More precise localization was determined by electron microscopy. RESULTS: Immunoblot analysis revealed expression of nectin-2 and nectin-3 but not nectin-1 in ovarian granulosa cells. Immunohistochemistry demonstrated expression of nectin-2 at cell-cell adhesion sites of granulosa cell layer in the primary and preantral follicles. Especially, intense immunoreactivity of nectin-2 accumulated around the zona pellucida. In antral follicles, the intensity of nectin-2 expression on granulosa cells was decreased. By electron microscopy nectin-2 was detected not only on thin extensions of granulosa cells penetrating the zona pellucida, but also on the attachment sites between thin extensions of granulosa cells and oocyte surface. CONCLUSION: The restricted expression of nectin-2 in the granulosa cells of primary and preantral follicles might reflect some of the molecular changes in cell-cell adhesion during early follicular development.

RhoA/Rho-kinase cascade is involved in oxytocin-induced rat uterine contraction.

The RhoA/Rho-kinase cascade is involved in various cellular functions, including migration, proliferation, and smooth muscle contraction. We examined the potential role of this pathway in oxytocin-induced uterine contraction. The specific Rho-kinase inhibitor Y-27632 inhibited oxytocin-induced rat uterine contraction on d 21 of pregnancy in a concentration-dependent manner, whereas the extent of this inhibition was reduced in the nonpregnant uterus. Y-27632 had no effect on oxytocin-induced intracellular Ca(2+) mobilization in myometrial cells. Immunoblot analysis showed that oxytocin increased the level of myosin light chain phosphorylation, and this increase was attenuated by Y-27632. Oxytocin increased the phosphorylation of myosin-binding subunit of myosin phosphatase, one of the major substrates of Rho-kinase, and this increase was reduced by Y-27632. The expression of Rho-kinase protein was shown to increase in the uterus during pregnancy compared with the nonpregnant uterus, whereas the expression of RhoA protein remained at the same level during pregnancy. RT-PCR and Northern blot analysis showed that the expression of Rho-kinase was up-regulated at the transcriptional level during pregnancy. These results suggest that the RhoA/Rho-kinase pathway may have an important role in oxytocin-induced uterine contraction, and that up-regulation of Rho-kinase is involved in the mechanism underlying the increased contractility of the pregnant myometrium.

Regulation of the PRL promoter by Akt through cAMP response element binding protein.

Regulation of the PI3K-protein kinase B/Akt (serine/threonine kinase) cascade by PRL-releasing peptide (PrRP) and insulin in GH3 rat pituitary tumor cells was investigated. PrRP and insulin rapidly and transiently stimulated the activation of Akt, and the PI3K inhibitor wortmannin blocked the PrRP- or insulin-induced activation of Akt. Both pertussis toxin (10 ng/ml), which inactivates Gi/Go proteins, and expression of a peptide derived from the carboxyl terminus of the beta-adrenergic receptor kinase I, which specifically blocks signaling mediated by the betagamma subunits of G proteins, completely blocked the PrRP-induced Akt activation, suggesting that Gi/Go proteins are involved in PrRP-induced Akt activation, as they are in the activation of ERK by PrRP. Moreover, to determine whether a PI3K-Akt cascade regulates rat PRL (rPRL) promoter activity, we transfected the intact rPRL promoter ligated to the firefly luciferase reporter gene into GH3 cells. PrRP and insulin activated the rPRL promoter activity. Pretreatment with wortmannin or cotransfection with a dominant-negative Akt partially but significantly inhibited the induction of the rPRL promoter by PrRP or insulin. Cotransfection with a constitutively active Akt induced the rPRL promoter activity and cotransfection with a dominant-negative cAMP response element-binding protein (CREB) completely abolished the response of the rPRL promoter to the constitutively active Akt. Furthermore, either treatment with PrRP and insulin or transfection with the constitutively active Akt induced the phosphorylation of CREB. These results suggest that PrRP and insulin activate a PI3K-Akt cascade that is necessary to elicit rPRL promoter activity via a CREB-dependent mechanism.

Tamoxifen inhibits cell proliferation via mitogen-activated protein kinase cascades in human ovarian cancer cell lines in a manner not dependent on the expression of estrogen receptor or the sensitivity to cisplatin.

Although tamoxifen (TAM), which is widely used in the treatment of breast cancer, also has a beneficial effect on cisplatin-refractory ovarian cancer, the biological mechanism of this effect has remained obscure. TAM, besides its action as an antiestrogen, also inhibits cell proliferation of estrogen receptor (ER)-negative cells by an unknown mechanism. Therefore, we examined the roles of the MAPK family in the antiproliferative effect of TAM on cisplatin-resistant Caov-3, which expresses ER and cisplatin-sensitive A2780, which does not express ER. The number of viable cells was reduced by TAM dose-dependently. TAM induced the activation of ERK, c-Jun N-terminal protein kinase (JNK), and p38 with different time courses. PD98059 canceled the reduction of the number of viable cells by 1 microM TAM and inhibited the TAM-induced cell-cycle arrest at the G(1) phase and dephosphorylation of the retinoblastoma protein. Either expression of dominant-negative JNK or pretreatment with SB203580 canceled the reduction of the number of viable cells by 5 microM TAM and inhibited the apoptotic nuclear changes and the cleavage of poly (ADP-ribose) polymerase induced by TAM. These results provide evidence that whereas the ERK cascade is involved in the induction of cell-cycle arrest at the G(1) phase by lower concentrations of TAM, the JNK or p38 cascade is involved in the induction of apoptosis by higher concentrations of TAM in both types of cells.