Keiichi Tasaka

Inhibition of phosphorylation of BAD and Raf-1 by Akt sensitizes human ovarian cancer cells to paclitaxel.

We studied the roles of the phosphatidylinositol 3-kinase (PI-3K)-Akt-BAD cascade, ERK-BAD cascade, and Akt-Raf-1 cascade in the paclitaxel-resistant SW626 human ovarian cancer cell line, which lacks functional p53. Treatment of SW626 cells with paclitaxel activates Akt and ERK with different time frames. Interference with the Akt cascade either by treatment with PI-3K inhibitor (wortmannin or LY294002) or by exogenous expression of a dominant negative Akt in SW626 cells caused decreased cell viability following treatment with paclitaxel. Interference with the ERK cascade by treatment with an MEK inhibitor, PD98059, in SW626 cells also caused decreased cell viability following treatment with paclitaxel. Treatment of cells with paclitaxel also stimulated the phosphorylation of BAD at both the Ser-112 and Ser-136 sites. The phosphorylation of BAD at Ser-136 was blocked by treatment with wortmannin or cotransfection with the dominant negative Akt. On the other hand, the phosphorylation of BAD at Ser-112 was blocked by PD98059. We further examined the role of BAD in the viability following paclitaxel treatment using BAD mutants. Exogenous expression of doubly substituted BAD2SA in SW626 cells caused decreased viability following treatment with paclitaxel. Moreover, because paclitaxel-induced apoptosis is mediated by activated Raf-1 and the region surrounding Ser-259 in Raf-1 conforms to a consensus sequence for phosphorylation by Akt, the regulation of Raf-1 by Akt was examined. We demonstrated an association between Akt and Raf-1 and showed that the phosphorylation of Raf-1 on Ser-259 induced by paclitaxel was blocked by treatment with wortmannin or LY294002. Furthermore, interference with the Akt cascade induced by paclitaxel up-regulated Raf-1 activity, and expression of constitutively active Akt inhibited Raf-1 activity, suggesting that Akt negatively regulates Raf-1. Our findings suggest that paclitaxel induces the phosphorylation of BAD Ser-112 via the ERK cascade, and the phosphorylation of both BAD Ser-136 and Raf-1 Ser-259 via the PI-3K-Akt cascade, and that inhibition of either of these cascades sensitizes ovarian cancer cells to paclitaxel.

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). 17β-Estradiol (E2) 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 E2-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 E2-induced activation of the hTERT promoter. In addition, estrogen induced the phosphorylation of IκB inhibitor protein via the Akt cascade, and cotransfection with a dominant-negative subunit of NFκB attenuated the response of the ERE-deleted hTERT promoter to E2. Moreover, E2 induced the phosphorylation of hTERT, the association of 14-3-3 protein and NFκB with hTERT, and nuclear accumulation of hTERT in an Akt-dependent manner. These results indicate that E2 induces telomerase activity not only by transcriptional regulation of hTERT via an ERE-dependent mechanism and a PI3K/Akt/NFκB 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 E2 in human ovarian cancer cells.

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

We investigated whether inhibition of nuclear factor-κB (NFκB) 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 IκB kinase (IKK), and the phosphorylation of inhibitor of NFκB (IκBα). Paclitaxel also caused a transient increase in NFκB activity, followed by a decrease in NFκB 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 IκBα phosphorylation and NFκB activity by paclitaxel. Inhibition of NFκB activity by treatment with an IκBα phosphorylation inhibitor (BAY 11-7085) attenuated both basal and transient induction of IκBα phosphorylation by paclitaxel. Treatment with BAY 11-7085 also enhanced the inhibition of NFκB 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 NFκB activity via the phosphatidylinositol 3-kinase/Akt cascade and that combination therapy with paclitaxel and an NFκB inhibitor would increase the therapeutic efficacy of paclitaxel.

Prevalence of premenstrual syndrome and premenstrual dysphoric disorder in Japanese women.

SummaryTo investigate the prevalence and impact of premenstrual symptoms in Japanese women, we developed the PSQ “The Premenstrual Symptoms Questionnaire” for the screening of premenstrual symptoms. The PSQ translates DSM-IV criteria into a rating scale with degrees of severity. One thousand one hundred and eighty-seven Japanese women between the ages of 20 and 49 yrs, who were seen at a clinic for uterine cancer screening, were assessed regarding their premenstrual symptoms using the PSQ. As many as 95% of these women were found to suffer from premenstrual symptoms. The rates of prevalence of moderate to severe PMS and PMDD in Japanese women were 5.3 and 1.2%, respectively, which are lower than those in Western women. Only 5.3% of women with moderate to severe PMS and PMDD were treated. The results of this study suggest that race and ethnicity influence the expression of premenstrual symptoms and that the current state of medical care for Japanese women with moderate to severe PMS and PMDD is not satisfactory.

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 Rhokinase 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. (Endocrinology 143: 920 –929, 2002)

Magnesium sulfate inhibits oxytocin-induced calcium mobilization in human puerperal myometrial cells: Possible involvement of intracellular free magnesium concentration

OBJECTIVE Our purpose was to elucidate the mechanisms of the tocolytic action of Mg2+ on the human myometrium. STUDY DESIGN The effects of extracellular Mg2+ on oxytocin-induced increases in the intracellular free Ca2+ concentration in human puerperal myometrial cells were examined by means of indo-1-AM. The changes in the intracellular free Mg2+ concentration under various conditions were also measured with Mg(2+)-fura-2-AM. RESULTS The increase in intracellular free Ca2+ concentration induced by oxytocin was reduced to 26% of that in the normal solution 20 minutes after replacement of the normal solution with an extracellular Mg2+ solution, 10 mmol/L. When extracellular Ca2+ was removed, the increase in intracellular free Ca2+ concentration was not suppressed even 20 minutes after the replacement. A solution of extracellular Mg2+ concentration, 10 mmol/L, raised the intracellular free Mg2+ concentration gradually, by approximately 150% in 20 minutes, concomitant with the suppression of the response to oxytocin in the intracellular free Ca2+ concentration. CONCLUSION High intracellular free Mg2+ concentration, caused by high extracellular Mg2+, is essential for suppression of oxytocin-induced Ca2+ influx across the cell membrane; this presumably results in inhibition of uterine contractions.

Changes of bioactive luteinizing hormone after laparoscopic ovarian cautery in patients with polycystic ovarian syndrome

The serum levels of bioactive luteinizing hormone (LH), immunoreactive LH, follicle-stimulating hormone, androstenedione (A), and testosterone (T) were determined in nine anovulatory women with polycystic ovarian syndrome (PCOS) before and after laparoscopic ovarian cautery. Eight ovulated spontaneously and three conceived after treatment. Before treatment, the mean (+/- SEM) levels of bioactive LH, immunoreactive LH, A, and T were 51.4 +/- 8.6 mIU/mL, 36.0 +/- 4.5 mIU/mL, 1.98 +/- 0.35 ng/mL, and 1.18 +/- 0.13 ng/mL, respectively, which were significantly higher than those of five control women (19.2 +/- 1.6 mIU/mL, 21.4 +/- 1.2 mIU/mL, 0.54 +/- 0.03 ng/mL, 0.28 +/- 0.03 ng/mL). After treatment, the mean levels of these hormones had significantly decreased. Decreases in the levels of these hormones by laparoscopic ovarian cautery in women with PCOS may result in both restoration of the ovulatory cycle and achievement of pregnancy.

Hydatidiform Mole in a Triplet Pregnancy Following Gonadotropin Therapy

A first case is reported of complete hydatidiform mole with two coexistent fetuses in a triple pregnancy following human menopausal gonadotropin human chorionic gonadotropin (hMG‐hCG) therapy. the molar mass and two fetuses were delivered separately at 17 weeks of gestation. the fetuses were female (155 g) and male (160 g) with individual placentae (85 g, 90 g). the hydatidiform mole (650 g) had a normal 46, XX karyotype. the sexes of the two fetuses and the karyotype of the mole are consistent with previous reports that the chromosomes of fetuses and moles are derived from both parents and the father, respectively.

SNAP-25 is essential for cortical granule exocytosis in mouse eggs

Synaptosome-associated protein of 25 kDa (SNAP-25) has been shown to play an important role in Ca2+-dependent exocytosis in neurons and endocrine cells. During fertilization, sperm-egg fusion induces cytosolic Ca2+ mobilization and subsequently Ca2+-dependent cortical granule (CG) exocytosis in eggs. However, it is not yet clear whether SNAP-25 is involved in this process. In this study, we determined the expression and function of SNAP-25 in mouse eggs. mRNA and SNAP-25 were detected in metaphase II (MII) mouse eggs by RT-PCR and immunoblot analysis, respectively. Next, to determine the function of SNAP-25, we evaluated the change in CG exocytosis with a membrane dye, tetramethylammonium-1,6-diphenyl-1,3,5-hexatriene, after microinjection of a botulinum neurotoxin A (BoNT/A), which selectively cleaves SNAP-25 in MII eggs. Sperm-induced CG exocytosis was significantly inhibited in the BoNT/A-treated eggs. The inhibition was attenuated by coinjection of SNAP-25. These results suggest that SNAP-25 may be involved in Ca2+-dependent CG exocytosis during fertilization in mouse eggs.

Effect of pituitary transplants on the LH-RH concentrations in the medial basal hypothalamus and hypophyseal portal blood

The effects of hyperprolactinemia on catecholamine turnover in the medial basal hypothalamus (MBH) and on the luteinizing hormone-releasing hormone (LH-RH) concentrations in MBH and hypophyseal portal blood were investigated in female Wistar rats. Chronic endogenous hyperprolactinemia was produced by implantation of anterior pituitary glands under the kidney capsule. Catecholamine turnover in the MBH was studied by inhibiting monoamine oxidase and then measuring the accumulation of catecholamines by high-performance liquid chromatography with electrochemical detection. Rats bearing pituitary transplants exhibited: (1) persistent vaginal diestrus within 3-6 days of the implantation; (2) increased serum concentrations of prolactin (PRL); (3) decreased serum concentrations of luteinizing hormone (LH); (4) increased pituitary concentrations of LH and follicle-stimulating hormone (FSH); (5) increased turnovers of dopamine in the MBH; and (6) decreased concentrations of LH-RH in the MBH and in plasma of hypophyseal portal blood. These findings suggest that chronic hyperprolactinemia may increase dopaminergic tone in the MBH that may inhibit LH-RH secretion from the MBH, and LH release from the pituitary. These processes may be responsible for disturbances of cyclic pituitary-ovarian activity.