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Vascular endothelial senescence is involved in human atherosclerosis. Telomerase activity is known to be critical in cellular senescence and its level is modulated by regulation of telomerase catalytic subunit (telomerase reverse transcriptase (TERT)) at both the transcriptional and post-transcriptional levels. Since the cardioprotective effect of estrogen itself has not been ruled out, we examined that of raloxifene, which has been classified as a selective estrogen receptor modulator, on the proliferation and telomerase activity of human umbilical vein endothelial cells (HUVECs). Raloxifene, like estrogen, clearly induced the telomerase activity and human TERT (hTERT) expression via estrogen receptor (ER) alpha and ERbeta. Treatment with raloxifene for 5 days significantly induced cell growth, and either cotreatment with a telomerase inhibitor, 3'-azido-3'-deoxythymidine, or transfection with hTERT-specific small interfering RNA significantly attenuated the raloxifene-induced cell growth. Raloxifene also induced the phosphorylation of Akt, and pretreatment with a phosphatidylinositol 3-kinase inhibitor, LY294002, significantly attenuated the raloxifene-induced telomerase activity. In addition, raloxifene induced both the phosphorylation of hTERT and IkappaB. Moreover, cotreatment with an IkappaBalpha phosphorylation inhibitor, BAY-11-7082, or a specific NFkappaB nuclear translocation inhibitor, SN50, significantly attenuated the raloxifene-induced telomerase activity and the association of NFkappaB with hTERT. These results show that raloxifene induced the up-regulation of telomerase activity not only by the transcriptional regulation of hTERT but also by post-translational regulation of the phosphorylation of Akt and hTERT and the association of hTERT with NFkappaB in HUVECs. Thus, the up-regulation of telomerase activity in vascular endothelial cells might be one mechanism contributing to the potential atheroprotective effect of raloxifene.

The expression of mitochondrial genes in granulosa cells was quantitated by real-time polymerase chain reaction. The expression ratio of mitochondrial genes in granulosa cells of patients with severe endometriosis showed no statistically significant difference compared with cases of tubal infertility without endometriosis.

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).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.The placebo group showed a significant decrease from the baseline in the trabecular BMD of the radius at 12 months (7.4+/-2.5%) (p0.05), 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 (p0.05). 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.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.

The mechanism of medroxyprogesterone acetate (MPA)-induced cell proliferation in human breast cancer cells remains elusive. We examined the mechanism by which MPA affects the cyclin D1 expression in progesterone receptor (PR)-positive T47D human breast cancer cells. MPA (10 nM) treatment for 48 h induced proliferation of the cells (1.6-fold induction). MPA induced cyclin D1 expression (3.3-fold induction), and RU486, a selective PR antagonist, blocked the MPA-induced cell proliferation and cyclin D1 expression (23% inhibition). MPA increased both the protein level (2.2-fold induction) and promoter activity (2.7-fold induction) of cyclin D1 in MCF-7 cells transfected with PRB but not with PRA. Although MPA transcriptionally activated cyclin D1 expression, cyclin D1 promoter does not have progesterone-responsive element-related sequence. We further examined the mechanism for the regulation of the cyclin D1 expression. Because the cyclin D1 promoter contains three putative nuclear factor-kappaB (NFkappaB)-binding motifs and NFkappaB is a substrate of Akt, we investigated the effect of the phosphatidylinositol 3-kinase (PI3K)/Akt/NFkappaB cascade on the responses of cyclin D1 to MPA. MPA induced the transient phosphorylation of Akt (2.7-fold induction at 5 min), and treatment with PI3K inhibitor (wortmannin) attenuated the MPA-induced up-regulation of cyclin D1 expression (40% inhibition) and cell proliferation (40% inhibition). MPA also induced phosphorylation of inhibitor of NFkappaBalpha (IkappaBalpha) (2.3-fold induction), and treatment with wortmannin attenuated the MPA-induced IkappaBalpha phosphorylation (60% inhibition). Treatment with an IkappaBalpha phosphorylation inhibitor (BAY 11-7085) or a specific NFkappaB nuclear translocation inhibitor (SN-50) attenuated the MPA-induced up-regulation of both cyclin D1 expression (80 and 50% inhibition, respectively) and cell proliferation (55 and 34% inhibition, respectively). Because MPA induced a transient phosphorylation of Akt and the cyclin D1 promoter contains no progesterone-responsive element-related sequence, the MPA-induced cell proliferation through PRB by up-regulation of cyclin D1 expression via the PI3K/Akt/NFkappaB cascade may be a nongenomic mechanism.

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.

Objective We longitudinally examined the changes of brachial to ankle distensibility using pulse wave velocity (PWV) throughout pregnancy and its difference between normal pregnancy and pregnancy-induced hypertension (PIH) groups. Study design One hundred and eighty-three pregnant women were included in this study. The PWV examinations were performed in a longitudinal way during the first, second, and third trimesters of pregnancy, and immediately and 1 month after delivery. Results In normal pregnancies, the PWV significantly decreased at the second trimester, increased from the third trimester through immediately after delivery, and decreased again at 1 month after delivery. In PIH patients, it increased in proportion to the progression of gestation. Conclusion We monitored the longitudinal changes in PWV and constructed a PWV normogram during pregnancy. The predictive value of PWV and blood pressure for PIH was higher than that of blood pressure alone, suggesting the usefulness of measuring PWV to predict PIH.

Estrogen deficiency is one significant cause of accelerated bone loss in women during and after menopause. Postmenopausal osteoporosis is a major health problem, primarily because of the severe morbidity and mortality associated with osteoporotic fractures. The Women's Health Initiative Study (WHI) reported that hormone replacement therapy (HRT) prevented osteoporotic fractures. The risks and benefits of HRT are complex and require the individual assessment of each woman considering taking HRT. Use of HRT can be considered as a treatment option for osteoporosis but the risks and benefits should be discussed with each individual woman before starting treatment.

Raloxifene has an estrogenic effect on the cardio-vascular system. In vascular endothelium, both clinical and basic studies show that raloxifene induces the synthesis and release of nitric oxide. In vascular smooth muscle, basic study shows that raloxifene attenuates the PDGF-induced cell proliferation by both induction of apoptosis and arrest of the cell cycle. We now await the results of currently ongoing prospective large scale randomized control trial, Raloxifene Use of The Heart.

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.

Objective: Since ovarian borderline tumor with invasive implant behaves as carcinoma, it should be managed like carcinoma. Since its characteristic features have not been reported, its preoperative diagnosis was thought to be impossible. We evaluated the features of MRI and macroscopic appearance in two cases of ovarian borderline tumor with invasive implant. Methods: Borderline tumor with invasive implant was evaluated in two patients by MRI and macroscopic examination. Results: In these patients, MRI revealed profuse papillary projections. Although the lesion showed high signal intensity on contrast-enhanced T1-weighted images compared with that on T1-weighted ones, most of the signal intensity on T2-weighted images was high, suggesting that the lesion is an assembly of vesicles and an obvious solid part is absent. The macroscopic appearance of the tumor showed profuse papillary projections consisting of many vesicles perforating and extending far beyond the ovarian capsule, without a solid part. The histological findings indicated serous borderline tumors with invasive implant. Conclusion: In these two cases, we found the characteristic features of serous borderline tumor with invasive implant by MRI and macroscopic examination. Our findings may be of clinical value since the preoperative information about the possibility of invasive implant may be quite important for the management of borderline tumor with invasive implant, especially for young patients wishing to bear children.

Background/Aims: Aortic stiffness, determined by pulse wave velocity (PWV), is an independent marker of cardiovascular risk. PWV is mainly influenced by age-associated alterations in arterial wall structure and blood pressure. The present study was conducted to assess the impact of menopause on the brachial-ankle PWV (baPWV) in healthy women. Methods: Fifty premenopausal women aged 22–54 years and 40 postmenopausal women aged 40–73 years were recruited for this study. Subjects with hypertension, diabetes, and hyperlipidemia were strictly excluded. The results of baPWV were analyzed chronologically by 10- or 5-year age intervals. Results: There was no significant difference in baPWV between premenopausal and postmenopausal women in their 40s and 50s. The baPWV of postmenopausal women aged over 60 years was significantly higher than that of postmenopausal women in their 50s. To clarify the age-dependent elevation in baPWV in detail, women their 50s and 60s were divided into subgroups by 5-year age intervals. There was no significant difference in baPWV among the 50–54-, 55–59- and 60–64-year subgroups. baPWV significantly increased in the 65–69- year subgroup (p< 0.05). There was a significant relationship between baPWV and age in premenopausal (r = 0.452, p = 0.001) and postmenopausal (r = 0.581, p < 0.0001) women. The slope of the regression line for baPWV plotted against age was steeper in postmenopausal than in premenopausal women. Conclusions: This study produces suggestive evidence that menopause amplifies the age-dependent increase in arterial stiffness.

We report the novel observation that medroxyprogesterone acetate (MPA) attenuates the induction by 17beta estradiol (E2) of both nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) activity in human umbilical vein endothelial cells. Although MPA had no effect on basal NO production or basal eNOS phosphorylation or activity, it attenuated the E2-induced NO production and eNOS phosphorylation and activity. Moreover, we examined the mechanism by which MPA attenuated the E2-induced NO production and eNOS phosphorylation. MPA attenuated the E2-induced phosphorylation of Akt, a kinase that phosphorylates eNOS. Treatment with pure progesterone receptor (PR) antagonist RU486 completely abolished the inhibitory effect of MPA on E2-induced Akt phosphorylation and eNOS phosphorylation. In addition, the effects of actinomycin D were tested to rule out the influence of genomic events mediated by nuclear PRs. Actinomycin D did not affect the inhibitory effect of MPA on E2-induced Akt phosphorylation. Furthermore, the potential roles of PRA and PRB were evaluated. In COS cells transfected with either PRA or PRB, MPA attenuated E2-induced Akt phosphorylation. These results indicate that MPA attenuated E2-induced NO production via an Akt cascade through PRA or PRB in a non-genomic manner.

The trophoblast, an important component of the mammalian placenta, has several essential biological roles in the maintenance of pregnancy. First, trophoblast cells must attach to the uterine endometrium, and then they must invade to a depth at which the vascular network exists. Here, we investigated the effects of epidermal growth factor (EGF) on alpha2 integrin expression, adhesiveness to collagen, and invasive activity using human BeWo choriocarcinoma cells. EGF induced the expression of alpha2 integrin mRNA and protein, as shown by Northern blotting, Western blotting, and flow cytometry. Human chorionic gonadotropin (hCG) secretion was enhanced by the addition of EGF, which suggests that the BeWo cells functionally differentiated similarly to normal trophoblasts. EGF also dose-dependently stimulated the invasiveness of BeWo cells. Antibody against alpha2 integrin inhibited this effect, suggesting that it may be mediated by an increase of cell surface integrin. EGF had no effect on the adhesiveness of BeWo cells to collagen, whereas it stimulated the chemokinetic activity in a dose-dependent manner. The increase of chemokinetic activity was suppressed by antibody against alpha2 integrin. These results suggest that EGF may induce alpha2 integrin expression in trophoblast cells, thereby enhancing their invasiveness into the endometrium via an increase of their chemokinetic activity.

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.

The influence of postoperative estrogen replacement therapy on the sensitivity of ovarian cancer to paclitaxel remains elusive. We examined whether estrogen affects paclitaxel-induced apoptosis in the Caov-3 human ovarian cancer cell line, which expresses estrogen receptor. 17beta-Estradiol (E2) significantly reversed the paclitaxel-induced apoptosis and reduction of cell viability, and a highly selective estrogen receptor antagonist, ICI182,780, and a phosphatidylinositol 3-kinase inhibitor, LY294002, attenuated the reversal effect of E2 on paclitaxel-induced apoptosis and reduction of cell viability. E2 significantly induced the phosphorylation of Akt. Akt and apoptosis signal-regulating kinase 1 (ASK1) were physically associated, and E2 induced the phosphorylation of ASK1 at serine-83, which is a consensus Akt phosphorylation site. We confirmed a previous report showing that paclitaxel induces cell damage via the ASK1-c-Jun N-terminal protein kinase (JNK) cascade. E2 inhibited the paclitaxel-induced JNK activation, and the E2-induced inhibition of the paclitaxel-induced JNK activation was attenuated in cells treated with either ICI182,780 or LY294002 or transfected with ASK1S83A, in which a consensus Akt phosphorylation site at serine-83 was converted to alanine. The inhibitory effect of E2 on the paclitaxel-induced reduction of cell viability and apoptosis was diminished in cells transfected with ASK1S83A. These results indicate that E2 inhibits paclitaxel-induced cell damage by inhibiting JNK activity via phosphorylation of Akt-ASK1. Thus, treatment of ovarian cancer with paclitaxel might be less effective in the setting of postoperative estrogen replacement therapy.

Objectives: Carotid intima-media thickness (IMT) is an appropriate intermediate end point to investigate clinically relevant effects on atherogenesis. The study objective was to clarify whether long-term hormone replacement therapy (HRT) modifies the progress of age-related IMT in healthy postmenopausal Japanese women. Methods: One hundred and eighty-eight healthy postmenopausal women aged 42–69 years were recruited into the retrospective study. IMT was measured by B-mode real-time ultrasound in the following three groups of patients. One hundred and fifteen women who were prescribed estrogen plus progestin or estrogen alone were classified into two groups according to the HRT treated period: short-term (

The activating transcription factor 2 (ATF2) is a member of the ATF/cAMP-response element-binding protein family of basic-leucine zipper proteins involved in cellular stress response. The transcription potential of ATF2 is enhanced markedly by NH2-terminal phosphorylation by c-Jun NH2-terminal kinase (JNK) and mediates stress responses including DNA-damaging events. We have observed that four DNA-damaging agents (cisplatin, actinomycin D, MMS, and etoposide), but not the cisplatin isomer, transplatin, which does not readily damage DNA, strongly activate JNK, p38, and extracellular signal-regulated kinase (ERK), and strongly increase phosphorylation and ATF2-dependent transcriptional activity. Selective inhibition studies with PD98059, SB202190, SP600125, and the dominant negative JNK indicate that activation of JNK but not p38 kinase or ERK kinase is required for the phosphorylation and transcriptional activation of ATF2. Stable expression of ATF2 in human breast carcinoma BT474 cells increases transcriptional activity and confers resistance to the four DNA-damaging agents, but not to transplatin. Conversely, stable expression of a dominant negative ATF2 (dnATF2) quantitatively blocks phosphorylation of endogenous ATF2 leading to a marked decrease in transcriptional activity by endogenous ATF2 and a markedly increased sensitivity to the four agents as judged by decreased cell viability. Similarly, application of SB202190 at 50 micro m or SP600125 inhibited JNK activity, blocked transactivation, and sensitized parental cells to the four DNA-damaging drugs. Moreover, the wild type ATF2-expressing clones exhibited rapid DNA repair after treatment with the four DNA-damaging agents but not transplatin. Conversely, expression of dnATF2 quantitatively blocks DNA repair. These results indicate that JNK-dependent phosphorylation of ATF2 plays an important role in the drug resistance phenotype likely by mediating enhanced DNA repair by a p53-independent mechanism. JNK may be a rational target for sensitizing tumor cells to DNA-damaging chemotherapy agents.

Objectives: Estrogen acts directly on endothelial nitric oxide synthase through a non-genomic mechanism, resulting in rapid dilatation of blood vessels. In this study, we examined the change of endothelial function after surgical menopause. Methods: In 20 subjects who underwent gynecological operations (ovariectomy (OVX) 12, sham (SHAM) operation 8), postoperative changes of flow-mediated dilatation (FMD) of the brachial artery were examined using ultrasonography. Postoperative changes of the response to nitroglycerin (NTG) were also studied in these patients. Results: In the OVX group, significant decreases of FMD were observed 1 week after the operation, although no changes were observed in the response to NTG. In the SHAM group, no remarkable changes of FMD or the response to NTG were observed after the operation. Conclusions: OVX influences endothelium-dependent vasodilatation within as little as 1 week. Therefore, it may be important to address the rapid changes of circulation after surgical menopause in order to prevent cardiovascular disease.

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.

Hormone replacement therapy (HRT) is effective treatment for postmenopausal bone loss and osteoporosis. However, postmenopausal women with low bone mass fail to respond after a few years of HRT and some postmenopausal women show no bone response to HRT. In these cases, additive therapy is necessary. Bisphosphonates are highly effective in postmenopausal osteoporosis. We investigated the effects of adding etidronate, a bisphosphonate, to HRT. The addition of etidronate to HRT improved bone density (4.40+/-1.12% increase per year) in patients showing no increase or even a decrease after at least 1 year of HRT (5.39+/-2.40% decrease per year).

Raloxifene is a tissue-selective estrogen receptor modulator. The effect of estrogen on cardiovascular disease is mainly dependent on direct actions on the vascular wall involving activation of endothelial nitric oxide synthase (eNOS) via Akt and extracellular signal-regulated protein kinase (ERK) cascades. Although raloxifene is also known to activate eNOS in the vascular endothelium, the molecular mechanism responsible for this effect remains to be elucidated. In studies of both human umbilical vein endothelial cells and simian virus 40-transformed rat lung vascular endothelial cells (TRLECs), the raloxifene analog LY117018 caused acute phosphorylation of eNOS that was unaffected by actinomycin D and was blocked by the pure estrogen receptor antagonist ICI182,780. Activation of Akt by raloxifene reached a plateau at 15-30 min and declined thereafter, a similar time frame to that of Akt activation by 17beta-estradiol. On the other hand, both activation and phosphorylation of ERK by raloxifene showed a biphasic pattern (peaks at 5 min and 1 h), whereas ERK activation and phosphorylation by 17beta-estradiol reached a plateau at 5 min and declined thereafter. A MEK inhibitor, PD98059, had no effect on the raloxifene-induced Akt activity, suggesting an absence of cross-talk between the ERK and Akt cascades. Either exogenous expression of a dominant-negative Akt or pretreatment of TRLECs with PD98059 decreased the raloxifene-induced eNOS phosphorylation. Moreover, raloxifene stimulated the activation of Akt, ERK, and eNOS in Chinese hamster ovary cells expressing estrogen receptor alpha but not Chinese hamster ovary cells expressing estrogen receptor beta. Our findings suggest that raloxifene-induced eNOS phosphorylation is mediated by estrogen receptor alpha via a nongenomic mechanism and is differentially mediated by Akt- and ERK-dependent cascades.

Tamoxifen is widely applied as an antiestrogenic agent for adjuvant therapy in the treatment of breast cancer, while its estrogen-agonistic activity occasionally causes proliferative disorders or carcinogenesis at other sites, such as the uterus. We reported that estrogen activates telomerase in breast and endometrial cancer cells. The present study examines the effects of tamoxifen on the gene expression of human telomerase reverse transcriptase (hTERT) in breast and endometrial cancer cells. Tamoxifen inhibited the cell growth of MCF-7 cells, as well as hTERT mRNA expression in the presence of estrogen (E2), antagonizing the E2 effects. In contrast, tamoxifen stimulated the growth of Ishikawa cells and activated hTERT mRNA expression in the absence or presence of E2, exhibiting estrogen-agonistic action. Transient expression assays revealed that these actions of tamoxifen are achieved by transcriptional regulation of the hTERT promoter. An estrogen responsive element (ERE) in the hTERT 5' regulatory region was partly responsible for both the E2-antagonistic and -agonistic actions of tamoxifen. Tamoxifen activated the MAP kinase cascade in Ishikawa cells, but not in MCF-7 cells, and the activation of hTERT mRNA expression was effectively blocked by MEK inhibitor, suggesting that the MAP kinase pathway is involved in the tamoxifen-induced activation of hTERT. These findings indicate that tamoxifen regulates hTERT expression in a cell-type specific manner. Tamoxifen-induced activation of hTERT may be one component of estrogen agonistic function of tamoxifen that is involved in endometrial carcinogenesis induced by this agent.

Objectives: Estrogen replacement therapy has favorable effects on serum lipoprotein levels in postmenopausal women with hypercholesterolemia. However, there are some patients who fail to respond to hormone replacement therapy (HRT) to lower the serum cholesterol level. In these cases, a conventional lipid-lowering therapy will be applied in addition to HRT, while the effects of these drugs are not well understood. In this study, we studied the effects of simvastatin and bezafibrate administered in addition to HRT. Methods: Patients who were hypercholesterolemic even after HRT were randomly assigned to three treatment groups: HRT only (control group, n =10), HRT+simvastatin (10 mg/day, n =10), or HRT+bezafibrate (400 mg/day, n =10). Serum lipids and lipoprotein levels were measured throughout 12 weeks. Results: The serum triglyceride levels were decreased by 24±28 and 38±13% in the HRT+simvastatin and HRT+bezafibrate groups, respectively. HRT+simvastatin decreased the total cholesterol (21±10%) and low-density lipoprotein cholesterol (28±12%) levels without affecting the high-density lipoprotein cholesterol (HDL-C) level, while HRT+bezafibrate increased the HDL-C level (12±11%). Conclusions: Treatment with simvastatin or bezafibrate in addition to HRT should be considered in cases of postmenopausal hypercholesterolemia in which HRT alone fails to lower the serum lipoprotein levels.

Brain reperfusion may be of particular importance in the etiology of periventricular leukomalacia, of which the common findings are gliosis and ventricular dilatation. To investigate the mechanism of this pathogenesis, we used a metabolic inhibition (MI) model using cyanide plus deoxyglucose treatment of cultured glia isolated from fetal rat brain and examined the activity of extracellular signal-regulated protein kinase (ERK) during MI and also during the recovery from MI of 30 min. ERK activation was stimulated during MI and the recovery from MI. The time course and extent of activation of ERK during MI and the recovery from MI, however, were distinctly different. Activation of ERK was stimulated within 5 min of MI and declined thereafter. Activation of ERK was sustained during the recovery phase from MI and the extent of the activation was much greater than that during MI. Pretreatment with EGTA to eliminate extracellular Ca(2+), or with APV, an NMDA receptor antagonist, to inhibit Ca(2+) influx through the NMDA receptor, attenuated the activation of ERK. Moreover, pretreatment with PMA to downregulate PKC abolished the activation of ERK. PD98059, an inhibitor of ERK kinase, attenuated the cell proliferation induced by MI followed by recovery from MI. These results suggest that ERK is involved in gliosis during the recovery phase from MI and may play a role in the etiology of periventricular leukomalacia.

Regulation of the mitogen-activated protein kinase (MAPK) family by prolactin-releasing peptide (PrRP) in both GH3 rat pituitary tumor cells and primary cultures of rat anterior pituitary cells was investigated. PrRP rapidly and transiently activated extracellular signal-regulated protein kinase (ERK) in both types of cells. Both pertussis toxin, which inactivates G(i)/G(o) proteins, and exogenous 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 ERK activation, suggesting the involvement of G(i)/G(o) proteins in the PrRP-induced ERK activation. Down-regulation of cellular protein kinase C did not significantly inhibit the PrRP-induced ERK activation, suggesting that a protein kinase C-independent pathway is mainly involved. PrRP-induced ERK activation was not dependent on either extracellular Ca(2+) or intracellular Ca(2+). However, the ERK cascade was not the only route by which PrRP communicated with the nucleus. JNK was also shown to be significantly activated in response to PrRP. JNK activation in response to PrRP was slower than ERK activation. Moreover, to determine whether a MAPK family cascade regulates rat prolactin (rPRL) promoter activity, we transfected the intact rPRL promoter ligated to the firefly luciferase reporter gene into GH3 cells. PrRP activated the rPRL promoter activity in a time-dependent manner. Co-transfection with a catalytically inactive form of a MAPK construct or a dominant negative JNK, partially but significantly inhibited the induction of the rPRL promoter by PrRP. Furthermore, co-transfection with a dominant negative Ets completely abolished the response of the rPRL promoter to PrRP. These results suggest that PrRP differentially activates ERK and JNK, and both cascades are necessary to elicit rPRL promoter activity in an Ets-dependent mechanism.

We have studied the roles of c-Jun N-terminal protein kinase (JNK) and extracellular signal-regulated protein kinase (ERK) cascade in both the cisplatin-resistant Caov-3 and the cisplatin-sensitive A2780 human ovarian cancer cell lines. Treatment of both cells with cisplatin but not transplatin isomer activates JNK and ERK. Activation of JNK by cisplatin occurred at 30 min, reached a plateau at 3 h, and declined thereafter, whereas activation of ERK by cisplatin showed a biphasic pattern, indicating the different time frame. Activation of JNK by cisplatin was maximal at 1000 microM, whereas activation of ERK was maximal at 100 microM and was less at higher concentrations, indicating the different dose dependence. Cisplatin-induced JNK activation was neither extracellular and intracellular Ca(2+)- nor protein kinase C-dependent, whereas cisplatin-induced ERK activation was extracellular and intracellular Ca(2+)- dependent and protein kinase C-dependent. A mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor, PD98059, had no effect on the cisplatin-induced JNK activity, suggesting an absence of cross-talk between the ERK and JNK cascades. We further examined the effect of each cascade on the viability following cisplatin treatment. Either exogenous expression of dominant negative c-Jun or the treatment by PD98059 induced sensitivity to cisplatin in both cells. Our findings suggest that cisplatin-induced DNA damage differentially activates JNK and ERK cascades and that inhibition of either of these cascades sensitizes ovarian cancer cells to cisplatin.

The regulation of mitogen-activated protein (MAP) kinase by endothelin-1 (ET-1) in cultured rat puerperal uterine myometrial cells was investigated. ET-1 caused the rapid stimulation of MAP kinase activity. ET-1-induced MAP kinase activation is neither extracellular Ca2+- nor intracellular Ca2+-dependent. ET-1 stimulation also led to an increase in phosphorylation of son-of-sevenless (SOS), and transfection of dominant negative SOS attenuated the ET-1-induced MAP kinase activity. Phorbol-12-myristate 13-acetate (PMA) also induced the MAP kinase activity, but pretreatment of the cultured cells with PMA, to down-regulate protein kinase C (PKC), did not abolish the activation of MAP kinase by ET-1. In addition, down-regulation of PKC had no effect on ET-1-induced SOS phosphorylation. Pertussis toxin, which inactivates Gi/Go proteins, blocked the ET-1-induced MAP kinase activation but not the PMA-induced MAP kinase activation. The results suggested that MAP kinase is acutely activated by ET-1 through a pertussis toxin-sensitive G protein and SOS, not through the PMA-sensitive PKC. In addition, although reverse-transcriptase PCR assays detected messenger RNA for both ET- 1 receptor subtypes in cultured rat puerperal uterine myometrial cells, ET-1-induced MAP kinase activity and uterine contraction were blocked by treatment with BQ485, an antagonist selective for an ET type A receptor (but not by BQ788, an ET type B receptor antagonist). Ritodrine, which is known to relax uterine muscle contraction, attenuated ET-1-induced MAP kinase activity. We further examined the role of MAP kinase pathway in uterine contraction using an inhibitor of MEK activity, PD098059. This inhibitor completely inhibited the ET-1-induced MAP kinase activation and partially, but significantly, inhibited the ET-1-induced uterine contraction. These results indicate that ET-1-induced MAP kinase signaling cascade may play an important role in the ET-1-induced uterine contraction.

The effects of prolactin (PRL) on proliferation of cultured human uterine leiomyoma-derived smooth muscle cells (SMC) and its mechanism of action were investigated. PRL stimulated DNA synthesis and the expression of PRL receptor was identified by ribonuclease protection assay. Moreover, the regulation of mitogen-activated protein (MAP) kinase by PRL in leiomyoma-derived SMC was investigated. PRL stimulated MAP kinase activity, as detected by 32 P incorporation into MAP-2, in a dose-dependent manner. PRL also rapidly stimulated MAP kinase phosphorylation as detected by in vivo phosphorylation using 32 P labeling and phosphotyrosine immunoblotting. These results suggest that PRL stimulates the proliferation of human leiomyoma cells via the MAP kinase cascade.

The regulation of mitogen-activated protein (MAP) kinase by oxytocin in cultured human uterine myometrial cells was investigated. Oxytocin caused the rapid stimulation of MAP kinase activity detected in 32P incorporation of MAP-2. Oxytocin also stimulated the phosphorylation of MAP kinase detected in incorporation of [32P]orthophosphate into MAP kinase. Furthermore, oxytocin induced the tyrosine phosphorylation of MAP kinase. The oxytocin-dependent increase in the tyrosine phosphorylation of MAP kinase displayed a transient time course and was dependent on the concentration of oxytocin applied to the cells. Furthermore, we examined the mechanism by which oxytocin induced MAP kinase phosphorylation. Islet-activating protein (100 ng/ml), which inactivates Gi/Go proteins, blocked the oxytocin-induced phosphorylation of MAP kinase. Moreover, 1 microM ritodrine, which is known to relax uterine muscle contraction, attenuated oxytocin-induced MAP kinase activity and phosphorylation. These results provide evidence that oxytocin acutely activates MAP kinase through an islet-activating protein-sensitive G-protein in human uterine myometrial cells, suggesting that this new pathway may play an important role in the biological action of oxytocin on these cells.

We recently reported the existence of two separate pathways for thyrotropin-releasing hormone (TRH)-induced mitogen-activated protein (MAP) kinase activation in GH3 pituitary tumor cells. To test the role of MAP kinase in TRH action, we examined the effect of dopamine (DA) on TRH-induced MAP kinase in primary cultures of rat anterior pituitary cells. 1 μM of DA attenuated 1 μM TRH-induced MAP kinase activity and phosphorylation. 100 ng/ml of islet-activating protein (IAP) blocked these inhibitory effects of DA. These results suggest that crosstalk exists between the DA signaling pathway and the TRH-stimulated MAP kinase activating pathway in rat anterior pituitary cells.

We investigated the effects of CINC/gro on IL-6 secretion by rat posterior pituitary cells. CINC/gro immunoreactivity was already detected in 1-h conditioned medium of normal posterior pituitary cells, and it increased significantly in a time-dependent manner during the first 24 h of culture. This immunoreactivity could be induced by TNF-alpha in a dose-dependent manner. On the other hand, CINC/gro stimulated IL-6 secretion by posterior pituitary monolayer cultures in a concentration dependent manner. Thus, CINC/gro significantly (P < 0.01) increased the secretion of IL-6 within 13 h of incubation, and this effect continued throughout 24 h of incubation. The stimulatory effect of 100 ng/ml CINC/gro on IL-6 secretion was completely blocked by 24-h incubation with 100 ng/ml IAP. These data demonstrate a new biological activity for CINC/gro in the posterior pituitary system.

In order to investigate the involvement of prolactin-dopamine and dopamine-gonadotropin interactions in the hypothalamo-pituitary axis of hyperprolactinemia, in vitro studies were performed using primary cultures of dispersed rat hypothalamic heterogeneous cells containing tubero-infundibular dopaminergic neurons or gonadotropin-releasing hormone (GnRH) neurons. We observed that prolactin caused dose-dependent stimulation of [3H]dopamine release after a 16-h incubation. Staurosporin (10 nmol/l), an inhibitor of protein kinase C, significantly reduced the [3H]dopamine release induced by prolactin (1 mg/l). Incubation of tubero-infundibular dopaminergic neurons with prolactin (1 mg/l) had no effect on intracellular cyclic adenosine monophosphate accumulation. Dopamine (1 μmol/l) significantly (p < 0.01) reduced the release of GnRH induced by 50 μmol/l calcium ionophore from dispersed hypothalamic cells from the preoptic area, while prolactin had no effect on GnRH release. These data support the hypothesis that the antigonadotropic effect of prolactin on the hypothalamus is mediated by an inhibitory effect of dopamine on GnRH release.

Immunocytochemical double-staining analysis revealed that in the rat anterior pituitary 86% of cells containing the beta II-subspecies of protein kinase C also contained follicle stimulating hormone (FSH), and that 22% of these FSH cells expressed the beta II-subspecies. These findings suggest a close relationship between the beta II-subspecies of protein kinase C and FSH regulation.

The effect of vasoactive intestinal peptide (VIP) and PHI-27 on dopamine accumulation in cultured rat hypothalamic cells was investigated. VIP enhanced [3H]dopamine accumulation dose dependently. This effect was significant at 10(-8)-10(-5) M VIP with a concomitant increase in intracellular cyclic AMP (cAMP), and reached its plateau level at 10(-6) M VIP. VIP increased [3H]dopamine accumulation significantly within 15 min. PHI-27 and dibutyryl cAMP ((Bu)2-cAMP) also enhanced [3H]dopamine accumulation. These results suggest that VIP enhances dopamine accumulation in hypothalamic cells by increasing intracellular cAMP.

Objective To report a case of a methotrexate (MTX)-resistant heterotopic interstitial pregnancy in which dactinomycin was necessary as a second-line chemotherapy. Design Case report. Setting Medical college–affiliated hospital. Patient(s) A 39-year-old woman with vaginal bleeding after the transfer of two thawed 8-cell embryos. Intervention(s) MTX 50 mg/m 2 was given to the patient to treat a concomitant pregnancy in which an interstitial pregnancy and an intrauterine blighted ovum were identified. However, the patient's hCG level rose despite two MTX administrations. As a result, dactinomycin was then administered as a second-line chemotherapy. The patient's serum hCG level declined to a normal range after two administrations of dactinomycin. Conclusion(s) Dactinomycin could be an alternative in cases for which MTX is not effective and persistently high hCG levels exist.

We investigated the effect of tumor necrosis factor-alpha (TNF alpha), a product of activated macrophages, on the release of arachidonate from dispersed anterior pituitary cells. Primary cultures of anterior pituitary cells from rats were preincubated with [3H]arachidonate to label their phospholipid-containing components. The cells were then washed and incubated with vehicle or test agents, and PRL release into the medium and [3H]arachidonate cleaved from phospholipid were measured. TNF alpha significantly increased the release of both PRL and [3H] arachidonate release in a time- and dose-dependent manner. Other cytokines, such as interleukin-1 alpha, interleukin-1 beta, and gamma-interferon, had no effect on [3H]arachidonate release. To define the role of calcium in TNF alpha-induced arachidonate release, dispersed pituitary cells were incubated with low calcium medium, which decreased arachidonate release in response to TNF alpha. TNF alpha potentiated the release of [3H]arachidonate and PRL promoted by phospholipase-A2 and melittin, and markedly shifted the dose-response curve to the left. Inhibitors of phospholipase-A2, such as p-bromophenacyl bromide and quinacrine, had no effect on TNF alpha-induced [3H]arachidonate and PRL release. BW755C, an inhibitor of the conversion of arachidonate to its metabolites, decreased TNF alpha-induced PRL release, while indomethacin, a prostaglandin synthesis inhibitor, had no effect on TNF alpha-induced PRL release. These data indicate that arachidonate metabolites may be involved in the process of TNF alpha-induced PRL release.