Takashi Minegishi

Expression of an activated mammalian target of rapamycin in adenocarcinoma of the cervix: A potential biomarker and molecular target therapy

Alterations of the Akt/mTOR pathway have been observed in numerous types of cancer, thus this pathway represents an exciting new target for molecular therapeutics. We investigated the expression of activated Akt (p‐Akt) and mTOR (p‐mTOR) in patients with adenocarcinoma of the cervix and the involvement of the p‐Akt/p‐mTOR pathway in response to combination of inhibitor agents, rapamycin and LY294002, with conventional therapy, cisplatin, in vitro. Immunohistochemistry analysis of p‐Akt and p‐mTOR was conducted in 26 patients with adenocarcinoma of the cervix. Western blot analysis was performed to determine the protein expression involved in response to chemotherapy in cervical cancer cell lines. The results showed that p‐Akt and p‐mTOR were identified in 50% and 53.8% of adenocarcinoma of the cervix. The expression of p‐mTOR was a significant independent marker for prognosis. A significant correlation between p‐Akt and p‐mTOR was observed. There was no correlation between their expressions with any of clinicopathological factors. In the in vitro study, cisplatin at CPI50 targets both the apoptosis and survival pathway by activating the caspase‐cascade; inhibiting Akt, mTOR, p70S6K, and 4EBP1. Combination of rapamycin with cisplatin induced synergistic interaction. On the other hand, combination with LY294002 resulted in either synergistic or antagonistic effect depending on the doses given. Rapamycin pretreatment potentiated cisplatin‐induced apoptosis cell death and enhanced blocking of the survival pathway. Overall, the expression of p‐mTOR is a significant prognostic marker of adenocarcinoma of the cervix and a potential molecular target for the treatment of cervical cancer. Inhibition of the mTOR pathway contributes to cisplatin‐induced apoptosis in cervical cancer cell lines.

Effect of Estrogen on the Expression of Luteinizing Hormone-Human Chorionic Gonadotropin Receptor Messenger Ribonucleic Acid in Cultured Rat Granulosa Cells

Estrogen has been considered to enhance FSH actions in the ovary, including the induction of the LH receptor (LHR). In this study, we elucidated the mechanism underlying the effect of estrogen on the induction of LHR by FSH in rat granulosa cells. Estradiol clearly enhanced the FSH-induced LHR mRNA increase in a time- and dose-dependent manner, with a maximum increase of approximately 3.5-fold at 72 h, compared with the level of LHR mRNA solely induced by FSH. We then investigated whether the effect of estrogen on LHR mRNA was due to increased transcription and/or altered mRNA stability. A luciferase assay with the plasmid containing the LHR 5-flanking region did not show that estradiol increased the promoter activity induced by FSH. In contrast, the decay curves for LHR mRNA showed a significant increase in half-life with FSH and estradiol, suggesting that the increased stability of LHR mRNA is at least responsible for the regulation of LHR mRNA by estrogen. Recently mevalonate kinase (Mvk) was identified as a trans-factor that binds to LHR mRNA and alters LHR mRNA stability in the ovary. We found that estradiol, with FSH, decreased Mvk mRNA levels in rat granulosa cell culture, resulting in up-regulation of LHR mRNA that was inversely correlated to Mvk mRNA expression. Furthermore, the augmentation of FSH-induced LHR expression in the presence of estrogen was erased with the overexpression of Mvk by transient transfection. Taken together, these data indicate that LHR mRNA is up-regulated due to increased stability when estrogen negatively controls Mvk.

The effect of splice variant of the human luteinizing hormone (LH) receptor on the expression of gonadotropin receptor.

A splice variant of human lutropin/choriogonadotropin-receptor [hLHR (exon 9)] that lacks exon 9 was previously cloned in the corpus luteum of a woman with a regular menstrual-cycle. Supported by detergent soluble binding assay and receptor biotinylation experiment, the receptor binding assay shows hLHR (exon 9) is neither expressed at the cell surface nor have the capability of binding to hCG. Interactions between hLHR (exon 9) with the immature bands of gonadotropin receptors not with the mature bands were seen. This phenomenon is specific among gonadotropin receptors since human thyrotropin-receptor (hTSHR) failed to be coimmunoprecipitated. Furthermore, this receptor complex attenuated the receptor protein level within the cells. To elucidate the mechanism underlying the decrease in receptor protein by this receptor complex, we performed a Percoll-fractionation experiment, which indicated the receptor complex drove hLHR to the lysosome instead of the plasma-membrane. Moreover, the expression of hLHR (exon 9) mRNA was seen at all phases of the menstrual cycle and relatively increased as the luteal phase progressed. These results reveal a novel mechanism of regulation for gonadotropin receptor expression.

Identification of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible genes in human amniotic epithelial cells.

BackgroundExposure to dioxins results in a broad range of pathophysiological disorders in human fetuses. In order to evaluate the effects of dioxins on the feto-placental tissues, we analyzed the gene expression in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated primary cultures of human amniotic epithelial cells.MethodsHuman amniotic epithelial cells were dispersed by trypsin from amniotic membranes and cultured in DME/Hams F12 medium supplemented with 10% FBS. Two weeks after plating, cells were treated with 50 nM TCDD or DMSO (control), further incubated for 48 hrs, and the gene expression was analyzed by DNA microarray technology and quantitative real-time PCR.ResultsThirty eight TCDD-inducible genes, including cytochromeP4501A1 and cytochromeP4501B1, were identified. One of the remarkable profiles of the gene expression was the prominent up-regulation of interferon-inducible genes. The genes involved in the interferon gene expression and interferon signaling pathways were also up-regulated. Furthermore, the expression of genes related to collagen synthesis or degradation was enhanced by TCDD.ConclusionUsing DNA microarray and quantitative real-time PCR analyses, we identified TCDD-inducible genes, including interferon-inducible genes and genes related to collagen synthesis or degradation, in human amniotic epithelial cells.

HOX cofactors expression and regulation in the human ovary

BackgroundHOX cofactors enhance HOX binding affinities and specificities and increase HOXs unique functional activities. The expression and the regulation of HOX cofactors in human ovaries are unknown.MethodsIn this study, the expression of HOX cofactors, PBX1, PBX2, and MEIS1/2, were examined by using RT-PCR, immunofluorescence in cultured immortalized human granulosa (SVOG) cells. The distribution of these HOX cofactors in human ovaries was examined by immunohistochemistry. The effects of growth differentiation factor-9 (GDF-9) and follicle-stimulating hormone (FSH) on PBX2 in SVOG cells were investigated by western blot analysis. Binding activities of HOXA7 and PBX2 to the specific sequences in granulosa cells were determined by electrophoretic mobility shift assay (EMSA).Results and conclusionIn SVOG cells, PBX1, PBX2 and MEIS1/2 were expressed during cell culture. In normal human ovaries, PBX1 and MEIS1/2 were expressed in granulosa cells at essentially all stages of follicular development. These cofactors were expressed in the nuclei of the granulosa cells from the primordial to the secondary follicles, whereas beyond multilayered follicles was observed in the cytoplasm. The co-expression of PBX1 and MEIS1/2 in granulosa cells in normal human ovaries suggested that MEIS1/2 might control PBX1 sublocalization, as seen in other systems. PBX2 was not expressed or weakly expressed in the primordial follicles. From the primary follicles to the preovulatory follicles, PBX2 expression was inconsistent and the expression was found in the granulosa cell nuclei. The PBX2 expression pattern is similar to HOXA7 expression in ovarian follicular development. Furthermore, FSH down-regulated, GDF-9 did not change PBX2 expression, but co-treatment of the granulosa cells with FSH and GDF-9 up-regulated PBX2 expression. These results implicated a role for PBX2 expression in the steroidogenic activities of granulosa cells in humans. Moreover, PBX2 and HOXA7 bound together to the Pbx sequence, but not to the EMX2 promoter sequence, in SVOG cells. Our findings indicate that HOX cofactors expression in normal human ovary is temporally and spatially specific and regulated by FSH and GDF-9 in granulosa cells. HOX proteins may use different HOX cofactors, depending on DNA sequences that are specific to the granulosa cells.

Primary hypothyroidism presenting as multiple ovarian cysts in an adult woman: A case report

A 21-year-old woman was referred because of abdominal pain. On physical examination, her abdomen was distended up to the umbilical region. Ultrasound and computer tomography of the abdomen revealed bilateral multiple ovarian cysts. Laboratory studies revealed increased liver function, total cholesterol and creatine phosphokinase. Further clinical investigations determined that the patient suffered from primary hypothyroidism due to autoimmune thyroiditis. The cysts resolved spontaneously after the simple replacement of a thyroid hormone. Some reports have been published of primary hypothyroidism presenting as ovarian cysts and precocious puberty in prepubertal girls. However, the case presented herein indicates that an ovarian tumor as a result of hypothyroidism may also occur in adult females. To avoid inadvertent surgery to remove an ovarian tumor, it is essential that a patient with multiple ovarian cysts and hypothyroidism be properly managed, as the simple replacement of a thyroid hormone could resolve the ovarian cysts.

Compartment syndrome with thrombosis of common iliac artery after gynecologic surgery.

BACKGROUND: Compartment syndrome is a potentially devastating complication with possible permanent neuromuscular and kidney damage. CASE: A woman who had undergone radical hysterectomy with pelvic and paraaortic lymphadenectomy was diagnosed with compartment syndrome of the lower left limb. Thrombosis of the left common iliac artery was also found after emergency fasciotomy. CONCLUSION: Arterial thrombosis is less common than deep vein thrombosis during gynecologic operations, but the lithotomy position may cause insufficient arterial circulation in both the pelvis and legs.

Regulation of human luteinizing hormone receptor in the ovary

The luteinizing hormone receptor (LHR) is essential for elevated levels of progesterone to maintain pregnancy during the first trimester; the maintenance of the expression of LHR is a key factor controlling the duration of luteal function. Therefore, as the expression of LHR is most likely to be regulated by the stability of the receptor mRNA at the luteal phase of the human menstrual cycle, we focused on studies examining the stability of mRNA rather than the production of mRNA. In addition, LHR (exon 9), one of the splice variants of human LHR (hLHR), was cloned in the corpus luteum of a patient with a regular menstrual cycle. The results of Western blots using Percoll gradient fractionation indicated that hLHR formed complexes with hLHR (exon 9), which are transferred to the lysosome, where they are eventually degraded, instead of being translocated from the endoplasmic reticulum to the transducing organelle. These results showed that hLHR (exon 9) caused a reduction in the expression of functional receptor number and affected the signaling condition of wild-type hLHR. As the luteal phase progressed hLHR (exon 9) increased relative to hLHR, demonstrating that hLHR (exon 9) was expressed more than hLHR in the late luteal phase. This work reveals the essential function of the regulatory and structural elements involved in human LH receptor splicing, and that hLHR (exon 9) can negatively control the function of wild-type receptors. Moreover, this finding presented a novel mechanism of regulation of LHR in the human corpus luteum.