Gastrointestinal stromal tumor arising in the rectovaginal septum.

We report herein a rare case of malignant gastrointestinal stromal tumor (GIST) originated from the rectal wall, which presented as a tumor on the rectovaginal septum. A 54-year-old Japanese woman, gravida 4, para 3, was admitted complaining of anuresis and severe constipation. She had a history of hysterectomy and right salpingo-oophorectomy for uterine leiomyoma 11 years previously. Pelvic examination revealed an 8.5 x 7.5 x 7.5 cm hard mass in the rectovaginal space. The inferior border of the tumor was 2 cm from the vaginal introitus and 2 cm from the anus. Computed tomography and magnetic resonance imaging showed a well-circumscribed soft-tissue mass filling the rectovaginal space. Urinary bladder and rectum were markedly compressed and displaced. Colon fiberscopy revealed invasion of the tumor into the rectal mucosa. An abdominoperineal resection of the rectum with posterior vaginal wall resection and pelvic lymphadenectomy was performed. The resected specimen showed a rectal submucosal tumor that was 8 x 8 x 7 cm in size. The tumor was diagnosed as a malignant GIST. Immunohistochemical analysis confirmed this diagnosis. The patient is now healthy without evidence of recurrence at 13 months after surgery. Gynecologists should be aware of rectal GIST arising in the rectovaginal space as a differential diagnosis of vaginal submucosal tumor.

The clinical role of interleukin-6 and interleukin-6 soluble receptor in human follicular fluids.

In order to investigate the role of interleukin-6 (IL-6) and interleukin-6 soluble receptor (sR) in human ovulation, we evaluated the concentrations in human follicular fluid and analyzed the correlation of IL-6 and IL-6 sR with oocyte maturation. The oocytes were obtained from the follicular fluid of 45 women undergoing in vitro fertilization and embryo transfer. The concentrations of IL-6 and IL-6 sR in follicular fluid were measured by ELISA. In addition, granulosa cells obtained from the follicular fluid were cultured and treated with forskolin and 12- o-tetradecanoylphorbol 13-acetate for 24-48 h. The concentration of IL-6 was significantly higher in the follicular fluid than in the serum (P<0.01). In contrast, the concentration of IL-6 sR was significantly lower in the follicular fluid than in the serum (P<0.001). The concentrations of IL-6 and IL-6 sR were significantly higher in the follicular fluid containing mature oocytes than in fluid containing immature oocytes (P<0.05). The production of IL-6 was markedly increased over the basal level after 24 h of treatment with forskolin (P<0.001) and 48 h of treatment (P<0.01) with cultured granulosa cells. Our data suggest that IL-6 and IL-6 sR may play an important role in follicular growth and development in human preovulatory processes. It is possible that IL-6 in particular may be regulated by cAMP. IL-6 and IL-6 sR might also be valuable biochemical markers in the evaluation of oocyte maturation.

Complete response to radiation therapy in a patient with chemotherapy-resistant ovarian clear cell adenocarcinoma.

Recurrent ovarian cancer after front-line chemotherapy is incurable. In most institutions, chemotherapy is continued as salvage therapy after primary chemotherapy failure and despite the fact that long-term survival and complete responses are infrequent. Radiation therapy for patients with recurrent ovarian cancer has often been done with palliative intent. A patient with ovarian clear cell adenocarcinoma received irradiation with palliative intent to the whole pelvis after chemotherapy (paclitaxel, carboplatin, and irinotecan) produced no effect. Although she developed a rectovaginal fistula due to cancer invasion during radiation therapy. One year and half after the therapy, she is still alive with no evidence of disease. In an effort to maximize salvage potential and quality of life while minimizing toxicity, selected patients with ovarian cancer should be treated with radiation therapy directed to residual or recurrent sites.

The effect of epidermal growth factor on vascular endothelial growth factor secretion by endometrial stromal cells.

Endometrial stromal cells undergo morphological and functional changes to facilitate oocyte implantation under regulation of various hormones and growth factors. We studied physiological induction by epidermal growth factor (EGF) of vascular endothelial growth factor (VEGF) in these cells. In human endometrial stromal cells, the effect of EGF, genistein, tryphostin AG1478 (a tyrosine kinase inhibitor), and wortmannin (a phosphatidylinositol 3-kinase inhibitor) on production of VEGF was examined. Total RNA was extracted and VEGF mRNA expression was quantified by Northern analysis. EGF induced production of VEGF by stromal cells in a time-dependent manner; the effect became significant after 12 h and increased further between 24 and 48 h (P<0.05). Dose dependency was also significant (P<0.01). Genistein, tryphostin AG1478, and wortmannin partially suppressed the increase in production induced by EGF (P<0.01, P<0.01, P<0.01), respectively. Production of EGF by fertilized oocytes and trophoblasts has been reported in early pregnancy. VEGF is believed to be induced by EGF through mechanisms involving tyrosine kinase and phosphatidylinositol 3-kinase. The increase in VEGF may contribute to neovascularization that promotes proliferation of endometrium and placentation.

c-Ets1 is a promising marker in epithelial ovarian cancer.

c-Ets1 controls the expression of some genes involved in extracellular matrix remodeling. To elucidate the involvement of c-Ets1 in epithelial ovarian carcinogenesis, we investigated the role of the proto-oncogene c-ets1 in the regulation of physiological processes such as cell proliferation, differentiation, and tumor invasion. Using fluorescent immunohistochemistry, we analyzed serial frozen sections for c-Ets1 protein expression in 26 patients with ovarian epithelial carcinoma, 10 patients with benign cystadenoma of the ovary, and 10 premenopausal patients with normal ovaries. We analyzed the relationship between the percentage of c-Ets1 stained cells in a patient and characteristics of the patient including histological classification, clinical stage, histological grade, and clinical outcome. c-Ets1 was not detected in any cases of benign ovarian cystadenoma. Most of the c-Ets1 proteins were found in the cytoplasm and some in the nucleus of epithelial ovarian cancer tissues. Moreover, c-Ets1 was strongly expressed in the head portion of papillary cancer tissues that had invaded the stroma. c-Ets1 expression was significantly associated with clinical stage (p<0.01), histological grade (p<0.01), and clinical outcome (p<0.01). Survival data were available for all patients and univariate Cox regression analysis showed that c-Ets1 expression was significantly associated with a poor prognosis (p<0.05). Our results demonstrate that c-Ets1 expression in epithelial ovarian cancer correlates to the malignant potential of the tumor.

Expression of cathepsin L in normal endometrium and endometrial cancer.

OBJECTIVE: To evaluate the expression of cathepsin L in normal endometrium and endometrial adenocarcinoma. STUDY DESIGN: Tissue from eight cases of G1 and eight of G2 endometrioid adenocarcinoma, and 15 normal endometrial specimens were examined by immunohistochemistry. RESULTS: In the normal endometrium, cathepsin L was expressed in a few cell layers of the apical part of the glandular epithelium throughout the menstrual cycle. In the carcinomas, there was an inverse correlation between the grade of tumor and the cathepsin L expression. CONCLUSION: Cathepsin L expression may cease during endometrial carcinogenesis and its expression may be less important in tumor progression than it is in tumors of other tissues.

Atypical polypoid adenomyoma in a patient with hyperprolactinemia.

We report a case of an atypical polypoid adenomyoma in a patient with hyperprolactinemia. A 23-year-old Japanese woman was admitted complaining of atypical genital bleeding. Specula examination revealed a walnut-size polypoid mass extruding from the cervix. The patient was oligomenorrheac, and endocrine analysis showed hyperprolactinemia. Transvaginal ultrasonography and magnetic resonance imaging revealed an endometrial polypoid mass (4 x 3 x 3 cm) arising from the lower segment of the uterine corpus. The pathologic diagnosis of the tumor after polypectomy was atypical polypoid adenomyoma. It is suggested that ovarian dysfunction caused by hyperprolactinemia may be involved in the pathogenesis of atypical polypoid adenomyoma in the present case.

The effect of dexamethasone on expression of mitogen-induced cyclooxygenase-2 mRNA by amnion-derived (WISH) cells.

OBJECTIVES: It has been reported that prostaglandin E(2) (PGE(2)) is synthesized in the amnion and that this synthesis increases during labor. The purpose of this study was to clarify the mechanism for the expression of cyclooxygenase-2 (COX-2) mRNA and the PGE(2) synthesis of amnion-derived (WISH) cells. STUDY DESIGN: Cells were cultured and treated by 12-O-tetradecanoylphorbol-13-acetate (TPA) and dexamethasone (DEX). PGE(2) in the culture medium was measured by ELISA. Total RNA was extracted from the cells, and COX-2 mRNA expression was analyzed by Northern blot analysis. RESULTS: During the time course of PGE(2) production in response to TPA stimulation, the PGE(2) production could not be detected until incubation had continued for 2h, but this production appeared to continue after 4h of incubation. PGE(2) production was significantly increased by TPA and suppressed by treatment with TPA and DEX. During the time course of COX-2 mRNA expression in response to treatment with TPA, the COX-2 mRNA band was detected after 1.5h. The strongest expression of COX-2 mRNA was observed at 2h incubation. After pre-treatment with TPA for 1h, the TPA-induced COX-2 mRNA was suppressed by treatment with DEX for 1 or 2h incubation in a dose-dependent manner. CONCLUSIONS: These results suggest that COX-2 mRNA is induced by TPA which activate protein kinase C, and suppressed by DEX in WISH cells.

Expression and regulation of growth-regulated oncogene alpha in human endometrial stromal cells.

Growth-regulated oncogene alpha (GROalpha), a potent chemoattractant for neutrophils, has previously been detected in the endometrial stromal cells (ESC) of human endometrium. In this study, the mRNA expression of GROalpha in the endometrium was evaluated by reverse transcription-polymerase chain reaction analysis, while the localization of GROalpha protein was studied by immunohistochemistry and the concentrations of GROalpha were measured using an enzyme-linked immunosorbent assay (ELISA). The effects of known modulators of endometrial function on the production of GROalpha by ESC were also examined by ELISA and Northern blot analysis. The expression of both GROalpha mRNA and GROalpha protein was detected in the cycling endometrium. GROalpha protein was localized mainly in the stroma, and endometrial tissues in the secretory phase contained higher amounts of GROalpha protein than did those in the proliferative phase. The production of GROalpha by ESC was enhanced by in-vitro decidualization. Lipopolysaccharide, tumour necrosis factor-alpha and interleukin-1beta also stimulated the expression of GROalpha mRNA and protein by ESC. These results suggest that the production of GROalpha by ESC is regulated by ovarian steroid hormones as well as by inflammatory mediators. The modulation of GROalpha concentrations in the local environment may contribute to normal and pathological processes in the uterus by regulating leukocyte trafficking in the endometrium.

Polo-like kinase (PLK) expression in endometrial carcinoma.

Polo-like kinase (PLK) is a cell cycle-regulated, cyclin-independent serine/threonine protein kinase. Recent reports have shown a critical role for PLK during tumorigenesis. To explore whether PLK plays a general role as a tumor marker of endometrial carcinomas, we examined the expression of PLK mRNA and protein in endometrial carcinomas and normal endometrium, and analyzed the relationship between PLK protein expression and malignant potential. We found that PLK mRNA was expressed in all specimens from endometrial carcinoma patients using RT-PCR methods, although some specimens from normal endometria were negative. Immunohistochemically, most of the PLK was found in the cytoplasm (around the nucleus), and partly in the nucleus of endometrial carcinoma glands and also secreted tissues from endometrial carcinoma glands. PLK was expressed at the basement membrane of carcinoma glands and partly expressed in the head portion of papillary carcinoma tissues. There was a significant correlation between percentages of PLK-positive cells and histological grade of endometrial carcinoma (P<0.0001). However, the expression of proliferating cell nuclear antigen and Ki-67 was independent of PLK expression. Moreover, we noted that PLK is strongly expressed in invading carcinoma cells. PLK expression could reflect the degree of malignancy and proliferation in endometrial carcinoma. Thus, in addition to being of diagnostic value, modulation of PLK activity in the tumors by chemotherapeutic agents or gene therapy may prove to be of therapeutic value.

Expression of epithelial neutrophil-activating peptide 78 in cultured human endometrial stromal cells.

It has been demonstrated that human endometrial stromal cells (ESC) produce a variety of chemokines in vivo and in vitro. To evaluate the expression of epithelial neutrophil-activating peptide 78 (ENA-78) in the endometrium, concentrations of ENA-78 in cyclic endometrial tissues were measured using enzyme-linked immunosorbent assay. The expression of ENA-78 was also detected in cyclic endometrium by immunohistochemistry. Endometrial tissues in the secretory phase contained higher amounts of ENA-78 protein than did those in the proliferative phase. Immunofluorescence staining revealed that ENA-78 protein was localized mainly in the stroma of endometrium. In addition, to evaluate the involvement of inflammatory mediators and ovarian steroid hormones in the production of ENA-78 by ESC was evaluated by in-vitro studies. Unstimulated ESC constitutively secreted ENA-78. Progesterone, lipopolysaccharide, tumour necrosis factor-alpha, and interleukin-1beta significantly stimulated the expression of ENA-78 by ESC. It is suggested that the production of ENA-78 by ESC is regulated by progesterone as well as by the inflammatory mediators. The modulation of ENA-78 concentration in the local environment by these mediators may contribute to the normal and pathological processes of human reproduction through regulation of leukocyte trafficking into the endometrium.

Effects of platelet-activating factor on cytokine production by human uterine cervical fibroblasts.

Platelet-activating factor (PAF), a lipid that acts as a potent proinflammatory mediator, is involved in several reproductive processes including parturition. To investigate the effects of PAF on expression of various cytokines by cultured human uterine cervical fibroblasts obtained at term prior to labour, Northern blot analyses and enzyme-linked immunosorbent assays were performed. C-PAF, a stable analogue of PAF, increased expression of interleukin-6 and -8 mRNA in a dose-dependent manner (10(-10) to 10(-8) mol/l of C-PAF), and the expression peaked within 4 h. The corresponding protein concentrations were increased in culture media. Monocyte chemoattractant protein-1 mRNA showed marked induction by 10(-8) mol/l of C-PAF; this peaked by 4 h and was followed by an increase in the protein concentration. Another cytokine, RANTES (regulated upon activation, normal T cell expressed and secreted) showed marked mRNA induction by 10(-8) mol/l of C-PAF, and continued to increase in a time-dependent manner until 24 h. The protein concentration was correspondingly increased in the medium. The PAF-induced cytokine production was abolished by co-incubation with WEB 2170, a specific PAF receptor antagonist. PAF may stimulate local production of cytokines which may induce migration of leukocytes and accelerate collagenolysis in the uterine cervix, thus contributing to cervical ripening during parturition.

Expression of receptor tyrosine kinase EphB4 and its ligand ephrin-B2 is associated with malignant potential in endometrial cancer.

The protein kinases includes many oncogenes and growth-factor receptors, as well as genes that are involved in cell cycle regulation. EphB4 receptors are a subfamily of receptor tyrosine kinases that are activated by ephrin-B2 ligands and are thought to play an important role in the development and oncogenesis of various tissues. However, very little experimental evidence exists to support this hypothesis. To elucidate the involvement of EphB4 and ephrin-B2 in endometrial carcinogenesis, we used fluorescent immunohistochemistry to analyze serial frozen sections of 20 endometrial carcinomas and 20 normal endometria for EphB4 and ephrin-B2 protein expression. We analyzed the relationship between the patient's characteristics and the percentages of EphB4- and ephrin-B2-stained cells. EphB4 expression was significantly associated with histological grade (p < 0.001) and certain clinical stages. Ephrin-B2 Expression was significantly associated with the presence of invasion to > 1/2 myometrium (p = 0.002). Our results demonstrate that increased EphB4 and ephrin-B2 expression may reflect or induce in endometrial carcinomas increased potential for growth and tumorigenicity. Furthermore, these results suggest that EphB4 receptor kinase may modulate the biological behavior of endometrial carcinomas through autocrine and/or paracrine activation, which is caused by ephrin-B2 ligands that are expressed in the same or neighbouring cells by immunohistochemistry.

Id1 expression is associated with histological grade and invasive behavior in endometrial carcinoma.

Basic helix-loop-helix (bHLH) DNA-binding proteins have been reported to regulate tissue-specific transcription of cellular differentiation within multiple cell lineages. The Id family of helix-loop-helix proteins does not possess a basic DNA-binding domain and functions as a negative regulator of bHLH proteins by forming high-affinity heterodimers with bHLH proteins. Id proteins were originally characterized as inhibitors of DNA binding and cell differentiation. Thus, overexpression of Id proteins correlates with cell proliferation and arrested differentiation in many cell lineages. To elucidate the involvement of Id1 in endometrial carcinogenesis, we analyzed serial frozen sections for Id1 protein expression in 20 cases of endometrial carcinoma and 20 cases of normal endometria by fluorescent immunohistochemistry. We analyzed the relationship between the percentages of Id1-stained cells and the patient's characteristics, including histological grade, clinical stage, presence of invasion to >1/2 myometrium, and clinical outcome. In normal endometria, Id1 was not detected in either the proliferative or the secretory phase. There was, however, abundant Id1 immunoreactivity in the endometrial carcinoma cells. Moreover, Id1 was strongly expressed in the inflammatory cells. Scoring on the basis of the percentage of positive cells indicated that Id1 expression is significantly associated with histological grade (P<0.05) and the presence of invasion to >1/2 myometrium (P<0.05). Our results demonstrate that increased Id1 expression in endometrial carcinoma correlates with the malignant potential of this tumor.

Effects of interleukin-4 on the in-vitro production of cytokines by human endometrial stromal cells.

A T helper (Th)1/Th2 model has been applied to as a system regulating the cytokine network during pregnancy. To evaluate the effects of interleukin (IL)-4, a Th2 cytokine, on the cytokine production by endometrial stromal cells (ESC), an enzyme-linked immunosorbent assay was used to measure the concentrations of IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1), and macrophage colony-stimulating factor (M-CSF) in the culture media of ESC and of an endometrial stromal sarcoma cell line, MaMi, following the addition of recombinant human IL-4. The expression of mRNAs for IL-6 and IL-8 in ESC after stimulation with IL-4 was also evaluated by Northern blot analysis. Increases in the concentrations of IL-6, IL-8, MCP-1, and M-CSF in the culture media of ESC and MaMi cells were observed on the addition of increasing amounts of IL-4. This cytokine also stimulated the transcription of IL-6 and IL-8 in ESC in a dose-dependent manner. It is suggested that IL-4 secreted by the maternal decidual tissue as well as by the developing embryo may stimulate the production of IL-6, IL-8, MCP-1, and M-CSF by ESC. The increased concentration of these cytokines in the local environment may contribute to the maintenance of early pregnancy by modulating the immune reaction at the feto-maternal interface.

Expression of polo-like kinase in ovarian cancer is associated with histological grade and clinical stage.

Polo-like kinase (PLK), a cell cycle-regulated, cyclin-independent serine/threonine protein kinase, has been shown in recent reports to play a critical role during tumorigenesis. To investigate whether PLK plays a general role as a tumor marker of ovarian cancers, we examined the expression of PLK protein in ovarian cancers, and analyzed the relationship between PLK protein expression and histological grade. Immunohistochemically, the majority of PLK was found in the cytoplasm (around the nucleus), and a portion was found in the nucleus of ovarian cancer glands and also in the fluid secreted from these glands. PLK was expressed at the basement membrane of cancer glands and partly expressed in the head portion of papillary cancer tissues. A significant correlation was found between percentages of PLK-positive cells and histological grade of ovarian cancer (P<0.001). However, the expression of proliferating cell nuclear antigen, Ki-67, and cyclin B1 was independent of PLK expression. Taken together, these findings suggest that PLK expression may reflect the degree of malignancy rather than the degree of proliferation in ovarian cancer. Thus, in addition to being of diagnostic value, PLK activity in ovarian tumors may be modulated by chemotherapeutic agents or gene therapy to therapeutic effect.

The production and clinical evaluation of macrophage colony-stimulating factor and macrophage chemoattractant protein-1 in human follicular fluids.

PROBLEM: In order to investigate the role of macrophage colony-stimulating factor (M-CSF) and macrophage chemoattractant protein-1 (MCP-1) in human ovulation, we measured the concentrations of M-CSF and MCP-1 in human follicular fluids (FFs) and correlated them with oocyte maturation. METHOD OF STUDY: The oocytes were obtained from the FFs of 46 women undergoing in vitro fertilization and embryo transfer (IVF ET). The concentrations of M-CSF and MCP-1 in the FFs were measured by enzyme-linked immunosorbent assay. In addition, granulosa cells obtained from the FFs of IVF patients were cultured and treated with forskolin and 12-O-tetradecanoylphorbol 13-acetate (TPA) for 24 48 hr. RESULTS: Concentrations of M-CSF and MCP-1 were significantly higher in the FFs than in the serum (P < 0.01). M-CSF concentrations tended to be higher, while MCP-1 concentrations were significantly higher in the FFs containing mature oocytes than in FFs containing immature oocytes (P < 0.05). The production of M-CSF was markedly increased over the basal level after treatment with forskolin (10 microM) for 24 (P < 0.02) and 48 hr (P < 0.01); however, the production of MCP-1 was unchanged. CONCLUSIONS: Our data suggest that M-CSF and MCP-1 may play an important role in human preovulatory processes and that M-CSF, in particular, may be regulated by cyclic adenosine monophosphate. M-CSF and MCP-1 may also be valuable biochemical markers in the evaluation of oocyte maturation.

Synergistic effect of interleukin-1alpha and ceramide analogue on production of prostaglandin E2 and F2alpha by endometrial stromal cells.

PROBLEM: Prostaglandins (PGs) are synthesized in the endometrium. Our objective was to evaluate interleukin (IL)-1alpha-induced production of PGE2 and PGF2alpha in endometrial stromal cells (ESC) following treatment with ceramide analogues. METHODS OF STUDY: ESC were obtained from human uterine endometrium by enzymic digestion and filtration. ESC were treated with IL-1alpha, IL-1 receptor antagonist (ra), C2-ceramide and C6-ceramide. The concentrations of PGE2 and PGF2alpha in media were determined using ELISA. The induction of prostaglandin H synthase (PGHS)-2 mRNA was also ascertained by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The production of PGE2 and PGF2alpha was significantly increased by IL-lalpha and suppressed by IL-1 ra, in a dose-dependent manner. PGF2alpha production was further increased by treatment with the combination of IL-1alpha and C2-ceramide as compared with IL-1alpha treatment alone. There was no significant difference in PGE2 production between cells treated with IL-1alpha and C2-ceramide and those treated with IL-1alpha alone. Both PGE2 and PGF2alpha production were significantly increased by treatment with IL-1alpha and C6-ceramide as compared with IL-1alpha treatment alone. Treatment of ESC with IL-1alpha stimulated PGHS-2 mRNA. PGHS-2 mRNA was decreased when IL-1 ra was added to the IL-1alpha-stimulated cells. CONCLUSIONS: These results suggest that IL-1alpha stimulates the production of PGE2 and PGF2alpha by a mechanism that involves the sphingomyelin-ceramide system, and thus that ceramide may be important in increasing the production of PGE2 and PGF2alpha in the human endometrium.