Tomomitsu Okamoto

Placental leucine aminopeptidase/oxytocinase in maternal serum and placenta during normal pregnancy

Placental leucine aminopeptidase (P-LAP), which is identical with cystine aminopeptidase as oxytocinase, was found to be homologous with rat insulin-regulated membrane aminopeptidase (IRAP) by sequence comparison. In the current study, we determined the P-LAP levels in maternal serum and placenta during healthy pregnancy. P-LAP activities in maternal serum increased with gestation and rose to the peak of 80 IU/ml at 38 weeks of gestation. Northern blot analysis revealed the increase of P-LAP mRNA levels in placenta in the third trimester compared to the first trimester. P-LAP protein and related activities could be detected in the conditioned medium of placental tissue, while they could not be detected in that of human umbilical vein endothelial cells. Immunohistochemically P-LAP was positively stained in the apical membrane of syncytiotrophoblast cells throughout the gestation. These results established the normal range of serum and tissue P-LAP levels during pregnancy and the possible source of serum P-LAP, which will be helpful to elucidate the physiological and clinical roles of P-LAP/oxytocinase/IRAP.

Expression of midkine and pleiotropin in ovarian tumors.

Objective To compare the expression of midkine and pleiotropin in malignant ovarian tumors with that in normal and benign ovarian tissue. Methods Total RNA was isolated from 23 samples of normal ovaries, 15 benign ovarian tumors, and 36 malignant ovarian tumors. Midkine and pleiotropin gene expression was examined by using Northern blot analysis. To confirm the localization of midkine expression, in situ hybridization and immunohistochemical analyses were performed. The truncated midkine messenger RNA was examined using polymerase chain reaction with complementary DNA synthesized from total RNA with reverse transcriptase. Results Expression of midkine gene was observed in 19 of 23 normal ovary samples and in 51 of 53 ovarian tumors (13 of 15 benign, both of the two borderline tumors, and all 36 malignant tumors). Pleiotropin gene was expressed in six normal ovaries and in 24 tumors (nine benign, two borderline, and 13 malignant tumors). The expression of midkine in germ cell tumors was significantly lower than in epithelial tumors, whereas expression in malignant epithelial tumors was significantly higher than in benign ones. In germ cell tumors, two samples with differentiated neural tissues showed high levels of pleiotropin gene expression. In situ hybridization and immunohistochemical analysis showed strong expression of midkine in cancer cells. The truncated midkine messenger RNA was not found in any of the normal, benign, or malignant tissues examined. Conclusion These results suggest an association between midkine and carcinogenesis. Expression of pleiotropin is more restricted, and high levels of its expression may be correlated with neural differentiation.

Quantitative analysis of matrix metalloproteinases-2 and -9, and their tissue inhibitors-1 and -2 in human placenta throughout gestation

To elucidate the implication of type IV collagenases(MMP-2 and MMP-9) and their tissue inhibitors (TIMP-1 and TIMP-2) for placental development, we quantified their levels in the conditioned media of placental organ culture and primary culture of the trophoblast as well as in the tissue extracts of placentas from different stages of gestation using specific enzyme-linked immunosorbent assays. First trimester villous tissue secreted about 10 times more pro-MMP-2 than pro-MMP-9, and pro-MMP-2 levels dramatically decreased in the second trimester. On the other hand, pro-MMP-9 levels were more than 10 times higher than those of pro-MMP-2 in the primary culture of the first trimester trophoblast, indicating the involvement of stromal cells for prominent pro-MMP-2 secretion from first trimester villous tissue described above. Levels of TIMPs, especially those of TIMP-2, remained constant throughout gestation both in the culture media and tissue extracts. Gelatin zymography revealed abundant secretion of the active form of MMP-2 as well as pro-MMP-2 from first trimester villous tissue. Western immunoblot analysis confirmed the presence of both TIMP-1 and TIMP-2 in placental tissue. These results suggest that active secretion of MMP-2 from villous tissue in the first trimester and constant production of TIMPs throughout gestation are characteristic of placental development.

Levels of hepatocyte growth factor in maternal serum and amniotic fluid.

OBJECTIVE The purpose of our study was to investigate hepatocyte growth factor levels in maternal serum and amniotic fluid during pregnancy. We also demonstrated production and secretion of hepatocyte growth factor by placenta and amnion at different stages of gestation. STUDY DESIGN Hepatocyte growth factor levels in maternal serum (n = 219), cord blood (n = 20), and amniotic fluid samples (n = 90) were measured by an enzyme-linked immunosorbent assay. The secretion of hepatocyte growth factor by placenta and amnion was evaluated by measuring the amount released into the culture supernatant. RESULTS Most hepatocyte growth factor levels in maternal serum were below the detection limit before 10 weeks of pregnancy. Levels increased significantly thereafter and continued to increase until term. On the other hand, levels in amniotic fluid were significantly higher between 20 and 29 weeks of gestation than after 30 weeks. Hepatocyte growth factor secretion from the placental tissue per weight seemed unchanged throughout pregnancy. Its secretion from amnion was, however, approximately 300 to 400-fold higher in the second trimester compared with that at term. CONCLUSION Both placenta and amnion produce and secrete hepatocyte growth factor, suggesting its role in fetal growth and the growth and differentiation of placenta.

Expression of Aminopeptidase N on Human Choriocarcinoma Cells and Cell Growth Suppression by the Inhibition of Aminopeptidase N Activity

We previously found that an aminopeptidase inhibitor, ubenimex (bestatin), had a growth‐suppressive effect on Choriocarcinoma cell lines in vitro. To clarify the mechanism of this action, we investigated the expression of aminopeptidase N (AP‐N/CD13) on Choriocarcinoma cells and other human tumor cells. Two Choriocarcinoma cell lines, NaUCC‐4 and Be Wo, had higher AP‐N activity than other cell lines (358.8 and 340.2 nmol/h/106 cells, respectively), as did the human myeloid leukemia cell line, HL‐60 (373.8 nmol/h/106 cells). These Choriocarcinoma and leukemia cell lines with abundant AP‐N activity showed much higher sensitivity to bestatin (IC50 = 0.5, 2.1 and l.0μg/ml, respectively) than the other cell lines. By imniuiioblotting and immunocytochemical staining, AP‐N was detected as an approximately 165‐kDa protein and localized on the cell membrane in Choriocarcinoma cells. We also examined the effects of two other aminopeptidase inhibitors and three anti‐CD13 monoclonal antibodies (MAbs) (WM15, MCS2 and MY7) on the growth of NaUCC‐4 cells. Cell growth was markedly suppressed by the AP‐N inhibitor actinonin as well as bestatin, but not by the AP‐B inhibitor arphamenine. Of the three MAbs, only WM15, which is able to inhibit AP‐N activity, suppressed cell growth in a dose‐dependent manner. These results indicate that AP‐N inhibitors show a growth‐suppressive effect, presumably through inhibition of the enzymatic activity of AP‐N on tumor cells, and suggest that AP‐N may play important roles in the growth of certain tumors, such as Choriocarcinoma and leukemia.

Combination effect of anti-Fas antibody and chemotherapeutic drugs in ovarian cancer cells in vitro.

To clarify the significance of the Fas antigen (Ag) in gynecologic tumors, its expression in gynecologic cancer cell lines was examined. The Fas Ag was expressed in 6 of 15 cell lines. Five of 8 ovarian cancer cell lines but none of 4 choriocarcinoma cell lines expressed the Fas Ag. In drug-resistant cell lines derived from one of the Fas-positive cells, its expression was not lost after development of resistance to cisplatin, SN-38 or etoposide, but its expression was absent in the cell line resistant to Adriamycin. The effect of the anti-Fas antibody (Ab) was then studied. Apoptosis was induced in 7 of 9 Fas-positive cell lines, whereas the remaining two cell lines were unaffected. Furthermore, the combination effect of the anti-Fas Ab and drugs was examined in an ovarian cancer cell line and its drug-resistant variants, and a synergistic effect was observed. These results suggest important roles of the Fas Ag in ovarian cancer and the potential for overcoming drug resistance by a combination of the anti-Fas Ab and various drugs.

Expression of human placenta alkaline phosphatase in placenta during pregnancy

To clarify the expression of PLAP during the course of pregnancy, the amount of PLAP mRNA and its activity in normal placental villi were measured. Both PLAP and its mRNA were found in placentae of as early as 7 weeks of gestation, and they continued to increase throughout pregnancy. But they showed different patterns of increase. The amount of PLAP mRNA began to increase dramatically around 13th week and probably continued to increase gradually until term. PLAP activity per gram of villi showed a gradual increase from around 13th week and a marked increase was observed after about 20th week. PLAP levels in sera from pregnant women were also measured, and they showed a pattern of increase imilar to that of PLAP activity per gram of villi. The continuous increase in the expression of PLAP throughout pregnancy suggests that PLAP may play a role in feto-maternal metabolism and placental differentiation.

Suppression of gelatinase production with decreased invasiveness of choriocarcinoma cells by human recombinant interferon beta

OBJECTIVE Choriocarcinoma is a highly invasive gynecologic tumor, and hematogenous metastases frequently develop. To establish a molecular basis for antiinvasion therapy of choriocarcinoma, we examined the effects of human recombinant interferons on gelatinase production and invasion by choriocarcinoma cells. STUDY DESIGN Using five choriocarcinoma cell lines, we measured gelatinase activity by gelatin zymography. The effects of recombinant interferons (rIFN-alpha, rIFN-beta, and rIFN-gamma) were then analyzed by Western blot analysis and chemoinvasion assay. RESULTS High levels of 72 kd gelatinase activity were detected in the highly invasive choriocarcinoma cell lines, two of which also contained an active form of 72 kd gelatinase with an apparent molecular mass of 68 kd. Gelatinase production was decreased by incubation with rIFN-beta. In the chemoinvasion assay, only rIFN-beta had an inhibitory effect on the invasiveness of tumor cells without a cytotoxic effect. CONCLUSION Choriocarcinoma cells showed high 72 kd gelatinase activity, which suggested a role for the enzyme in vascular metastasis. Studies on the use of rIFN-beta to inhibit metastasis of choriocarcinoma via suppression of gelatinase production are warranted.

The Expression and Localization of Neutral Endopeptidase 24.11/CD10 in Human Gestational Trophoblastic Diseases

Neutral endopeptidase 24.11 (NEP)/CD10 is a cell-surface peptidase that hydrolyzes various bioactive peptides. NEP is distributed in both normal and neoplastic cells and plays a functional role by modulating cellular responses to peptide substrates. Recently, NEP has been shown to be expressed in normal placental trophoblasts, suggesting its physiological role during pregnancy. In the present study, we investigated the expression of NEP in hyperplastic and anaplastic trophoblasts in gestational trophoblastic diseases (GTDs). Flow cytometric analysis demonstrated that NEP was expressed in all choriocarcinoma cell lines examined. The NEP enzyme activity in these cell lines correlated with cell-surface protein levels and was abolished by the NEP inhibitor phosphoramidon. On immunoblot analysis, NEP protein was detected in both hydatidiform mole and choriocarcinoma tissues as a double band of 95 and 100 kDa similar to that of the normal placental tissues. Immunohistochemical analysis revealed that NEP was present on syncytiotrophoblasts, while no or very faint NEP immunoreactivity was observed on cytotrophoblasts in the normal placenta. Similarly, NEP in hydatidiform mole and invasive mole was localized on the membrane of syncytiotrophoblasts, but not on hyperplastic cytotrophoblasts. In contrast, in choriocarcinoma, NEP was highly expressed not only on syncytiotrophoblastic cells but also on invading anaplastic cytotrophoblasts. In addition, NEP was also expressed on intermediate trophoblasts in placental site trophoblastic tumors. In summary, this is the first study demonstrating the expression of NEP/CD10 in GTDs. The differential localization of NEP among various trophoblastic tumors suggests that NEP may play a functional role in the regulation of trophoblast transformation and human chorionic gonadotropin secretion.

Pain relief of oral ulcer by dibucaine-film.

A water-soluble three-layered oral mucosa-adhesive film made from hydroxypropyl cellulose containing dibucaine (0.25 mg of drug/cm(2)) was designed for alleviation of severe pain due to oral ulcers, caused by chemotherapy and/or radiotherapy. We report two patients with constant severe pain ulcers treated with the dibucaine film. Patients were asked to record the time that pain was relieved while chewing following first application of the film. Pain relief lasted for 2-5 h after application of the dibucaine film.