Mesothelin is a glycosylphosphatidylinositol-linked cell surface molecule expressed in the mesothelial lining of the body cavities and in many tumor cells. Based on the finding that a soluble form of mesothelin specifically binds to ovarian carcinoma cell line OVCAR-3, we isolated cDNAs encoding a mesothelin-binding protein by expression cloning. The polypeptides encoded by the two cloned cDNA fragments matched to portions of CA125, an ovarian cancer antigen and a giant mucin-like glycoprotein present at the surface of tumor cells. By flow cytometric analysis and immunoprecipitation, we demonstrate that CA125 binds to mesothelin in a specific manner. Binding of CA125 to membrane-bound mesothelin mediates heterotypic cell adhesion as anti-mesothelin antibody blocks binding of OVCAR-3 cells expressing CA125 to an endothelial-like cell line expressing mesothelin. Finally, we show that CA125 and mesothelin are co-expressed in advanced grade ovarian adenocarcinoma. Taken together, our data indicate that mesothelin is a novel CA125-binding protein and that CA125 might contribute to the metastasis of ovarian cancer to the peritoneum by initiating cell attachment to the mesothelial epithelium via binding to mesothelin.

One of the distinguishing features of the gonadotropin and thyrotropin hormone family is their heterodimeric structure, consisting of a common a subunit and a hormone-specific f8 subunit. Subunit assembly is vital to the function of these hormones: The conformation of the heterodimer is essential for controlling secretion, hormonespecific posttranslational modifications, and signal transduction. To address whether a and ,3 subunits can be synthesized as one chain and also maintain biological activity, a chimera composed of the human chorionic gonadotropin (hCG) ,3 subunit genetically fused to the a subunit was constructed. question of whether the common a subunit and a hormonespecific (3 subunit can be synthesized as a single chain to achieve the biological response. For structure-function studies, the ability to express a heterodimer as a single biologically active chain would likely avoid mutagenesis-induced defects in combination and secretion of individual subunits. In addition, since subunit dissociation will inactivate the in vivo activity of the heterodimer, single-chain analogs could have a longer biological half-life. A chimera composed of the C-terminal end of hCG(3 genetically fused to the N-terminal end of the a subunit was constructed because these regions can be modified without significant effects on the biological activity of the heterodimer (7,8). Expression of this chimeric gene in CHO cells produced a single polypeptide hCG molecule that was biologically active in vitro and in vivo. These results demonstrate that the a and hCG,B subunits encoded as single chain can fold into an appropriate conformation and that a noncovalent linkage of the a and 13 subunits is not required for biological activity. This approach can be used to further investigate structure-function relationships of hCG and related glycoprotein hormones that were previously not tractable because of the absolute dependence on subunit assembly for biological activity.

Hormonal regulation of apoptosis has been studied in cultured preovulatory follicles. Because early antral follicles are most vulnerable to undergo atretic degeneration under physiological conditions in vivo, the present studies were designed to investigate the hormonal regulation of apoptosis using in vitro culture of early antral follicles. Rats were implanted with diethylstilbestrol at 24 days of age to stimulate the development of early antral follicles, and ovaries were collected at day 27 of age. Early antral follicles were dissected and cultured (four per vial) for 24 h with or without hormonal treatments. After culture, DNA was extracted from follicles, and the degree of apoptotic DNA fragmentation was determined using 3'-end labeling and gel electrophoresis. In situ analysis of apoptotic DNA fragmentation revealed that granulosa cells in these follicles are the main cell type undergoing apoptosis. Follicles cultured in the absence of hormones showed a 12-fold increase in the level of apoptotic DNA fragmentation which was prevented by treatment with FSH in a dose-dependent manner (60% maximal suppression and apparent ED50 of 30 ng/ml). Similarly, treatment with (Bu)2cAMP also suppressed follicle apoptosis. Treatment with LH or human CG, however, minimally suppressed apoptotic DNA fragmentation (35% maximal suppression). Insulin-like growth factor-I (IGF-I) also suppressed apoptosis by 45%. Moreover, the suppressive effect of FSH on apoptosis was partially reversed by coincubation with IGF-binding protein-3, suggesting a potential mediatory role of endogenous IGF-I. However, recombinant bovine GH had no effect on follicle apoptosis despite its ability to stimulate IGF-I messenger RNA (mRNA) levels. Incubation of follicles with epidermal growth factor (EGF) and basic fibroblast growth factor maximally suppressed follicle apoptosis by only 32% and 42%, respectively. Ligand binding analysis indicated the minimal effectiveness of EGF on apoptosis in early antral follicles, as compared with its potent action in preovulatory follicles reported earlier, may be due to a 3.5 fold increase in EGF receptor concentration in the mature follicles. High doses (150 or 500 ng/ml) of interleukin-1beta also suppressed apoptosis by 48% whereas treatment with an NO generator, sodium nitroprusside, or a cyclic GMP analog suppressed apoptosis as effectively as that of FSH. Furthermore, treatment with activin resulted in a dose-related suppression of follicle apoptosis, reaching a maximal 40% suppression. In contrast, cotreatment of activin with its binding protein, follistatin, abolished this effect. Collectively, these data demonstrated a stage-dependent difference in the hormonal regulation of follicle apoptosis. Although FSH, LH/human CG, GH, IGF-I, EGF, basic fibroblast growth factor, and interleukin-1beta are all effective survival factors for preovulatory follicles, FSH is a major survival factor for early antral follicles, the stage during which a majority of follicle undergo atresia under physiological conditions.

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that has immunoregulatory functions. Our prior study showed that tumoral IDO overexpression is involved in disease progression and impaired patient survival in human ovarian cancer, although its mechanism remains unclear. The purpose of the present study is to clarify the role of IDO during the process of peritoneal dissemination of ovarian cancer. Indoleamine 2,3-dioxygenase cDNA was transfected into the murine ovarian carcinoma cell line OV2944-HM-1, establishing stable clones of IDO-overexpressing cells (HM-1-IDO). Then HM-1-IDO or control vector-transfected cells (HM-1-mock) were i.p. transplanted into syngeneic immunocompetent mice. The HM-1-IDO-transplanted mice showed significantly shortened survival compared with HM-1-mock-transplanted (control) mice. On days 11 and 14 following transplantation, the tumor weight of peritoneal dissemination and ascites volume were significantly increased in HM-1-IDO-transplanted mice compared with those of control mice. This tumor-progressive effect was coincident with significantly reduced numbers of CD8+ T cells and natural killer cells within tumors as well as increased levels of transforming growth factor-β and interleukin-10 in ascites. Finally, treatment with the IDO inhibitor 1-methyl-tryptophan significantly suppressed tumor dissemination and ascites with reduced transforming growth factor-β secretion. These findings showed that tumor-derived IDO promotes the peritoneal dissemination of ovarian cancer through suppression of tumor-infiltrating effector T cell and natural killer cell recruitment and reciprocal enhancement of immunosuppressive cytokines in ascites, creating an immunotolerogenic environment within the peritoneal cavity. Therefore, IDO may be a promising molecular target for the therapeutic strategy of ovarian cancer.

Progression of preantral follicle development is essential to further follicle maturation and ovulation, but there are few models for studying the regulation of preantral follicle survival and growth. We have evaluated preantral follicle survival in vivo and in vitro, and have developed a serum-free rat follicle culture system that can be used to characterize the regulation of preantral follicle growth and differentiation. Analysis of ovarian cell DNA fragmentation during the first wave of follicle growth in the infantile rat indicated negligible apoptosis up to day 16 of age. However, a major increase in apoptosis was found by day 18, a time point associated with the appearance of large antral follicles. In situ analysis confirmed that apoptotic DNA fragments were limited to antral follicles. Culture of individual preantral follicles mechanically dissected from ovaries of 12- or 14-day-old rats in serum-free conditions led to major increases in follicle cell apoptosis, similar to that seen in cultures of antral and preovulatory follicles. In contrast to antral and preovulatory follicles, treatment of preantral follicles with gonadotropins or cAMP analogs did not prevent apoptosis. However, treatment with 8-bromo-cGMP or 10% serum suppressed apoptosis by 75% in cultured preantral follicles. In situ analysis identified granulosa cells as the cell type susceptible to apoptosis regulation. Taking advantage of the ability of the cGMP analog to suppress apoptosis, we evaluated the potential of FSH as a growth factor. In the absence of serum, FSH treatment for 48 h did not affect follicle size compared to controls; however, treatment with the cGMP analog together with FSH increased follicle diameter (13%; P < 0.01) and viable cells (2.4-fold; P < 0.01) compared to control values. Immunoblot analysis further indicated that the inhibin-alpha content of the cultured follicles was increased by treatment with the combination of FSH and 8-bromo-cGMP, demonstrating the induction of follicle cell differentiation during culture. Therefore, we demonstrated that activation of the cGMP pathway promotes the survival of cultured preantral follicles and that in the presence of alpha cGMP analog, FSH is a growth and differentiation factor for preantral follicles. The present serum-free follicle culture model system will be useful in further evaluation of the regulation of growth and differentiation of preantral follicles.