The Anti-Mullerian hormone and ovarian cancer.

The Anti-Mullerian hormone (AMH), which is produced by fetal Sertoli cells, is responsible for regression of Mullerian ducts, the anlagen for uterus and Fallopian tubes, during male sex differentiation. Ovarian granulosa cells also secrete AMH from late in fetal life. The patterns of expression of AMH and its type II receptor in the post-natal ovary indicate that AMH may play an important role in ovarian folliculogenesis. Recent advances in the physiological role of AMH has stimulated interest in the significance of AMH as a diagnostic marker and therapeutic agent for ovarian cancer. Currently, AMH has been shown to be a circulating marker specifically for granulosa cell tumour (GCT). Its diagnostic performance seems to be very good, with a sensitivity ranging between 76 and 93%. In patients treated for GCT, AMH may be used post-operatively as marker for the efficacy of surgery and for disease recurrence. Based on the physiological inhibitory role of AMH in the Mullerian ducts, it has been proposed that AMH may inhibit epithelial ovarian cancer cell both in vitro and in vivo. These observations will be the basis for future research aiming to investigate the possible clinical role of AMH as neo-adjuvant, or most probably adjuvant, therapy for ovarian cancer.

Mullerian Inhibiting Substance enhances subclinical doses of chemotherapeutic agents to inhibit human and mouse ovarian cancer.

Mullerian Inhibiting Substance (MIS), a biological modifier that causes regression of Mullerian ducts in male embryos, is effective as a single agent in vitro and in vivo against human and mouse ovarian cancer cell lines expressing MIS type II receptor; however, little is known about how recombinant human MIS (rhMIS), now being scaled for preclinical trials, could be used in combination with cytotoxic or targeted chemotherapeutic agents. Mouse serous and endometrioid ovarian carcinoma cell lines were tested in vitro against rhMIS alone and with doxorubicin, paclitaxel, or cisplatin as agents in clinical use. Because MIS releases FK506 binding protein (FKBP12), which activates the mammalian target of rapamycin (mTOR) downstream of Akt, rhMIS and rapamycin combinations were tested. MIS increases p16 protein levels, and 5'-Aza-2'-deoxycytidine (AzadC) induces p16 mRNA; therefore, they were used in combination in vitro and in vivo with a human ovarian cancer cell line. A paclitaxel-resistant human ovarian cancer cell line and its parental line both respond to rhMIS in vitro. Additivity, synergy, or competition was observed with MIS and rapamycin, AzadC, doxorubicin, cisplatin, and paclitaxel, suggesting that MIS in combination with selective targeted therapies might achieve greater activity against ovarian cancer than the use of each individual agent alone. These assays and statistical analyses could be useful in selecting rhMIS and chemotherapeutic agent combinations that enhance clinical efficacy and reduce toxicity.

Recombinant human Mullerian inhibiting substance inhibits long-term growth of MIS type II receptor-directed transgenic mouse ovarian cancers in vivo.

PURPOSE: Mullerian inhibiting substance (MIS) is a glycoprotein hormone that causes Mullerian duct regression in male embryos. In short-term experiments, recombinant human MIS (rhMIS) inhibits xenotransplanted human ovarian cancer cell lines that are thought to be of Mullerian origin. Because this highly lethal cancer has a high recurrence rate after conventional chemotherapy, new treatments are warranted. We examined whether rhMIS as a novel, nontoxic, naturally occurring growth inhibitor can be an effective anticancer drug in long-term studies in vivo against allograft tumors that recapitulate human ovarian carcinoma. EXPERIMENTAL DESIGN: Mouse ovarian carcinoma (MOVCAR) cell lines expressing the early region of the SV40 virus, including the large and small T-antigen genes under transcriptional control of a portion of the murine MIS receptor type II (MISRII) gene promoter, were derived from TgMISIIR-TAg transgenic mice. rhMIS was tested against MOVCAR cells in growth inhibition assays in vitro, and in vivo in 6-week-old female nude mice. Tumor growth in animals was measured at weekly intervals for up to 20 weeks. RESULTS: MOVCAR cells and tumors express MISRII by Western blot, immunohistochemical, and Northern blot analyses. rhMIS significantly inhibited MOVCAR cell growth in vitro and in vivo in three separate long-term allotransplantation experiments. CONCLUSIONS: Because rhMIS is an effective anticancer agent in in vitro and in long-term in vivo preclinical experiments against MISRII-positive tumors, we predict that rhMIS can be used safely and effectively to treat human ovarian malignancies.

Enhanced purification and production of Müllerian inhibiting substance for therapeutic applications.

It is almost 60 years since Prof. Alfred Jost reported the seminal observations regarding Müllerian inhibiting substance (MIS). His experiments clearly showed that a testicular product other than testosterone, a Müllerian inhibitor, was responsible for Müllerian duct regression. Twenty-five years later Dr. Picon established an organ culture assay which paved the way for the initial studies into the biochemistry and biology of Müllerian inhibiting substance, also known as Anti-Müllerian hormone (AMH), undertaken first in Dr. Nathalie Josso's Laboratory in Paris then in our own laboratory in Boston. Purification of MIS led to cloning the human gene and production of recombinant human (rhMIS). MIS is a 140 kDa glycoprotein homodimer which is activated by a biosynthetic protease, cleaving MIS into an aminoterminus (110 kDa) and a carboxyterminus (25 kDa). The latter domain is sufficient for biological activities. MIS functions by interacting with two receptors; a type II binds the hormone and at type I that initiates downstream signaling. The MIS type II receptor has been cloned and functionally confirmed as distinct from that of other members of the TGFbeta superfamily. MIS can employ a number of type I receptors (ALK2, ALK3, ALK6) and BMP receptor specific SMADS 1, 5, and 8 in various tissue specific contexts. Cell lines derived from human ovarian, breast, and prostate tumors, and from rodent Leydig cell tumors, which respond to MIS in growth inhibition assays, all express the MIS type II receptor. A variety of signal transduction pathways are associated with the grown inhibition mediated by MIS. For example, breast and prostate cancer cell lines use a MIS-mediated NFkappaB pathway leading to G1 arrest and apoptosis. The ovarian cancer cell lines employ a pathway which enhances p16, modulates the E2Fs, and induces apoptosis. These signal transduction events can establish new rational treatment strategies to complement the growth inhibitory effects mediated by MIS. These combination strategies are being tested in vitro, and where appropriate will be tested in vivo using the highly purified MIS preparations, prior to use in early human clinical trials.

Highly purified müllerian inhibiting substance inhibits human ovarian cancer in vivo.

PURPOSE: Müllerian inhibiting substance (MIS) causes Müllerianduct regression in mammalian, avian, and reptilian embryos; MIS also inhibits growth in vitro of Müllerian-derived cell lines and primary cells from human ovarian carcinomas. We hypothesize that highly purified MIS delivered parenterally inhibits ovarian cancer in vivo. EXPERIMENTAL DESIGN: To test the efficacy of highly purified MIS against ovarian cancer cell lines in vivo, we treated immunosuppressed mice with MIS after implanting OVCAR 8 or IGROV 1 human ovarian cancer cells beneath the renal capsules and measured tumor volume over time. Animals were treated with daily injections of 10 micro g of purified exogenous recombinant human MIS or by endogenous MIS secreted from cells growing on biodegradable mesh. RESULTS: The average graft size ratio (change in size compared with starting size) of the OVCAR 8 tumor implants was larger in the control animals than in animals treated for 2 weeks (P < 0.019) or 3 weeks (P < 0.001) with parenteral MIS, or after treating with MIS produced from transfected cells, which impregnated the biodegradable mesh (P = 0.02). The average graft size ratio of the IGROV 1 tumors was also larger in the control animals than in those treated with injected MIS (P = 0.0174). CONCLUSIONS: Highly purified recombinant human MIS, delivered parenterally, or MIS produced endogenously causes inhibition of human ovarian cancer cell lines in vivo, providing convincing preclinical evidence to support the use of MIS as a parenteral agent for the treatment of ovarian cancer.

Mullerian inhibiting substance: an update.

The decades long study of Mullerian Inhibiting Substance by numerous laboratories around the world has been driven, in large part, by pediatric surgeons and pediatric endocrinologists who have a keen interest in the molecular pathophysiology of genital tract defects that are visited upon their patients. A better understanding of the genes involved in the development of the normal reproductive tract in males and females should lead to a more rational analysis of the diseases caused by their abnormal function. Furthermore, a translation of this knowledge from the bench to the bedside may lead to clinically useful advances in the diagnosis and management of intersex patients. The molecular analyses of MIS and MIS receptor gene mutations and persistent Mullerian duct syndrome and the development of MIS ELISAs to evaluate testicular function as well as to follow the progress of gonadal tumors are several clear examples of successes over the years. It will be interesting to see what lies ahead.

Müllerian inhibiting substance: an instructive developmental hormone with diagnostic and possible therapeutic applications.

Dr. Alfred Jost pioneered the field of reproductive endocrinology with his seminal observation that two hormones produced by the testes are required for the male embryo to develop a normal internal reproductive tract. T induces the Wolffian ducts to differentiate into epididymides, vasa deferens, and seminal vesicles. Müllerian inhibiting substance (MIS) causes regression of the Müllerian ducts, which in its absence would normally develop into the Fallopian tubes, uterus, and upper vagina as is observed in female embryos. This review will summarize our current understanding of molecular mechanisms underlying the function of MIS both as a fetal gonadal hormone that causes Müllerian duct regression and as an adult hormone, the roles for which are currently being investigated, i.e., inhibition of steroidogenesis, germ cell development, and cancer. We will also address the regulation of MIS expression as one of the first genes expressed after the commitment of the bipotential gonads to differentiate into testes under the influence of SRY, the gene on the sex-determining region of the Y chromosome. We will discuss what is known regarding MIS signal transduction, which as with other members of the TGFbeta family of growth and differentiation factors, occurs through a heteromeric complex of single transmembrane serine/threonine kinase receptors to effect downstream signaling events, including Smad, nuclear factor-kappaB, beta-catenin, and p16 activation. Finally, we will assess the clinical relevance of studying MIS in patients with persistent Müllerian duct syndrome and our efforts to determine the therapeutic value of MIS for patients with ovarian and other MIS receptor-expressing cancers.

An immunoassay to detect human müllerian inhibiting substance in males and females during normal development.

An enzyme-linked immunosorbent assay has been developed to measure human Müllerian inhibiting substance (MIS) in biological fluids. The enzyme-linked immunosorbent assay is specific for MIS, with a sensitivity in human serum to 0.5 ng/ml and does not recognize transforming growth factor-beta 1 or -beta 2, LH, or FSH. It similarly fails to recognize other proteins secreted from the cell type into which the MIS gene was cloned. MIS was detected in the serum of normal newborns, infants, children, and adults. In males the serum level of MIS is 10-70 ng/mL at birth. The level increases slightly after birth, and then decreases to a basal level of 2-5 ng/mL after the first 10 yr of life. Newborn male urine contains minimal amounts of MIS (0.5 ng/mL). In females MIS is barely detectable in serum at birth, but rises to the basal level equal to that seen in males after 10 yr of age. Similar basal levels of MIS were found in adult ovarian follicular fluid. MIS levels were high in the serum of a female patient with a sex cord tumor (3200 ng/mL), but fell to 100 ng/mL after multiple excisional operations. In addition, a serum MIS level of 20 ng/mL was detected in a patient with an ovarian granulosa cell tumor. A sensitive assay for MIS could be useful in the diagnosis of patients with congenital abnormalities of sexual development and patients with Sertoli cell and/or other MIS-producing neoplasms. Other applications may also be recognized as the biology of MIS in both males and females is further elucidated.

Mullerian inhibiting substance: a fetal hormone with surgical implications.

Mullerian Inhibiting Substance (MIS) is secreted from the fetal (and postnatal) testis and is known to cause regression of the Mullerian ducts, the anlage of the fallopian tubes, uterus and upper vagina. It is a large glycoprotein hormone, the action of which appears to be modulated by sex steroids: mainly testosterone in mammals and oestrogen in birds. Recent evidence has raised the possibility that its action may be to diminish cell surface phosphorylation and thereby change the direction of differentiation of the Mullerian duct towards regression. Other suspected functions for MIS include control of testicular descent and inhibition of malignant tumours of the female genital tract.

Mullerian inhibiting substance reduction of colony growth of human gynecologic cancers in a stem cell assay.

Mullerian Inhibiting Substance (MIS), a fetal testicular product that causes regression of the Mullerian duct in the male mammalian embryo, was evaluated for its antitumor effect on the premise that a substance active against this genital precursor in the fetus might also be active against tumors derived from these tissues. Increasingly pure fractions of biologically active MIS, prepared from newborn calf testes, were tested in the soft agar colony inhibition assay against single cell suspensions of fresh tumors derived in ascitic or solid form from patients with gynecologic malignancies. Twenty-eight tumor specimens placed in soft agar culture have provided sufficient growth to assess an MIS effect. Twenty-five of these 28 tumors showed significant colony inhibition after incubation with MIS. Increased antitumor response correlated with increased purification of MIS when the same tumor was treated with preparations of different purity. Samples obtained from the same patient at different times, from both ascites and solid tumor sources, produced nearly identical responses to MIS. MIS preparations, previously shown to be active in microcytotoxicity and colony inhibition assays against established human ovarian and endometrial carcinoma lines demonstrate consistent antitumor activity against fresh human gynecologic cancers removed at surgery.

Subrenal capsule assay to test the viability of parenterally delivered müllerian inhibiting substance.

Production of bovine müllerian inhibiting substance (MIS) has been increased to allow generation of large quantities of biologically active purified material. The limited MIS previously available allowed only pretreatment of tumors prior to colony inhibition or implanting in nude mice. In preparation for posttransplantation tumor treatment, a subrenal capsule assay, which was first used against human tumors heterotransplanted into nude mice and subsequently against those heterotransplanted into immunocompetent mice, was adapted to determine (1) if MIS preparations could traverse the bloodstream without degradation and (2) the optimal dose required to produce a biologic effect. Urogenital ridges from female 14-day-old rat embryos were transferred atraumatically to small pouches beneath the renal capsule of the immunocompetent male CDF1 mice. The cranial-caudal orientation of the ridge with its müllerian duct was maintained. Over the next 72 hours, the mice were injected via the tail vein with 0.1 mL of an MIS-containing solution over a 100-fold concentration range. After three days, the kidneys were removed and shaved just below the ridge, which was then placed in soft agar for orientation and subsequent serial sectioning. After fixation, dehydration, and paraffin embedding, sections were stained and regression of the müllerian duct was graded and compared according to concentration and number of MIS doses administered. Regression diminished from almost complete (4+) at the highest dose, to minimal (1 to 2+) at 1/100 of that dose. Heat-inactivated and vehicle controls caused no regression of the müllerian ducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Müllerian inhibiting substance inhibition of a human endometrial carcinoma cell line xenografted in nude mice.

Müllerian inhibiting substance (MIS) is a fetal testicular product which causes regression of the müllerian duct in the male mammalian embryo. This material has been partially purified from a neonatal bovine source and in cruder fractions has shown antitumor effects when tested against the HOC-21 ovarian carcinoma line in monolayer cytotoxicity, in soft agar colony inhibition assay, and in nude mouse xenografts. The glycoprotein used for the present studies was more highly purified by sequential ion exchange, carbohydrate affinity, and dye affinity chromatography. After a 1-hr exposure with 1.0 X 10(6) tumor cells prior to heterotransplantation, this more purified preparation with MIS biological activity as determined by organ culture assay of embryonic urogenital ridges delayed the appearance of palpable tumor nodules. That this response may be specific for tumors derived from the coelomic epithelium of the embryo is further supported by the absence of any antitumor effect when this substance was tested against the SW-48 colon carcinoma line. It is of interest that the antitumor activity followed the biological activity as the preparation was further purified to 30,000-fold.

Mullerian inhibiting substance inhibits growth of a human ovarian cancer in nude mice.

Mullerian inhibiting substance (MIS) was investigated for its ability to inhibit growth of a human ovarian cancer in nude mice. Biologically active preparations from newborn calf testes, obtained after sequential ion exchange chromatography, delayed or prevented growth of a human ovarian cancer (HOC-21) when 2 X 10(6) cells were preincubated with them prior to subcutaneous injection of the tumor cells into Balb/C homozygous nude mice. Preincubation of a human colon carcinoma cells (SW-48) with similar preparations of MIS failed to inhibit growth of the tumor cells in nude mice. Human serous carcinomas are thought to arise from the ovarian surface epithelium, a derivative of the coelomic epithelium of the urogenital ridge, which invaginates to form the mullerian duct early in embryonic life. The neoplastic cells of serous tumors simulate morphologically the lining cells of the fallopian tube, which are derivatives of mullerian duct epithelium. This study provides physiologic confirmation of the mullerian nature of this type of tumor and suggests that MIS may ultimately prove to be effective in its therapy.