Jennie P. Mather

Activins, Inhibins, and Follistatins: Further Thoughts on a Growing Family of Regulators

Abstract Inhibin, a feedback inhibitor of pituitary FSH secretion, and its homodimer, activin, have been the subject of a growing body of literature in the last 5 years. These factors play a role not only in endocrine feedback in the reproductive system but also in paracrine and autocrine regulation of both reproductive and nonreproductive organs, including the liver, kidney, and brain. Additionally, the messages coding for both subunits and their receptors are exquisitely regulated, both spatially and temporally, during embryonic development. The cloning of a family of activin receptors; the development of specific immunoassays for inhibins A and B, and activins A and B; the description of α subunit, β subunit, and receptor loss of function transgenic mouse models; and the cloning of two new β subunit homologs have increased our understanding of the possible roles this complex family of proteins plays in development and endocrine function. This review largely confines itself to the roles of inhibins and activins in the male and female reproductive system, and is intended as an update to a 1992 review published in this journal.

[6] The growth of cells in serum-free hormone-supplemented media

Publisher Summary This chapter presents the practical details for the growth of cells in serum free hormone supplemented media. In the absence of serum, greater than usual care must be taken in preparation of the synthetic portion of the medium. Careful preparation of water is essential for consistent results with serum-free medium. Serum and even dialyzed serum can mask nutritional requirements of cells in culture. Commercially available powdered media are adequate for serum-free work although each batch must be checked for suitability. Serum also serves the function of a trypsin inhibitor in conventional tissue culture procedures. The serum may be necessary for the repair of trypsinization damage after subculture; or the residual serum left after its removal, even after one or more washes, may be furnishing unknown growth factors.

Effects of temperature shift on cell cycle, apoptosis and nucleotide pools in CHO cell batch cultues

Temperature reduction in CHO cell batch culture may be beneficial in the production of recombinant protein and in maintenance of viability. The effects on cell cycle, apoptosis and nucleotide pools were studied in cultures initiated at 37°C and temperature shifted to 30 °C after 48 hours. In control cultures maintained at 37 °C, viable cells continued to proliferate until the termination of the culture, however, temperature reduction caused a rapid decrease in the percent of cells in S phase and accumulation of cells in G-1. This was accompanied by a concurrent reduction in U ratio (UTO/UDP-GNAc), previously shown to be a sensitive indicator of growth rate. Culture viability was extended following temperature shift, as a result of delayed onset of apoptosis, however, once initiated, the rate and manner of cell death was similar to that observed at 37 °C. All nucleotide pools were similarly degraded at the time of apoptotic cell death. Temperature reduction to 30 °C did not decrease the energy charge of the cells, however, the overall rate of metabolism was reduced. The latter may be sufficient to extend culture viability via a reduction in toxic metabolites and/or limitation of nutrient deprivation. However, the possibility remains that the benefits of temperature reduction in terms of both viability and productivity are more directly associated with cultures spending extended time in G-1.

Paracrine regulation of reproductive function by inhibin and activin.

Conclusions Inhibin has been confirmed as a factor produced in the gonads and capable of specifically regulating FSH release by the pituitary. However, inhibin and the related activins seem to play important paracrine roles in locally regulating testicular and ovarian function. They are also involved in paracrine regulation in the placenta, the pituitary, and other nonreproductive tissues. The local effects of these factors may be modified by the presence of binding proteins and the receptor or receptors expressed. The specific type of activin or inhibin produced and the net effect locally may vary as a function of the sexual development of the organism or the stage of the reproductive cycle. A more complete understanding of these complex relationships will develop with advances in our ability to measure the proteins, their binding proteins, and cell surface receptors.

Development of an Fc-Enhanced Anti–B7-H3 Monoclonal Antibody with Potent Antitumor Activity

Purpose: The goal of this research was to harness a monoclonal antibody (mAb) discovery platform to identify cell-surface antigens highly expressed on cancer and develop, through Fc optimization, potent mAb therapies toward these tumor-specific antigens. Experimental Design: Fifty independent mAbs targeting the cell-surface immunoregulatory B7-H3 protein were obtained through independent intact cell-based immunizations using human tissue progenitor cells, cancer cell lines, or cell lines displaying cancer stem cell properties. Binding studies revealed this natively reactive B7-H3 mAb panel to bind a range of independent B7-H3 epitopes. Immunohistochemical analyses showed that a subset displayed strong reactivity to a broad range of human cancers while exhibiting limited binding to normal human tissues. A B7-H3 mAb displaying exquisite tumor/normal differential binding was selected for humanization and incorporation of an Fc domain modified to enhance effector-mediated antitumor function via increased affinity for the activating receptor CD16A and decreased binding to the inhibitory receptor CD32B. Results: MGA271, the resulting engineered anti–B7-H3 mAb, mediates potent antibody-dependent cellular cytotoxicity against a broad range of tumor cell types. Furthermore, in human CD16A-bearing transgenic mice, MGA271 exhibited potent antitumor activity in B7-H3–expressing xenograft models of renal cell and bladder carcinoma. Toxicology studies carried out in cynomolgus monkeys revealed no significant test article-related safety findings. Conclusions: This data supports evaluation of MGA271 clinical utility in B7-H3–expressing cancer, while validating a combination of a nontarget biased approach of intact cell immunizations and immunohistochemistry to identify novel cancer antigens with Fc-based mAb engineering to enable potent antitumor activity. Clin Cancer Res; 18(14); 3834–45. ©2012 AACR.

The role of cultured Schwann cell grafts in the repair of gaps within the peripheral nervous system of primates

With recent advances in cell culture techniques it is possible to isolate human SCs from adult peripheral nerves, expand and purify their number in cell culture, and construct a cellular prosthesis from the cultured cells. The current study was designed to ascertain whether these techniques could be used to repair nonhuman primate nerve injuries. In 12 adult female cynomologous monkeys, the musculocutaneous (msk) nerve was divided and prevented from regenerating and the brachioradialis nerve (brach) was exposed bilaterally (n = 24 nerves) and injured so that a 15-mm gap existed within the nerve. The brach nerves were either repaired with sural nerve autografts (n = 6), guidance channels which contained monkey SCs (120 x 10(6) cells/ml; n = 6), or guidance channels without SCs (n = 6). The remaining brach nerves (n = 6) had either no injury or an injury to the nerve without a repair. Autologous expanded primate SCs were increased in number at least 10-fold over a 2-week period at which time the SC purity exceeded 99.9%. Monkeys in each group, including the control group, regained some degree of elbow flexion after 3 months despite sectioning both the mask nerve and the brach nerve; therefore, we were unable to determine simply on clinical grounds which repair was the most effective in promoting functional recovery. Brach nerves repaired with sural nerve grafts were superior to both the channels which contained SCs and empty channels in regards to the number of myelinated axons proximal, within, and distal to the repair site (P < 0.05). Electrophysiologic results closely paralleled the histologic data with evidence of reinnervation of the brachioradialis muscle based on the compound muscle action potential in both sural nerve graft and monkey SC channel repair groups.

IDENTIFICATION OF AN INHIBIN RECEPTOR IN GONADAL TUMORS FROM INHIBIN ALPHA-SUBUNIT KNOCKOUT MICE

Inhibins and activins are dimeric proteins that are functional antagonists and are structurally related to the transforming growth factor-β (TGFβ) family of growth and differentiation factors. Receptors for activin and TGFβ have been identified as dimers of serine-threonine kinase subunits that regulate cytoplasmic proteins known as Smads. Despite major advances in our understanding of activin and TGFβ receptors and signaling pathways, little is known about inhibin receptors or the mechanism by which this molecule provides a functionally antagonistic signal to activin. Studies described in this paper indicate that an independent inhibin receptor exists. Numerous tissues were examined for inhibin-specific binding sites, including the developing embryo, in which the spinal ganglion and trigeminal ganglion-bound iodinated inhibin A. Sex cord stromal tumors, derived from male and female inhibin α-subunit-deficient mice, were also identified as a source of inhibin receptor. Abundant inhibin and few activin binding sites were identified in tumor tissue sections by in situ ligand binding using iodinated recombinant human inhibin A and125I-labeled recombinant human inhibin A. Tumor cell binding was specific for each ligand (competed by excess unlabeled homologous ligand and not competed by heterologous ligand). Based on these results and the relative abundance and homogeneity of tumor tissues versus the embryonic ganglion, tumor tissues were homogenized, membrane proteins were purified, and putative inhibin receptors were isolated using an inhibin affinity column. Four proteins were eluted from the column that bind iodinated inhibin but not iodinated activin. These data suggest that inhibin-specific membrane-associated proteins (receptors) exist.

The glycotope-specific RAV12 monoclonal antibody induces oncosis in vitro and has antitumor activity against gastrointestinal adenocarcinoma tumor xenografts in vivo

RAV12 is a chimeric antibody that recognizes an N-linked carbohydrate antigen (RAAG12) strongly expressed on multiple solid organ cancers. More than 90% of tumors of colorectal, gastric, and pancreatic origin express RAAG12, and a majority of these tumors exhibit uniform RAAG12 expression. RAV12 exhibits potent cytotoxic activity in vitro against COLO 205 colon tumor cells via an oncotic cell death mechanism. RAV12-treated COLO 205 cells undergo morphologic changes consistent with oncosis, including cytoskeletal rearrangement, rapid plasma membrane swelling, and cell lysis. RAV12 inhibited the growth of RAAG12-expressing gastrointestinal tumor xenografts in athymic mice. In the case of SNU-16 tumor cells, twice weekly treatment of established s.c. tumors with 10 mg/kg RAV12 caused a ∼70% suppression of tumor growth at the end of the study. This preclinical data has led to the initiation of a phase I/IIA clinical study of RAV12 in patients with metastatic or recurrent adenocarcinoma. [Mol Cancer Ther 2007;6(3):856–65]

Apoptosis in CHO cell batch cultures: examination by flow cytometry.

Chinese hamster ovary cells grown under conditions which are optimal for the production of a genetically engineered protein in batch culture, lose significant viability shortly after entering the stationary phase. This cell death was investigated morphologically and was found to be almost exclusively via apoptosi. Furthermore, cells were analyzed by flow cytometry using a fluorescent DNA end-labeling assay to label apoptotic cells, in conjunction with cell cycle analysis using propidium iodide. Apoptotic cells could be detected by this method, and by the radioactive end-labeling of extracted DNA, on all days of culture from day 1 to day 7; however, the degree of apoptotic cell death increased dramatically when the cells entered the stationary phase, rising to 50–60% of the total cell number at the termination of the culture. Flow cytometric analysis showed that the majority of cells underwent apoptosis whilst in G1/G0 and formed an apoptotic population with high DNA FITC end-labeling and hypodiploid propidium iodide binding. Additionally, the ability or inability to secrete specific protein products did not appear to interfere with the development of the apoptotic population with time.

Inhibins, activins, their binding proteins and receptors: Interactions underlying paracrine activity in the testis

The inhibin-related peptides are present in the testis from early gestation through adulthood. They are produced from multiple testicular sites in a highly regulated manner, suggesting important paracrine roles. Similarly, receptors for these peptides are located in specific stages of the seminiferous tubule and on particular cell types, and an additional level of control is afforded by specific binding proteins, such as follistatin, which may regulate bioavailability. The actions of these factors include the modulation of interstitial cell function and the increase of spermatogonial proliferation in vitro. It thus appears that activin and inhibin are significant factors in the local control of testicular function.