Xenotransplantation of pre-pubertal ovarian cortex and prevention of follicle depletion with anti-Müllerian hormone (AMH)

Objective: To determine whether recombinant AMH (rAMH) could prevent post-transplant follicular depletion by acting on the stemness markers Oct-4, Sox2, and NANOG. Materials and methods: This was an experimental study where 12 ovariectomized nude mice were xenotransplanted with vitrified/warmed ovarian cortex obtained from a pre-pubertal girl and Alzet pumps delivering rAMH, or placebo (control), were inserted intra-abdominally. Previously vitrified/warmed ovarian cortex fragments were transplanted after 7 days and then harvested after 14 days from pump placement. We performed real-time RT-PCR analyses, ELISA for AMH, FSH, and estradiol, histologic measurement of ovarian follicles, and immunohistochemistry for Ki67 and TUNEL. The main outcome measures were serum levels and tissue expression of the parameters under investigation and follicle count. Results: Serum AMH, FSH, and estradiol reflected post-ovariectomy profiles and were mildly influenced by rAMH administration. Ovarian cortex expression of AMH, AMH-R2, VEGF, GDF9, Oct-4, and Sox2 was lower in rAMH mice than in controls, while NANOG was upregulated. There was a non-significant decrease in primordial follicles after vitrification-warming, and xenotransplantation further decreased this number. There were lower cell replication and depressed apoptosis in the rAMH group. Conclusions: Administration of recombinant AMH in the peri-transplant period did not protect the initial follicular depletion but decreased apoptosis and cellular activation and regulated stem cell markers\u27 tissue expression. These results aid our understanding of the inhibitory effects of AMH on follicular development and show the benefit of administering exogenous AMH at the time of pre-pubertal ovarian cortex transplant to protect the follicles from pre-activation and premature depletion

Interaction of the novel anthracycline antitumor agent N-benzyladriamycin- 14-valerate with the C1-regulatory domain of protein kinase C: Structural requirements, isoform specificity, and correlation with drug cytotoxicity

Anthracycline antibiotics like doxorubicin (DOX) are known to exert their antitumor effects primarily via DNA intercalation and topoisomerase II inhibition. By contrast, the noncross-resistant cytoplasmically localizing DOX analogue, N-benzyladriamycin-14-valerate (AD 198), only weakly binds DNA and does not inhibit topoisomerase II, yet it displays superior antitumor activity, strongly suggesting a distinct cytotoxic mechanism. In recent modeling studies, we reported a structural similarity between AD 198 and commonly accepted ligands for the C1-domain of protein kinase C (PKC), and we hypothesized that the unique biological activity of AD 198 may derive, in part, through this kinase. Consistent with this hypothesis, the present biochemical studies demonstrate that AD 198 competes with [3H]phorbol-12,13-dibutyrate ([ 3H]PDBu) for binding to phorbol-responsive PKC isoforms, the isolated C1b domain of PKC-δ (δ C1b), and the nonkinase phorbol ester receptor, β2-chimaerin. In NIH/3T3 cells, AD 198 competitively blocks PKC activation by C1-ligands. Importantly, neither DOX nor N-benzyladriamycin, the principal AD 198 metabolite, inhibits basal or phorbol-stimulated PKC activity or appreciably competes for [3H]PDBu binding. In CEM cells, structure activity studies with 14-acyl congeners indicate that the rapid induction of apoptosis correlates with competition for [3H]PDBu binding, strongly implicating phorbol-binding proteins in drug activity. Collectively, these studies support the conclusion that AD 198 is a C1-ligand and that C1-ligand receptors are selective drug targets. These studies provide the impetus for continuing efforts to understand the molecular basis for the unique biological activity of AD 198 and provide for the design of analogues with improved affinity for C1-domains and potentially greater antitumor activity. © 2002 American Association for Cancer Research