Cytotoxic activity of gonadotropin-releasing hormone (GnRH)-pokeweed antiviral protein conjugates in cell lines expressing GnRH receptors.

Pokeweed antiviral protein (PAP), a 29-kDa ribosome-inactivating protein isolated from the leaves of Phytolacca americana, has potent cytotoxic activity once it enters the cytoplasm of a cell. It is incapable of entering cells by itself. Therefore, our objective was to determine whether a GnRH analog could be used to deliver PAP specifically to cells expressing GnRH receptors. D-Lys(6)-GnRH-Pro(9)-ethylamide was conjugated to PAP (GnRH-PAP). Chinese hamster ovary cells stably transfected with cDNA for the murine GnRH receptor and a mouse gonadotroph tumor cell line that expresses endogenous GnRH receptors (alphaT3-1 cells) were used to evaluate the cytotoxic effects of GnRH-PAP. We also examined cytotoxicity of GnRH-PAP using human endometrial, breast, and prostate cancer cell lines. Treatment of GnRH receptor-positive cells with GnRH-PAP resulted in dose-dependent cytotoxicity. Cytotoxicity of GnRH-PAP was dependent on number of GnRH receptors (r(2) = 0.871, P < 0.05) and duration of exposure of GnRH-PAP to the cells. In contrast, GnRH-PAP was not cytotoxic to Chinese hamster ovary cells not harboring GnRH receptors. Moreover, the cytotoxic activity of GnRH-PAP could be inhibited by addition of excess GnRH analog. Neither PAP nor GnRH analog alone was cytotoxic. These results suggest that GnRH analogs can be used to specifically deliver toxin molecules to cells that express GnRH receptors. Thus, a new class of biomedicines that act as hormonotoxins against cells expressing GnRH receptors provides a novel approach for inhibiting reproduction and treating cancers that are dependent on reproductive hormones.

A sandwich enzyme-linked immunoabsorbent assay for measurement of gonadotropin-releasing hormone-toxin conjugates.

PROBLEM: Biological effectiveness of targeted cytotoxins is dependent on their stability, circulating half-life, receptor binding ability, and cytotoxicity. The objective of this study was to compare stability of gonadotropin-releasing hormone (GnRH)-toxin conjugates made with disulfide linkers to those using a maleimidodibutyryl (mb) linkage. METHOD OF STUDY: We developed a sandwich enzyme-linked immunoabsorbent assay recognizing both GnRH analog and cytotoxin to ensure the conjugate measured was intact. Anti-D-Leu(6)-GnRH was used for capture and anti-pokeweed antiviral protein (anti-PAP) or anti-RNase for quantification. Specificity was verified by lack of reactivity with ovine FSH and LH, PAP, RNase, and D-Lys(6)-GnRH. RESULTS: Conjugates prepared using disulfide linkages were not stable in serum in vitro (half-lives <10 min), whereas mb conjugates had half-lives >2 hr. Clearance of mbGnRH-PAP from the circulation of sheep was rapid (t(1/2) <20 min). CONCLUSION: The assays were found to be specific, sensitive and accurate for measurement of GnRH-toxin conjugates in vitro and in vivo.