Reanalysis of the involvement of gamma-glutamyl transpeptidase in the cell activation process.

The inhibitor of gamma-glutamyl transpeptidase (gamma-GT) acivicin modulates cellular responses including growth, myeloid maturation and apoptosis. Whether these effects result from the inhibition of gamma-GT enzyme activity remains unclear. We compared the cellular effects of acivicin against a more potent and specific inhibitor of gamma-GT (L-2-amino-4-boronobutanoic acid (L-ABBA)) in gamma-GT-negative (B lymphoblastoid Ramos) and gamma-GT-positive (myelomonocytic HL-60, gamma-GT-transfected Ramos) cell lines. Under non-oxidative stress conditions, acivicin-induced cell growth arrest, apoptosis and macrophage maturation occurred independent of gamma-GT while L-ABBA did not influence any of these processes. Acivicin triggered tyrosine phosphorylation and increased nuclear factor kappaB activity. Further insight into the role of gamma-GT in cellular processes is needed.

A new acivicin prodrug designed for tumor-targeted delivery.

Acivicin is an antitumor agent known to inhibit cell growth. A new prodrug 9b of acivicin 10 was synthesized, based on a p-hydroxybenzylcarbamate self-immolative spacer capable to release acivicin under esterase activity. The prodrug includes a maleimide-containing arm for linkage with thiol-containing macromolecules such as antibodies. This molecule is intended for the conception of bioconjugates to target an inactive acivicin precursor to tumor cells, when linked to a monoclonal antibody (mAb) which recognizes a tumor-specific antigen. Prodrug cleavage by plasmatic esterases will then restore the acivicin's activity toward tumor cells. We report here the synthesis and the in vitro characteristics of the prodrug. As expected, its inhibitory activity against the gamma-glutamyl transpeptidase (gamma-GT) enzyme and its cytotoxicity towards HL-60 cells were highly reduced compared to the parent drug. The chemical and plasmatic hydrolysis kinetics of the compound was studied by HPLC. The prodrug is stable, being slowly hydrolyzed in pH 7.6 buffer at 37 degrees C with a half-life of 37 h. It is converted into an active acivicin under the effect of pig liver esterase, and its half-life in human plasma is 3 h. These results indicate this compound may be further used as a prodrug-antibody conjugate, to target acivicin to malignant cells.

Transmembrane proteases as disease markers and targets for therapy.

Transmembrane proteases (i.e. membrane-associated proteases, ectoproteases) are present in a wide variety of tissues and cell types including endothelial, epithelial and hematopoietic cells. Natural and synthetic inhibitors have been characterized and have revealed that certain ectoenzymes are able to modulate bioactive peptide responses and to influence major biological events such as cell proliferation, survival and invasiveness. Dysregulated expression of some of them in human diseases triggers research on their role in pathophysiology, on their value as disease markers and as putative targets for therapy.

Targeting of acivicin prodrugs as antibody conjugates.

The ectopeptidase gamma-glutamyltranspeptidase (gamma-GT) is overexpressed in myeloid leukemias. Its specific inhibitor, acivicin, was previously shown to induce an inhibitory growth effect associated with an induction of morphological features characteristic of macrophage maturation. We have considered a construction in which an antibody linked to a prodrug of acivicin will target acivicin to tumoral cells. In a first set of experiments we have synthesized a chromogenic model of this prodrug to validate this concept of prodrug, allowing an amine function to be released upon esterase action. Thereafter this model was applied to acivicin. The acivicin prodrug is inactive toward purified gamma-GT, and recovers its inhibitory activity under the effect of esterase.

Ectopeptidases in pathophysiology.

Ectopeptidases are transmembrane proteins present in a wide variety of tissues and cell types. Dysregulated expression of certain ectopeptidases in human malignancies suggests their value as clinical markers. Ectopeptidase interaction with agonistic antibodies or their inhibitors has revealed that these ectoenzymes are able to modulate bioactive peptide responses and to influence growth, apoptosis and differentiation, as well as adhesion and motility, all functions involved in normal and tumoral processes. There is evidence that ectopeptidase-mediated signal transduction frequently involves tyrosine phosphorylation. Combined analyses of gene organization and regulation of ectopeptidases by various physiological factors have provided insights into their structure-function relationships. Understanding the roles of ectopeptidases in pathophysiology may have implications in considering them as therapeutic targets.

Effects of estrogen and dexamethasone on a transgenic pituitary cell line. Regulation of hormone and chromogranin/secretogranin expression.

BACKGROUND: Recent studies with the growth hormone releasing hormone (GHRH) transgenic mouse model have shown that growth hormone (GH) and prolactin (PRL) cell hyperplasia and adenomas develop in a time-dependent manner after chronic stimulation by GHRH. However, the adenomatous foci have not been shown to be neoplastic with the ability to proliferate in vitro in the absence of GHRH stimulation. EXPERIMENTAL DESIGN: A cell line was established from an enlarged pituitary from a GHRH transgenic mouse. The cells proliferated readily in culture and were characterized with respect to PRL and GH production and response to estradiol and dexamethasone. The production of chromogranin/secretogranin (Cg/Sg) mRNA transcripts and the regulation of Cg/Sg expression was also analyzed in the newly established cell line to analyze the relationship between PRL, GH, and Cg/Sg production by this cell line. RESULTS: The tumor cells responded to 10(-7) M 17 beta-estradiol (estradiol) by increasing the percentage of immunoreactive PRL-positive cells, and to dexamethasone by decreasing the percentage of PRL-positive cells and mRNA levels. Dexamethasone (10(-7) M) treatment resulted in a 3-fold reduction in PRL mRNA and CgB mRNA, whereas GH and Sg II mRNAs were both increased after dexamethasone treatment. CgA mRNA level was not changed significantly by estradiol or dexamethasone in this cell line. CONCLUSIONS: This stable transgenic cell line is regulated by estradiol and dexamethasone with changes in PRL, GH, and Cg/Sg mRNA transcript levels. There is concordant regulation of PRL and CgB mRNAs as well as GH and SgII mRNAs. These findings indicate that this cell line can be used to study the regulation and possible functions of Cg/Sg in vitro.