RETINOIC ACID DISRUPTS THE GOLGI-APPARATUS AND INCREASES THE CYTOSOLIC ROUTING OF SPECIFIC PROTEIN TOXINS

All-trans retinoic acid can specifically increase receptor mediated in toxication of ricin A chain immunotoxins more than 10,000 times, where as fluid phase endocytosis of ricin A chain alone or ricin A chain imm unotoxins was not influenced by retinoic acid. The immunotoxin activat ion by retinoic acid does not require RNA or protein synthesis and is not a consequence of increased receptor binding of the immunotoxin. Vi tamin D-3 and thyroid hormone T-3, that activate retinoic acid recepto r (RAR) cognates, forming heterodimers with retinoid X receptor (RXR), do not affect the potency of immunotoxins. Among other retinoids test ed, 13-cis retinoic acid, which binds neither RAR nor RXR, also increa ses the potency of the ricin A chain immunotoxin. Therefore, retinoic acid receptor activation does not appear to be necessary for immunotox in activity. Retinoic acid potentiation of immunotoxins is prevented b y brefeldin A (BFA) indicating that in the presence of retinoic acid, the immunotoxin is efficiently routed through the Golgi apparatus en r oute to the cytoplasm. Directly examining cells with a monoclonal anti body (Mab) against mannosidase II, a Golgi apparatus marker enzyme, de monstrates that the Golgi apparatus changes upon treatment with retino ic acid from a perinuclear network to a diffuse aggregate. Within 60 m in after removal of retinoic acid the cell reassembles the perinuclear Golgi network indistinguishable with that of normal control cells. C- 6-NBD-ceramide, a vital stain for the Golgi apparatus, shows that reti noic acid prevents the fluorescent staining of the Golgi apparatus and eliminates fluorescence of C-6-NBD-ceramide prestained Golgi apparatu s. Electron microscopy of retinoic acid-treated cells demonstrates the specific absence of any normal looking Golgi apparatus and a perinucl ear vacuolar structure very similar to that seen in monensin-treated c ells. This vacuolization disappears after removal of the retinoic acid and a perinuclear Golgi stacking reappears. These results indicate th at retinoic acid alters intracellular routing, probably through the Go lgi apparatus, potentiating immunotoxin activity independently of new gene expression. Retinoic acid appears to be a new reagent to manipula te the Golgi apparatus and intracellular traffic. As retinoic acid and immunotoxins are both in clinical trials for cancer therapy, their co mbined activity in vivo would be interesting to examine.