Determination of apo and holo retinoic acid-binding protein levels in retinoid-responsive transformed cells by high-performance size-exclusion chromatography

Abstract

A method to measure the endogenous levels of apo and holo cellular retinoic acid-binding proteins was developed using calf testis cytosol as the source of retinoic acid-binding protein. [ 3H]Retinoic acid-retinoic acid-binding protein complexes were assayed by high-performance size-exclusion chromatography. Preincubation of cytosol with 10 m m p-hydroxymercuribenzoate at 4 °C resulted in complete inhibition of retinoic acid binding to apo retinoic acid-binding protein. In addition, total dissociation of preformed holo retinoic acid-binding protein complexes was noted within 20 min after mercurial addition. Thus, p-hydroxymercuribenzoate converted the total pool of cellular retinoic acid-binding protein (apo plus holo) to mercurial-protein complexes unable to bind retinoic acid in vitro. Mercurial inhibition of retinoic acid-retinoic acid-binding protein complex formation was totally reversed upon the addition of 50 m m dithiothreitol. Total cytosolic retinoic acid-binding protein was determined from specific retinoic acid binding after treatment with p-hydroxymercuribenzoate and dithiothreitol. Apo cellular retinoic acid-binding protein concentration was measured by determining specific radioligand binding prior to p-hydroxymercuribenzoate treatment, and correcting for exchange of endogenously bound retinoid with exogenous tritiated retinoic acid. Holo cellular retinoic acid-binding protein concentration was derived from the difference between total and apo retinoic acid-binding protein concentrations. Using this method, we have demonstrated that retinoid-responsive EJ and T24 human bladder carcinoma cell lines and AT 3A and AT 3B rat pancreatic acinar carcinoma cell lines lack detectable levels of either apo or holo cellular retinoic acid-binding protein. These results established that retinoid inhibition of transformed bladder and acinar cell proliferation in culture was mediated by a cellular retinoic acid-binding protein-independent mechanism.