Determination of apo and holo retinoic acid-binding protein levels in retinoid-responsive transformed cells by high-performance size-exclusion chromatography.
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 mM p-hydroxymercuribenzoate at 4 degrees 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 mM 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 AT3A and AT3B 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.
D000818:Animals; D001059:Apoproteins / Q000032:analysis*; D002352:Carrier Proteins / Q000032:analysis*; D002417:Cattle; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000187:drug effects; D002850:Chromatography, Gel / Q000379:methods; D003600:Cytosol / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D010190:Pancreatic Neoplasms / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D018168:Receptors, Retinoic Acid; D012176:Retinoids / Q000494:pharmacology; D013737:Testis / Q000378:metabolism; D001743:Urinary Bladder / Q000378:metabolism; D001749:Urinary Bladder Neoplasms / Q000378:metabolism
D001059:Apoproteins; D002352:Carrier Proteins; D018168:Receptors, Retinoic Acid; D012176:Retinoids