Identification and characterization of nuclear retinoic acid-binding activity in human myeloblastic leukemia HL-60 cells.

Abstract

Specific [3H]retinoic acid (RA)-binding sites in nuclear and cytosolic extracts prepared from human myeloblastic leukemia HL-60 cells have been detected by sucrose density gradient sedimentation and size-exclusion highperformance liquid chromatography (HPLC) analyses. This RA-binding activity migrated as a single peak with an apparent molecular weight of 50,000 and >95% of the total binding activity was associated with the nuclear extract. Nuclear extracts prepared from COS-1 cells transfected with an expression vector for the nuclear RA receptors RARa or RARE were enriched (20-to 100-fold) with a RA-binding activity that coeluted by size-exclusion HPLC with the putative RAR from HL-60 cells. The HL-60 nuclear receptor exhibited high affinity binding of RA and its benzoic acid analogs ChS5, Ch3O, Ro 13-7410, and SRI 6409-40 and low-affinity binding of retinol, Ro 8-8717, and SRI 5442-60, correlating well with the biological activity of these compounds in HL-60 cells. Saturation binding and Scatchard plot analyses of the binding of RA to the nuclear HL-60 receptor yielded an apparent dissociation constant of -0.46 nM and 1400 ± 100 receptor sites per cell. Northern blot analyses of poly(A)+ RNA with cDNA probes specific for RARa and RARf indicated that HL-60 cells contain predominantly transcripts encoded by the RARa gene. Our results suggest that the observed nuclear RA-binding activity in HL-60 cells might mediate the action of RA in these cells.Retinoids, analogs of vitamin A, are modulators of cellular proliferation and differentiation in many cell types in vitro as well as in vivo (1-3). In human myeloblastic leukemia HL-60 cells, precursor cells that are able to undergo either myeloid or monocytic differentiation (4-8), retinoids cause terminal differentiation into morphologically mature granulocytes (9-12). This induction of differentiation is accompanied by several molecular changes, including an increase in the expression of NAD glycohydrolase (13) and transglutaminase type II activity (14) and a reduction in the levels of the protooncogene c-myc (15, 16).The mechanism by which retinoids induce differentiation in HL-60 cells remains to be established. Several studies have demonstrated that these cells do not contain detectable levels of either cellular retinol (CRBP)-or cellular retinoic acid (CRABP)-binding proteins (refs. 17,18; A.M.J. and T. R. Breitman, unpublished observations). Additional studies with a specific series of benzoic acid analogs of RA, the Ch series, showed that these retinoids, which do not bind to CRABP or CRBP, are very effective inducers of differentiation in HL-60 cells (19-21). These observations indicate that CRBP and CRABP are not involved in the mechanism of action of retinoids in HL-60 cells. It was speculated that other, high affinity receptors are involved in this signal transduction of retinoids (21,22). Recently, cDNAs of two nuclear RA receptors, RARa and RARP3, were cloned and sequenced (23)(24)(25)(26). These RARs are part of a larger gene family of ligand-re...

Molecular Characterization of Peptidylarginine Deiminase in HL-60 Cells Induced by Retinoic Acid and 1α,25-Dihydroxyvitamin D3

“…Since both PAD mRNAs and proteins were simultaneously detectable during HL-60 cell differentiation, the expression of PAD is regulated at a transcriptional level. HL-60 cells have RA and D 3 receptors (36,37). It is still unknown whether these receptors can activate PAD gene expression during HL-60 cell differentiation.…”

Retinoid-induced G1 Arrest and Differentiation Activation Are Associated with a Switch to Cyclin-dependent Kinase-activating Kinase Hypophosphorylation of Retinoic Acid Receptor α

“…RAR␣ belongs to the superfamily of nuclear ligand-activated transcriptional regulators, the retinoic acid receptors. RAR␣ is a phosphoprotein (23)(24)(25)(26) and mediates the action of retinoids in myeloid differentiation (27)(28)(29). In HL60 leukemic cells, the all-trans retinoic acid (ATRA)-induced differentiation is mediated directly through the RAR␣ (30,31).…”

Induction of adipose conversion in 3T3‐L1 cells is associated with an early phosphorylation of a protein partly homologous with mouse vimentin

“…4). The 60 kDa protein could not be extracted from the nuclei-rich fraction by standard procedures for preparing nuclear extracts using extraction media described by Dignam et al or Nervi et al (containing 0.42 or 0.80 M NaCl, respectively) [7,8]. It was however fully recovered in the non-extractable nuclear residue.…”