Hyperacetylation enhances the growth-inhibitory effect of all-trans retinoic acid by the restoration of retinoic acid receptor β expression in head and neck squamous carcinoma (HNSCC) cells

all-trans-Retinoic acid, one of the hormonally active derivatives of vitamin A, occurs physiologically in plasma at a concentration below 10 nmol/l. The methods currently used for its quantification are based on HPLC, need about 1 ml of serum, are relatively laborious and thus not well suited for mass analysis. The affinity and specificity of retinoic acid receptors for all-trans-retinoic acid encouraged us to express both the entire human retinoic acid receptor beta (RAR-beta) and two versions of its retinoic acid-binding domain in Escherichia coli in the hope that these recombinant proteins might be used as binders in a ligand-binding assay for all-trans-retinoic acid. The recombinant receptors, the whole receptor [RAR-beta-(V7-Q448)], corresponding to domains A-F, and the ligand-binding domain [RAR-beta-(E149-Q448)], corresponding to domains D-F, were expressed in the vector pET 3d/BL21 (DE3) as inclusion bodies, solubilized with guanidinium chloride, renatured and purified by ion-exchange chromatography. RAR-beta-(P193-Q448), corresponding to domains E-F, was expressed in the vector pET 3d/BL21(DE3)pLysS, and purified by reversed-phase chromatography. Under non-denaturing conditions, the expressed whole receptor [RAR-beta-(V7-Q448)] and the D-F construct (RAR-beta-(E149-Q448)] behaved chromatographically as monomeric proteins whereas the E-F construct [RAR-beta-(P193-Q448)] had a strong tendency to aggregate. RAR-beta-(V7-Q448) and RAR-beta-(E149-Q448) had similar Kd values for all-trans-retinoic acid (1.4 and 0.6 nmol/l respectively) whereas RAR-beta-(P193-Q448) bound all-trans-retinoic acid less avidly (Kd 9.6 nmol/l). 9-cis-Retinoic acid bound to RAR-beta-(E149-Q448) and RAR-beta-(V7-Q448) as avidly as all-trans-retinoic acid. Competition experiments showed weak or no binding of 4-oxo-all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid, 13-cis-retinoic acid, acitretin and retinol by RAR-beta-(E149-Q448).

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Retinoic Acid Prevents Phosphorylation of pRB in Normal Human B Lymphocytes: Regulation of Cyclin E, Cyclin A, and p21Cip1

Blood ◽

10.1182/blood.v94.4.1348.416k29_1348_1358 ◽

1999 ◽

Vol 94 (4) ◽

pp. 1348-1358 ◽

Cited By ~ 2

Author(s):

Soheil Naderi ◽

Heidi Kiil Blomhoff

Keyword(s):

Retinoic Acid ◽

B Lymphocytes ◽

Retinoic Acid Receptor ◽

Cyclin E ◽

Cyclin A ◽

Inhibitory Effect ◽

Dependent Manner ◽

Growth Inhibitory Effect ◽

Growth Inhibitory ◽

Normal Human

The mechanisms underlying the growth-inhibitory effect of retinoids on normal human B lymphocytes are not well understood. We addressed this issue by examining the effect of retinoic acid on the cell cycle machinery involved in G1/S transition. When retinoic acid was administered to B cells stimulated into mid to late G1 by anti-IgM antibodies (anti-μ) and Staphylococcus aureus crude cell suspension (SAC), the phosphorylation of pRB required for S-phase entry was prevented in a time- and dose-dependent manner. Thus, 2-hour treatment with retinoic acid at the optimal concentration of 1 μmol/L prevented phosphorylation of pRB, and effects were noted at concentrations as low as 10 nmol/L. Based on our results, we suggest that the rapid effect of retinoic acid on pRB phosphorylation is due primarily to the reduced expression of cyclin E and cyclin A in late G1. This could lead to the diminished cyclin E– and cyclin A–associated kinase activities noted as early as 2 hours after addition of retinoic acid. Furthermore, our results imply that the transient induction of p21Cip1 could also be involved. Thus, retinoic acid induced a rapid, but transient increased binding of p21Cip1 to CDK2. The retinoic acid receptor (RAR) agonist TTNPB mimicked the key events affected by retinoic acid, such as pRB phosphorylation, cyclin E expression, and expression of p21Cip1, whereas the RAR-selective antagonist Ro 41-5253 counteracted the effects of retinoic acid. This implies that retinoic acid mediates its growth-inhibitory effect on B lymphocytes via the nuclear receptors.

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