Brahma P. Sani

Retinoic acid: a binding protein in chick embryo metatarsal skin.

Summary A protein which binds retinoic acid and which sediments with an S 20,w value of 2.2 is present in extracts of metatarsal skin from 12- to 13-day-old chick embryos. Competition experiments with unlabeled retinoic acid, retinol, retinal, and a cyclopentene analog of retinoic acid demonstrate that a terminal carboxyl group is required for binding. Since retinoic acid and the cyclopentene analog have potent biological action in preventing squamous metaplasia of chick skin explants, this binding protein may be involved in their effect.

Localization of retinoic acid-binding protein in nuclei.

Abstract Retinoic acid-binding component has been detected in the nuclei of chick embryo skin. The physicochemical properties of this macromolecule are in agreement with the properties of the retinoic acid-binding protein isolated from tissue cytosol. Although no binding protein could be detected in normal colon or lung tissue, nuclei isolated from a transplantable colon tumor and Lewis lung carcinoma contained this protein.

Subacute toxicity of all-trans- and 13-cis-isomers of N-ethyl retinamide, N-2-hydroxyethyl retinamide, and N-4-hydroxyphenyl retinamide

The major limitation for continuous administration of natural retinoids for chemoprevention of cancer is their high toxicity; however, synthetic retinamides have the desirable quality of reduced toxicity while retaining most of the biological activity. We have presently evaluated the comparative toxicity of all-trans- and 13-cis-isomers of N-ethyl retinamide (ER), N-2-hydroxyethyl retinamide (HER), and N-4-hydroxyphenyl retinamide (HPR) in mice and rats after po and ip administration. The computed LD90, DL50, and LD10 values for combined sexes of mice following 21 daily doses of the above retinoids were determined. Identical doses of the same retinoid by ip administration produced more toxicity and deaths than by the po route. The 13-cis-isomers exhibited comparatively less toxicity than the corresponding all-trans-isomer. Based on the lethality data, all-trans-retinoic acid was most toxic followed by all-trans-HER greater than all-trans-HPR greater than all-trans-ER. Changes in clinical chemistry and hematological parameters associated with administration of the retinamides include a dose-dependent peripheral anemia evidenced by erythrocytopenia and decreased hemoglobin concentration and packed cell volume. Retinoid treatment also caused increased plasma alkaline phosphatase activity and decreased serum albumin levels. Histopathological changes associated with retinoid administration primarily included liver lesions as characterized by degeneration and enlargement of hepatocytes. The present studies indicate that synthetic retinoids are less toxic than the natural ones.

Retinoyl β-glucuronide: lack of binding to receptor proteins of retinoic acid as related to biological activity

Retinoid beta-glucuronides have emerged as biologically active, water-soluble, natural retinoids with relatively few toxic and teratogenic effects. The mechanism of action of these glucuronides in the control of epithelial differentiation, growth, and tumorigenesis is unknown. Since retinoyl beta-glucuronide (RAG) contains a free carboxyl group, we studied the interactions of RAG with cellular retinoic acid-binding protein (CRABP) and nuclear receptors of retinoic acid (RARs), the possible mediators of the biological action of retinoic acid (RA). RAG did not exhibit any significant affinity to bind either CRABP or RARs. During 24- and 48-hr incubations of RAG in chick cytosol, detectable amounts of RA were generated which interacted with the RA receptors. In chick skin, the biological activity of RAG may be due to this slowly released RA. Other possible modes of action of RAG are suggested.

ISOLATION, PARTIAL PURIFICATION AND CHARACTERIZATION OF NUCLEAR RETINOIC ACID RECEPTORS FROM CHICK SKIN

Nuclear receptors (RARs) for retinoic acid (RA) are considered to be the ultimate mediators of the action of RA in the control of cell differentiation and inhibition of tumorigenesis. We have isolated and partially purified and characterized RAR from a RA-responsive tissue, chick embryo skin. The purification steps included Affi-Gel blue chromatography, ultrafiltration, size exclusion chromatography, and preparative isoelectric focusing. The electrofocusing of RAR-[3H]RA complex in ampholines (pH 3-10) revealed that the receptors have an isoelectric pH of 7.5. Whereas pronase-digested the RAR-[3H]RA complex completely, DNase showed 20-35% and RNase showed negligible digestive action on the complex. The ligand binding to RAR was completely inhibited by a mercury compound. RAR-alpha- and RAR-beta-specific antibodies, on Western blot analysis, immunoreacted with a protein having a molecular weight of 50,000, presumably RAR. Binding affinity studies revealed that biologically active analogs of RA with a free COOH group (e.g., 13-cis-RA, RO-13-7410, Ch 55, and Am 80) showed, like RA, high binding affinity for RAR, whereas biologically ineffective analogs of RA (e.g., furyl and pyridyl) were poor binders. Other groups of retinoids, in which the COOH group was either lacking or blocked, did not bind to RAR whether or not they were biologically active.

Retinoic acid‐binding protein in experimental and human colon tumors

Retinoic acid‐binding protein is present in metastatic murine colon tumors as well as in Lewis lung tumors and in lungs and brains of mice bearing these tumors; however, this protein is below the limits of detection in weakly‐metastatic carcinomas and in normal lung, colon, or brains. These observations are interesting since they concern the possibility of measuring the binding protein levels of colon tumors in clinical specimens as biochemical markers in human malignancy. A total of thirty‐three human colon tumors and related materials were analyzed for the presence of the binding protein. The interfering serum albumin, which nonspecifically binds retinoic acid, was eliminated by affinity chromatography. Of the twenty colon, cecum, and rectum tumors analyzed, 80% contained the binding protein in detectable amounts, and 20% showed nondetectable or marginally detectable amounts. Twenty‐two percent of the human colon segments isolated from patients suspected for colon tumors contained the binding protein in readily detectable amounts, whereas 78% revealed nondetectable to marginally detectable amounts. The retinoic acid‐binding protein of human colon tumor shared the same ligand specificity, thiol functions in ligand‐binding, and sedimentation coefficient as the binding protein isolated from chick embryo skin. However, the human protein exhibited altered isoelectric pH.

Retinoic acid-binding protein: a plasma membrane component.

Abstract Soluble protein extracts from lyophilized plasma membranes prepared from chick embryo skin and transplantable murine carcinomas were found to contain a specific retinoic acid-binding component. This binding component showed high affinity for biologically active analogs of retinoic acid. The plasma membrane component exhibited similar physicochemical properties to those of the retinoic acid-binding protein described earlier in the cytosol. The protein exhibited mercurial-sensitive thiol functions in ligand binding; the mercurial-inhibition was reversed on treatment with thiol compounds. The plasma membrane binding component may be involved in the cellular uptake of retinoic acid.

A convenient procedure for purification of thymidylate synthase from L1210 cells.

Abstract Several laboratories have described procedures for purification of thymidylate synthase (TMP synthase) that utilize folates or folate analogs covalently attached to a matrix. The principle of separation is the formation of a ternary complex between dUMP, TMP synthase, and the bound ligand and the subsequent elution of the enzyme with buffers that do not contain dUMP. We have successfully used 10-formylfolic acid as the bound ligand for the purification of TMP synthase. As compared to other ligands that have been used, 10-formylfolic acid has the advantages that it can be easily synthesized, it is stable, and the enzyme is eluted as a sharp peak. Application of this procedure to L1210 leukemia cells gave 1765-fold purification of TMP synthase with a recovery of 39%. The native enzyme had a molecular weight of 78,000, which is about the same as that reported.

Purification and properties of retinol- and retinoic acid-binding proteins from a transplantable mouse colon tumor

Cellular retinol-binding protein and retinoic acid-binding protein, the possible mediators of the action of retinoids in epithelial differentiation and control of tumorigenesis, have been reproducibly purified from mouse colon tumor 26, and some of their properties were studied. The main steps of purification involved acid-precipitation, DEAE-Sephadex, CM-cellulose and Sephadex G-100 chromatography. About 2 mg of the binding proteins were isolated from 60 g tumor. The purified preparations showed only two protein bands on polyacrylamide gel electrophoresis. The two binding proteins were partially resolved by sedimentation equilibrium technique; but was completely separable by preparative electrophoresis in the presence of sodium dodecyl sulfate. The retinol- and retinoic acid-binding proteins are presumably monomers with molecular weights of 15,500 and 14,600, respectively, as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. On gel filtration however, both the binding proteins retarded to the same molecular size of 17,800. On preparative columns, both the proteins expressed the same isoelectric pH, 4.5. Both proteins of the tumor possessed functional thiol groups. The mercurial inhibition of the binding capacity of the proteins for their ligands was reversible upon treatment with thiol compounds.

The presence of binding proteins for retinoic acid and dihydrotestosterone in murine and human colon tumors

Retinoic acid‐binding protein (RABP), which is distinctly present in embryonic colon and lung, is below the limits of detection in adult mouse colon and lung. The binding protein is present in malignant murine colon tumors as well as in lungs of animals bearing subcutaneously implanted tumors. Primary cell cultures from 1 g of colon tumor 26 gave rise to about 107 tumor cells and yielded 30 mg of extractable protein. The lower limit for detection of RABP, based on the appearance of its specific 2S peak after sucrose density gradient sedimentation, was 0.1 mg of protein, which corresponds to 3.3 × 104 tumor cells. After subcutaneous implantation of colon tumor 26 in mice, no RABP peak was evident in the lung extracts up to the fourth day. From the fifth day onwards RABP appeared in lung extracts, possibly as a consequence of pulmonary metastasis. Fragments of mouse lungs containing the metastatic tumor foci were reimplanted subcutaneously and produced tumors that contained RABP at levels comparable to those in colon tumor 26. The primary subcutaneous tumors and pulmonary metastatic tumors showed the same histologic appearance—an undifferentiated carcinoma. On the 15th day of subcutaneous implantation of colon tumor 26 in mice, RABP was detected in lung and brain but in none of the other tissues where the protein is normally undetectable. After intraperitoneal implantation of colon tumor 26 in mice, no well‐defined RABP peaks were detected from their liver extracts. None of the three normal human colon extracts analyzed for RABP or a dihydrotestosterone‐binding protein (DHTBP) contained any detectable amounts of either of the binding proteins. However, 70% of the human colon tumors contained RABP and 90% contained DHTBP. Both of these binding proteins were evident in the two human colon tissues adjoining colon tumors.