Recent developments in studies on biological functions of vitamin A in normal and transformed tissues

Abstract A biochemical pathway of phosphorylation and glycosylation of vitamin A has recently been found in hepatic, intestinal and epidermal tissues. More recent work suggests that mannosylretinylphosphate functions as a donor of mannose to membrane glycoconjugates. These reactions might ultimately explain the effects of vitamin A deficiency and some of the effects of excess vitamin A on biological systems. Studies of the effect of retinoids on cellular in vitro systems showed an increase in the adhesive properties of spontaneously-transformed mouse fibroblasts in culture (Balb/c 3T12–3 cells). These cells are usually detached from the culture dish surface in an EDTA adhesion assay. After culturing in presence of 3.3 x 10-6 to 3.3 x 10-5M retinol or retinoic acid the cells are no longer lifted from the plate and their morphology and adhesion resemble those of normal fibroblasts. This phenomenon of increased adhesion is observed as early as two days after exposure to the retinoid and it is readily reversible upon culturing in medium without exogenous retinoid. A variety of retinoids was tested in the adhesion assay. The most active compounds were retinol, retinyl-phosphate, retinoic acid, 5,6-epoxyretinoic acid and the TMMP and DACP derivatives of retinoic acid. All these compounds possess biological activity in other systems. Anhydroretinol, perhydromonoeneretinol, the phenyl derivative of retinoic acid, which do not have biological activity in other systems, did not increase adhesion of 3T12 cells. Other polyprenoid compounds without vitamin A activity were also tested in this assay. Dolichol, dolichylphosphate juvenile hormone, abscisic acid, β-ionone, dibutyryl cyclic adenosine monophosphate and sodium butyrate did not induce adhesion. The mechanism by which retinol and retinoic acid increase the adhesive properties of 3T12 cells was investigated. Cyclic adenosine monophosphate and guanosine monophosphate levels were not significantly altered by retinoid treatment at least at 6, 24, 48 and 72 hours after treatment with 3.3 x 10-5M retinoic acid, when most of the cells remain attached. Retinoic acid stimulated the incorporation of (2-3H) mannose into glycoproteins of 3T12 cells. (11, 123H and carboxyl-14C)Retinoic acid was incorporated into a compound (Metabolite I) which had chromatographic properties of a glycosylretinylphosphate. The synthesis of this compound was time-dependent and was not carried out by formalin -fixed 3T12 cells. Mild alkaline conditions which release anhydroretinol from retinylphosphate, also cleaved Metabolite I to yield a product with the polarity of a hydrocarbon, but slightly more polar than anhydroretinol. It is suggested that retinoic acid can be reduced to an alcohol, probably after metabolic modification. It is further suggested that such “retinol-like” compound would follow the same route of phosphorylation and glycosylation as shown for retinol in other systems. Microsomes from 3T12 cells were active as the intact cells in synthesizing mannosylretinylphosphate and dolichyl mannosylphosphate. Exogenous retinylphosphate specifically stimulated the synthesis of mannosylretinylphosphate. Thus it appears that vitamin A is involved in glycosyl transfer reactions in the 3T12 system, as well as in normal membranes. It remains to be established whether the observed increased adhesion is the result of such involvement. A novel reaction for retinol was found in 3T12 cells. Up to 55% of exogenously supplied retinol was converted to the hydrocarbon anhydroretinol in 48 hours. The same reaction was also carried out by microsomes from 3T12 cells, which converted 7% of retinol to anhydroretinol in 30 minutes at 37°C. This reaction may well represent a detoxification mechanism for the transformed cell.