Luigi M. De Luca

Retinoids in chemoprevention and differentiation therapy

Retinoids are essential for the maintenance of epithelial differentiation. As such, they play a fundamental role in chemoprevention of epithelial carcinogenesis and in differentiation therapy. Physiological retinoic acid is obtained through two oxidation steps from dietary retinol, i.e. retinol-->retinal-->retinoic acid. The latter retinal-->retinoic acid step is irreversible and eventually marks disposal of this essential nutrient, through cytochrome P450-dependent oxidative steps. Mutant mice deficient in aryl hydrocarbon receptor (AHR) accumulate retinyl palmitate, retinol and retinoic acid. This suggests a direct connection between the AHR and retinoid homeostasis. Retinoids control gene expression through the nuclear retinoic acid receptors (RARs) alpha, beta and gamma and 9-cis-retinoic acid receptors alpha, beta and gamma, which bind with high affinity the natural ligands all-trans-retinoic acid and 9-cis-retinoic acid, respectively. Retinoids are effective chemopreventive agents against skin, head and neck, breast, liver and other forms of cancer. Differentiation therapy of acute promyelocytic leukemia (APL) is based on the ability of retinoic acid to induce differentiation of leukemic promyelocytes. Patients with relapsed, retinoid-resistant APL are now being treated with arsenic oxide, which results in apoptosis of the leukemic cells. Interestingly, induction of differentiation in promyelocytes and consequent remission of APL following retinoid therapy depends on expression of a chimeric PML-RAR alpha fusion protein resulting from a t(15;17) chromosomal translocation. This protein functions as a dominant negative against the function of both PML and RARs and its overexpression is able to recreate the phenotypes of the disease in transgenic mice. The development of new, more effective and less toxic retinoids, alone or in combination with other drugs, may provide additional avenues for cancer chemoprevention and differentiation therapy.

The Direct Involvement of Vitamin A in Glycosyl Transfer Reactions of Mammalian Membranes

Publisher Summary This chapter discusses the direct involvement of vitamin A in glycosyl transfer reactions of mammalian membranes. The path to the understanding of the molecular mechanism of action of vitamin A, outside vision, has proved very difficult. The most commonly used approach has been that of trying to gain some insight into the molecular mechanism by studying biochemical changes that result from depletion of this essential nutrient, in the hope of finding a metabolic process that is affected profoundly and directly by the deficiency of the vitamin. The main difficulty in this approach is the very arduous task of singling out a biochemical change that is a direct consequence of vitamin A deficiency; a variety of enzymic reactions are, in fact, affected in the vitamin A-depleted animal, in which infection ensues and death occurs relatively soon after the onset of deficiency. Thus, what originally may have seemed to be a good lead often turns into a labyrinthine confusion with a variety of effects, all of which are precipitated by the status of vitamin A deficiency.

Biosynthetic studies on mannolipids and mannoproteins of normal and vitamin A-depleted hamster livers

The incorporation of [1-14C]mannose into hamster liver glycolipids and glycoproteins was studied in normal and vitamin A-depleted hamsters. Severly (25% weight loss) and mildly (no weight loss) deficient animals were compared to vitamin A-fed controls. The incorporation of [14C]mannose into glycolipids and glycoproteins decreased in mild and severe vitamin A deficiency by 63-90% compared to vitamin A-fed animals. These results were essentially the same whether expressed per g of wet liver, per DNA or per protein. The size of the pools of mannose, glucose and galactose and their specific radioactivity in liver were determined by gas-liquid chromatography of the boronates of the hexitols (Eisenberg, Jr, F. (1972) Methods Enzymol. XXVIIIB, 168-178) in normal and vitamin A-deficient conditions. It was found that the amount of free hexoses per g of liver was very similar in normal and vitamin A-deficient conditions. The specific radioactivities for mannose and glucose were greater in vitamin A deficiency, thus excluding the possibility that the observed severe decrease in glycopeptide and glycolipid synthesis is a reflection of a similar decrease in the specific radioactivity of the precursor pools. Quantitation of mannose in glycoprotein showed a 79% decrease in vitamin A deficiency. Specific radioactivity of mannose in glycoproteins, 20 min after injection of the label, was 187 dpm/mug of mannose in the normal and 48 kpm/mug of mannose in the vitamin A-deficient livers. It is concluded that vitamin A is necessary for the biosynthesis of liver mannose-containing glycoproteins and glycolipids.

Involvement of all-trans-retinoic acid in the breakdown of retinoic acid receptors α and γ through proteasomes in MCF-7 human breast cancer cells

Most studies have reported an up-regulation of retinoic acid receptor (RAR) mRNA expression by all-trans retinoic acid (RA). We aimed to study the effect of RA on RAR protein levels in MCF-7 human breast cancer cells. Incubation of these cells with 10(-6) M RA induced a rapid breakdown of both RARalpha and RARgamma in spite of the accumulation of their mRNAs. Proteasome specific inhibitors blocked the RA-induced breakdown of RARs. Furthermore, RA enhanced the formation of the complex between RARalpha and ubiquitin in a concentration- and time-dependent manner, suggesting the involvement of ubiquitin and proteasome in this reaction. Retinoid X receptor alpha (RXRalpha) was also decreased, albeit to a lesser extent, in RA-treated cells. Use of synthetic receptor agonists and antagonists clearly showed that the effect of the retinoid on the breakdown of the retinoid receptors is receptor-ligand agonist-dependent and blunted by the antagonist. An electrophoretic mobility shift assay, using nuclear extracts from RA-treated cells, showed that a reduction in complex formation with hormone response elements correlated with the reduction of RAR and RXR protein. These data suggest that RA induces the breakdown of RARs through a process involving ubiquitination and that this phenomenon causes a reduction in the formation of DNA-receptor complexes.

Vitamin adeficiency and keratin biosynthesis in cultured hamster trachea

SummaryTracheas from vitamin A-deficient hamsters in organ culture in vitamin A-free medium developed squamous metaplasia. Addition of retinyl acetate to the medium prevented squamous metaplasia and a mucociliary epithelium was maintained. Indirect immunofluorescent staining with antikeratin antibodies AE1 and AE3 indicated positive reactions with epithelium of tracheas either cultured in vitamin A-free or retinyl acetate (RAc)-containing medium. The “stratum corneum”-like squames in metaplastic tracheas were strongly stained by AE3.Immunoprecipitation of cytoskeletal extracts from [35S]methionine labeled tracheas with a multivalent keratin antiserum indicated that the concentration of keratins synthesized in tracheas cultured in vitamin A-free medium was greater than that observed in tracheas cultured in the presence of RAc. In addition, new species of keratin were expressed in tracheas cultured in RAc-free medium. Alterations in the program of keratin synthesis were clearly detectable after 1 d in vitamin A-free medium, even though squamous metaplasia was not yet obvious. Squamous tracheas were shown by immunoblot analysis to contain keratins of 50, 48, 46.5, and 45 kilodalton (kd) detected with AE1; and 58, 56, and 52 kd detected with AE3. Immunoblot analysis with monospecific antimouse keratin sera also demonstrated the presence of 60, 55, and 50 kd keratins in the metaplastic tracheas. All these various species of keratins were either absent or present in much reduced quantity in mucociliary tracheas in RAc-containing medium. Interestingly, the induction of squamous metaplasia in tracheal epithelium did not result in the expression of the 59 and 67 kd keratins which are characteristically expressed in the differentiated layers of the epidermis.Therefore, this study shows that squamous metaplasia of tracheas due to vitamin A-free cultivation is accompanied by an increase in keratin synthesis as well as by the appearance of keratin species not normally present in mucociliary tracheal epithelium.

Human breast cancer MDA-MB-231 cells fail to express the neurofibromin protein, lack its type I mRNA isoform and show accumulation of P-MAPK and activated Ras.

Neurofibromin is a tumor suppressor protein, which is similar in function to the GTPase activating protein (GAP), p120GAP, in that it accelerates inactivation of Ras. Mutations in the NF1 gene cause neurofibromatosis type 1, NF1, an autosomal dominant disease with a diverse spectrum of clinical manifestations, including neurofibromas. Ras activation (GTP binding) is induced by the GTP exchange factor Sos and its inactivation is regulated through the GAPs (p120GAP and neurofibromin). Strikingly, neurofibromin was nearly absent in MB-231 human breast cancer cells and present in the remaining four cell lines studied, with higher levels in BT-474 and MB-453 than in MCF-7 and BT-20 cells, as tested with polyclonal antibodies to both the N-terminal as well as the C-terminal peptides. Coordinated with the near absence of neurofibromin, these cells also presented with much greater levels of P-MAPK and activated Ras. Further, RT-PCR analysis demonstrated the absence of expression of NF1 mRNA type I isoform only in the MB-231 cell lines. This result documents for the first time an altered NF1 expression at the protein and mRNA levels in MDA-MB-231 breast cancer cells.

Effects of retinoic acid on the growth and morphology of hamster tracheal epithelial cells in primary culture

SummaryHamster tracheal epithelial cells were grown in primary culture on a collagen gel substrate in hormone-supplemented serum-free Ham’s F12 medium with 10-8 M retinoic acid (RA +), or without retinoic acid (RA -). On days 1 and 2, the colonies were composed of large (secretory) cells and lesser numbers of small (basal) cells; ciliated cells were rare. At these times, cell number, thymidine incorporation, and total labelling indices (small and large cells, combined) were similar in RA+ and RA-cultures, but the large cells became flat in RA-medium on day 2. On days 3–5, thymidine incorporation and total labelling indices were less in RA-than RA+ cultures, and on days 4–6, cell numbers were decreased in RA-cultures. On day 3, the large cells of the RA-colonies had flattened further and clusters of small basal cells had formed. On day 4, the RA+ colonies were composed of densely-packed cuboidal secretory cells, small basal cells were inconspicuous; the total labelling index was about 27%. The RA-colonies were composed of large flat secretory cells and numerous small basal cells which were clustered in groups; the total labelling index was about 7%. Since large and small cells could be discriminated by size in RA-colonies, a labelling index was generated based on cell size. On days 2, 3 and 4, the labelling index of the small basal cells in the RA-colonies was 44%, 43% and 24% respectively, whereas the labelling index of the large secretory cells fell rapidly over the same period (56%, 14% and 2%). On days 5 and 6, the cuboidal secretory cells in the RA + cultures had differentiated further and the cells were stratified focally. Some new ciliated cells had formed on day 6. In RA cultures, mucous granules were not observed in the large flat cells and ciliated cells were not seen. The total labelling indices were 11% and 0.35% in RA+ cultures, and 0.5% and 0.25% in RA-cultures on days 5 and 6, respectively.The study shows that the target cell for vitamin A in the hamster tracheal epithelium is the secretory (mucous) cell. When retinoic acid was deficient, the secretory cells flattened and their capacity to divide was greatly diminished. Since the basal cells continued to replicate when the secretory cells did not, the population density of the basal cells increased disproportionally, which could be interpreted erroneously as a “basal cell hyperplasia.” Real hyperplasia and epidermoid metaplasia were late secondary events which occurred in this study following focal disintegration of the epithelial cell sheet. Then the large secretory cells became keratinized and expressed an epidermoid phenotype.

Reverse phase high-pressure liquid chromatographic separation of retinoids, including retinylphosphate and mannosylretinylphosphate

Abstract Reverse phase hplc was used to separate retinylphosphate mannosylretinylphosphate from other retinoids. These phosphorylated retinoids constituted 12 and 42% of the extracted radioactivity in OEL mouse intestine at 3 and 24 h respectively after the intubation of [1- 3 H]retinol. A detailed procedure for the chemical synthesis of retinylphosphate is described.

Retinoic Acid Down-regulation of Fibronectin and Retinoic Acid Receptor Proteins in NIH-3T3 Cells BLOCK OF THIS RESPONSE BY ras TRANSFORMATION

All-trans-retinoic acid (RA) markedly reduced the level of intracellular fibronectin (FN) in a time- and concentration-dependent fashion in NIH-3T3 cells, but not in NIH-3T3 cells transformed by an activated Ha-ras oncogene. Pulse/chase experiments indicated that RA affects FN biosynthesis rather than its turnover rate. Steady state levels of FN transcripts did not change after treatment of the cells with RA for various times or concentrations, suggesting that RA acts at the translational level. Similar effects were observed in other fibroblasts. In NIH-3T3 cells, RA had distinct effects on different receptors; it down-modulated retinoic acid receptor (RAR) α protein and transcript levels, it up-regulated RARβ transcripts, and it had no effect on RAR. Transformation of NIH-3T3 cells with an activated Ha-ras oncogene down-modulated RAR expression and abolished responsiveness to RA. We identified the retinoid signal transduction pathways responsible for the effects of RA on FN and RARα proteins by the use of the retinoid X receptor-selective compound, SR11237, by stable overexpression of a truncated form of the RARα gene, RARα403, with strong RAR dominant negative activity, and by overexpression of RARα. We conclude that: 1) RA-dependent FN down-modulation is mediated by RARs, 2) retinoid X receptors mediate the observed reduction of RARα by RA, and 3) the block of RA responsiveness in Ha-ras cells cannot be overcome by overexpression of RARα. These studies have defined fibronectin and RARα as targets of RA in fibroblast cells and have shown that oncogenic transformation renders the cells resistant to RA action.

Reversal of liver fibrosis in aryl hydrocarbon receptor null mice by dietary vitamin A depletion

Aryl hydrocarbon receptor (AHR)‐null mice display a liver fibrosis phenotype that is associated with a concomitant increase in liver retinoid concentration, tissue transglutaminase type II (TGaseII) activity, transforming growth factorβ (TGFβ) overexpression, and accumulation of collagen. To test the hypothesis that this phenotype might be triggered by the observed increase in liver retinoid content, we induced the condition of retinoid depletion by feeding AHR‐null mice a vitamin A‐ deficient diet with the purpose to reverse the phenotype. Liver retinoid content decreased sharply within the first few weeks on the retinoid‐deficient diet. Analysis of TGFβ1, TGFβ2, and TGFβ3 expression revealed a reduction to control levels in the AHR ‐/‐ mice accompanied by parallel changes in TGaseII protein levels. In addition, we observed an increase in the TGFβ receptors, TGFβ RI and TGFβ RII, as well as in Smad4, and their reduction to wild‐type mouse liver levels in AHR ‐/‐ mice fed the retinoid‐deficient diet. Reduction of peroxisomal proliferator‐activated receptor γ (PPARγ) messenger RNA (mRNA) and protein levels in AHR ‐/‐ mice was consistent with the presence of hepatic stellate cell (HSC) activation and liver fibrosis. Vitamin A deficiency normalized PPARγ expression in AHR ‐/‐ mice. In conclusion, livers from AHR ‐/‐ mice fed the vitamin A‐deficient diet showed a decrease in collagen deposition, consistent with the absence of liver fibrosis. (HEPATOLOGY 2004;39:157–166.)