Retinoic acid treatment of fibroblasts causes a rapid decrease in [3H]inositol uptake

NIH 3T3 fibroblasts treated with all-trans-retinoic acid (RA) showed a dramatic decrease in the uptake of [3H]inositol compared to solvent-treated controls. The onset of RA-induced inhibition of [3H]inositol uptake was rapid with a 10-15% decrease occurring after 2-3 h of RA exposure and 60-70% reduction after 16 h of RA treatment. A progressive dose-dependent decrease in inositol uptake was found as the concentration of RA increased from 10(-8) to 10(-5) M and the effect was fully reversible within 48 h after RA removal. The Vmax and Kt for the controls were 10 nmol/2.5 x 10(6) cells/2 h and 51 microM; and for RA-treated cells the values were 4 nmol/2.5 x 10(6) cells/2 h and 52 microM. The decreased [3H]inositol uptake was not due to a change in the affinity (Kt) of the transporter for the inositol but to a decrease in the Vmax. The maximal effect on inositol uptake was dependent on RA treatment of the cells after they reached saturation density or if made quiescent by serum starvation. RA was the most active of the different retinoids examined in the order RA greater than 13-cis-RA = retinyl acetate greater than all-trans-retinol greater than 5,6-dihydroxyretinoic acid methyl ester greater than N-4-hydroxyphenyl retinamide. In contrast to this effect on inositol, the uptake of fucose, mannose, galactose, and glucose was either not affected or enhanced (for mannose and fucose) by RA treatment. RA inhibition of inositol uptake was also observed in 3T3-Swiss and Balb/3T3 cells but not in two virally transformed 3T3 cell lines. Phlorizin, amiloride, and monensin inhibited inositol uptake by 66, 74, and 58%, respectively, and this inhibition was additive when the cells were treated with RA as well as these inhibitors. A decreased incorporation of [3H]inositol into polyphosphoinositides was also observed in RA-treated cells but not to the same extent as for [3H]inositol uptake. In conclusion, RA treatment of 3T3 fibroblasts decreases the uptake of [3H]inositol by up to 70% within 8 to 10 h at near physiological concentrations in a reversible and specific manner.     

Effects of retinoic acid on the differentiation of THP-1 cell lines containing aneuploid or diploid chromosomes

The effects of retinoic acid on the differentiation of human monocytic leukemia cell lines containing aneuploid (THP-1-Cs5) or diploid chromosomes (THP-1-R) were studied and compared. The induction of cell adhesion to a substratum, phagocytosis of sheep red blood cells (SRBC) or IgG-coated SRBC, pinocytosis of dextran sulfate, and NBT dye reduction by the cells were examined. The occurrence of these processes was much greater in RA-treated THP-1-Cs5 cells than in RA-treated THP-1-R cells. Of all these functional activities, the most remarkable differences between the two cell types were seen for cell adhesion and phagocytosis of SRBC. Morphological changes in RA-treated THP-1-Cs5 cells were observed by light and electron microscopy. RA-treated THP-1-Cs5 cells had a moderately-developed Golgi apparatus, and abundant lysosomes, mitochondria and lipid droplets in the cytoplasm. Among various retinoids examined, RA was the strongest inducer of the differentiation of the THP-1-Cs5 cells into mature cells. These findings suggest that THP-1-Cs5 cells which contain aneuploid chromosomes are more efficiently functionally differentiated by RA than are THP-1-R cells.     

Down modulation of N-myc, heat-shock protein 70, and nucleolin during the differentiation of human neuroblastoma cells.

Cultured human neuroblastoma (GOTO) cells were induced to differentiate by dibutyryl cyclic AMP (Bt2cAMP) and/or retinoic acid (RA). A combination of Bt2cAMP (1 mM) and RA (1 microM) yielded the most significant networks of neurites after 3 to 4 days, this being associated with the reduction of N-myc mRNA levels. Next, we examined several cellular genes that were possibly linked with changes in N-myc gene expression under these conditions. Among the genes examined, both nucleolin and a major heat-shock protein (hsp70) mRNAs showed changes concomitant with those in N-myc mRNA levels when induced by Bt2cAMP and RA. Dibutyryl cAMP alone induced several short cellular processes and caused a marked decrease in N-myc mRNA within 2 days. RA alone induced a few long and straight neurites along the longitudinal axis of individual cells and a significant decrease in growth rate but showed neither network formation nor a decrease in N-myc gene expression. These results indicate differential effects of Bt2cAMP and RA on the regulatory mechanisms of both cell proliferation and differentiation and also indicate a possible association of expression of N-myc gene with those of hsp70 and nucleolin genes.     

Retinoic acid treated HL60 cells express CEACAM1 (CD66a) and phagocytose Neisseria gonorrhoeae

Neisseria gonorrhoeae (gonococci, GC) are phagocytosed by neutrophils through the interaction between opacity proteins (Opa) and the CEA (CD66) family of antigens. In order to study this interaction, we used the human myeloid leukemia HL60 cell line, which differentiates into granulocyte-like cells upon treatment with dimethylsulfoxide (DMSO) or retinoic acid (RA). We found that RA-, but not DMSO- or untreated-HL60 cells, can phagocytose OpaI-expressing gonococci as well as Escherichia coli. The interaction of OpaI E. coli with RA-treated HL60 cells was inhibited by antibodies against CEACAM1. Phagocytosis of OpaI E. coli was found to be a result of the expression of CEACAM1 in RA-treated HL60 cells. Our results indicate that the level of expression of CEACAM1 in HL60 cells can be regulated by treatment with RA in a differentiation-dependent manner, and that this is important for phagocytosis of OpaI-expressing gonococci or E. coli.     

Stimulation of sialyltransferase activity of melanoma cells by retinoic acid

Retinoic acid (RA) treatment of murine S91-C2 melanoma cells has been found to augment the activity of glycoprotein: sialyltransferase in a dose-dependent and time-dependent process. The enzymatic activity in cells treated with 10 microM RA reached a maximal level, 3-fold higher than in untreated cells, 72 h after initiation of treatment. In contrast, the addition of RA directly into the reaction mixture had no stimulatory effect on sialyltransferase. The endogenous glycoproteins to which sialic acid is transferred from cytidine monophosphate (CMP)-[14C] sialic acid by the action of sialyltransferase have been identified by fluorography after polyacrylamide gel electrophoresis. One of these acceptors, a glycoprotein of Mr 160 000, comigrated in gel electrophoresis with a cell surface sialoglycoprotein that can be labeled by the periodate-tritiated borohydrate procedure more intensely on intact RA-treated than on untreated cells. Removal of sialic acid residues exposed on the surface of either control or RA-treated cells enhanced 2- to 3-fold the transfer of sialic acid to endogenous acceptors. These results suggest that the increased sialyltransferase activity in RA-treated melanoma cells may be responsible for the enhanced sialylation of certain cell surface glycoproteins. RA treatment of several other tumor cell lines also resulted in stimulation of sialyltransferase activity indicating that this effect of RA is not limited to the S91-C2 melanoma cells.     

Calcium thresholds in the activation of DNA and RNA synthesis in cultured planarian cells: relationship with hormonal and DB cAMP effects

DNA and RNA syntheses were reduced to a basal level in dissociated planarian cells grown for 48 h in a Ca2+-free medium. These syntheses could be triggered anew by raising the Ca2+ concentration in the medium. Serotonin could be substituted for Ca2+ in stimulating DNA synthesis by Ca2+-depleted cells, while dopamine greatly enhanced RNA synthesis in these cells. When Ca2+ concentration was raised in hormone-treated cultures, DNA synthesis was again slightly increased but RNA synthesis was depressed. Both hormonal effects were completely inhibited by the anticalmodulin drug trifluoperazine. As serotonin and dopamine are both known to stimulate the adenylate cyclase system, it was further investigated whether the hormonal effects were mediated by cAMP. Indeed, a DB cAMP concentration of 1 microM increased DNA labelling when applied for 8 h to Ca2+-depleted cultures. However, when Ca2+ was present, the 8-h treatment with 1 microM DB cAMP was inhibiting. A 4-h pulse with 1 microM DB cAMP just after Ca2+ addition was a condition for a high stimulation of DNA labelling. The other DB cAMP concentrations used, 0.1 and 10 microM, reduced DNA labelling. In the absence of Ca2+, RNA labelling was only slightly increased by 0.1 microM DB cAMP, but was highly stimulated by a 4-h treatment of 1 microM DB cAMP in the presence of Ca2+. The noted effects with 1 or 0.1 microM DB cAMP on DNA or RNA labelling corresponded to true changes in synthesis rather than alterations of the specific activity of the nucleotide pool by DB cAMP. Besides, it was precluded that these effects were due to butyrate issued from DB cAMP degradation. It was further shown that DB cAMP at 1 microM increased Ca2+ uptake in planarian cells, whereas the other concentration reduced it. This observation might explain the stimulating effect on nucleic acid synthesis of 1 microM DB cAMP applied at the appropriate moment. Based on these results it seems that, for triggering RNA synthesis, the threshold value of Ca2+ was lower than for DNA synthesis. These Ca2+ thresholds might be reached, in the absence of Ca2+ in the medium, by treatments with DB cAMP or hormones at the appropriate doses and periods. This interpretation is in agreement with the succession of biochemical events described in regenerating planarians and suggests that these events might be causally related.     

Differential expression and biological activity of retinoic acid–induced TGFβ isoforms in embryonic palate mesenchymal cells

The effect of retinoic acid (RA) on TGF-β mRNA expression and protein production in murine embryonic palate mesenchymal (MEPM) cells was examined by Northern blotting and TGF-β bioassay in association with TGF-β isoform-specific neutralizing antibodies. Heat or acid activation was used to distinguish between latent and active TGF-β protein released into the culture medium. RA had little or no effect on TGF-β1 mRNA expression and protein production. In contrast, RA increased TGF-β2 and β3 protein released into the culture medium, the protein being mostly in an inactive or latent form. The amount of active TGF-β released was increased relative to the total increase in TGF-β released, suggesting that RA treatment stimulated activation of latent TGF-β. RA also increased TGF-β2 mRNA expression; we have previously shown that RA upregulates TGF-β3 mRNA in these cells. RA and TGF-β individually inhibited ³H-thymidine incorporation into MEPM cell DNA, while, when administered simultaneously, they inhibited proliferative activity to a greater extent. Heat- or acid-activated conditioned medium (CM) from MEPM cells treated with RA was able to inhibit ³H-thymidine incorporation into MEPM cell DNA to an extent greater than seen with RA treatment alone. Coincubation of heat-activated CM from RA-treated MEPM cells with pan-specific or TGF-β2 or β3-specific neutralizing antibodies partially relieved the inhibitory effect on ³H-thymidine incorporation, suggesting that this proliferative response was due to RA-induced TGF-β. Simultaneous treatment with RA and TGF-β also stimulated gycosaminoglycan (GAG) synthesis to an extent greater than that seen with TGF-β treatment alone, this despite the ability of RA to inhibit GAG synthesis. These data demonstrate a role for RA and RA-induced TGF-β in the regulation of palate cell proliferation and GAG synthesis and suggest a role for TGF-β in retinoid-induced cleft palate. J. Cell. Physiol. 177:36–46, 1998. © 1998 Wiley-Liss, Inc.     

Exogenous retinoic acid decreases in vivo and in vitro proliferative activity during the early migratory stage of neural crest cells

We have previously demonstrated that directional migration of neural crest cells (NCC) is associated with a high cell density, resulting from an active cell proliferation. It is also known that treatment with retinoic acid (RA) causes a dose-dependent inhibition of proliferation of some cell types, and that administration of RA during the early stages of embryonic development, induces cranio-facial abnormal patterns corresponding to NCC derivatives. In view of these findings, it was of interest to determine if exogenous RA is a potential modulator of the mitotic rate of NCC, and to explore the hypothesis of an inhibitory effect exerted by RA on the proliferative behaviour of NCC in vivo and in vitro. Homogenates of RA-treated chick embryos showed a low [³H]dT incorporation, indicating a generalized diminution of DNA synthesis. The labelling index (LI=number of labelled cells/total number of cells) revealed that NCC from RA-treated and control embryos had higher values of [³H]dT incorporation than neural tube cells (P < 0.0001). Autoradiographs of RA-treated chick embryos showed a significantly lower [³H]dT incorporation in NCC at the prosencephalic and mesencephalic levels, as well as in the neural tube cells at the prosencephalic, mesencephalic and rhombencephalic levels, than in control chick embryos (P < 0.0001). NCC cultures treated with 1 or 10 μm RA had a significantly lower LI than in cultures treated with 0.1 μm RA or control cultures (P < 0.04). In chick embryos, the mitotic index of NCC was 0.026 for RA-treated and 0.033 for controls, while the duration of the cell cycle was significantly longer in the NCC of RA-treated embryos (～ 40 h) than in controls (～ 25 h). The length of the cell cycle phases of NCC was similar in both experimental conditions, except for G₁ phase, which was significantly longer in the RA-treated group than in controls. These results show that RA blocks DNA synthesis and lengthens the proliferative behaviour of NCC both in early chick embryos and in vitro, effects that could modify the morphogenetic patterns of NCC distribution through a decreased cell population.     

Effects of growth medium and cyclic AMP analogues on the cAMP-induced differentiation of F9 teratocarcinoma cells

Summary F9 teratocarcinoma cells differentiate into parietal endodermlike cells when treated with retinoic acid (RA) and cyclic AMP (cAMP). We have previously found that F9 cells can be induced to differentiate by treatment with cAMP in the absence of RA. In the course of determining why other investigators had failed to observe cAMP-induced differentiation, we found that the growth medium played an important role in determining the response of F9 cells to differentiating agents. When F9 cells were grown in minimal essential medium (MEM) and treated with either 8-bromo-cAMP (8BrcA) + 1-methyl, 3-isobutylxanthine (MIX), or dibutyryl cAMP (DBcA) + theophylline (T), they differentiated to parietal endodermlike cells without any requirement for exogenous RA. However, when F9 cells were grown in Dulbecco’s modified Eagle’s medium (DME), DBcA/T failed to induce differentiation alone and required exogenous RA to induce formation of parietal endoderm-like cells. 8BrcA/MIX alone was still able to induce some differentiation, although the extent was not as great as those cells grown in MEM. These results could not be explained by the different growth-promoting properties of the two culture media because there was no difference in the doubling time of F9 cells grown in either medium. Likewise, RA and cAMP both inhibited growth to the same extent in either medium. Inasmuch as almost all published reports on F9 cell differentiation have used DME as a growth medium, and RA with or without DBcA/T as the differentiating agents, these studies would not have had the appropriate conditions to detect the cAMP-induced differentiation of F9 cells.     

Modulation of lysosomal-associated membrane glycoproteins during retinoic acid-induced embryonal carcinoma cell differentiation

Differentiation of the murine embryonal carcinoma (EC) cell lines F-9 and PC-13, induced by beta-all transretinoic acid (RA) resulted in an increased level of two lysosomal-associated membrane glycoproteins (LAMP-1 and LAMP-2). After differentiation, the levels of both LAMPs in the EC cells were comparable to those found in visceral and parietal endoderm cell lines (PSA-5E and PYS-2, respectively). RA treatment of the EC cells also resulted in an increase in the apparent Mr of both LAMPs apparently due to increased glycosylation because the deglycosylated LAMP-1 from undifferentiated and from differentiated cells had a similar electrophoretic migration. Indeed, the binding of 125I-labeled L-phytohemagglutinin (L-PHA) to glycoproteins with Mr or 90,000-130,000 increased after differentiation and about 24 times more 125I-labeled L-PHA bound to LAMP-1 isolated by immunoprecipitation from extracts of RA-treated F-9 cells than to LAMP-1 from undifferentiated cells. The increased level of the LAMPs was detected in F-9 cells treated with greater than 10(-7) M RA and required greater than 48 h of treatment as did the increased expression of the B1 chain of laminin, an established marker for differentiation in this system. LAMP-1- and L-PHA-reactive glycoproteins were localized by fluorescence techniques to intracellular vesicles, presumably lysosomes, and to the cell surface and both increased after RA treatment. LAMP-2 was barely detectable intracellularly in undifferentiated cells but could be detected clearly after differentiation. In contrast, no LAMP-2 could be detected on the cell surface either before or after differentiation of F-9 cells. The increased level and glycosylation of both LAMP-1 and LAMP-2 was observed also in cells treated with a synthetic chalcone carboxylic acid analog of RA and by combination of either retinoid with dibutyryl cyclic AMP. These results demonstrate that differentiation of EC cells is accompanied by changes in the synthesis and glycosylation of LAMP glycoproteins and that these changes are specific for the cell type that results after differentiation.     

Retinoic acid treatment enhances the acetylcholine contents in the human teratocarcinoma cell line NTera-2

Human NTera-2/clone D1 teratocarcinoma cells are induced by retinoic acid (RA) to differentiate into postmitotic cells with morphological and biochemical characteristics of embryonic human neurones. Currently only limited information concerning peptide-contents and neurotransmitter pools of these cells is available. Zeller and Strauss [Int. J. Dev. Neurosci. 1995;13(5):437] described an increase in choline acetyltransferase (ChAT) activity in RA-treated, but not in untreated NTera-2 cells, suggesting the induction of a cholinergic phenotype during treatment with RA. In the present study we investigated the effect of RA-differentiation on the amount of the neurotransmitters acetylcholine (ACh), and dopamine in NTera-2 in order to specify the transmitter phenotype induced by RA-differentiation. We found that a 4-week treatment of NTera-2 cells with 10 microM RA markedly increased the ACh-content of these cells, while dopamine levels were unchanged. Depolarisation with potassium (60 mM) enhanced ACh-outflow in the differentiated cells in a Ca(++) dependent way. Also neuropeptides like substance P and NPY were detectable in the undifferentiated NTera-2 cells, while vasointestinal peptide (VIP) could not be found in either precursor or RA-differentiated cells. Differentiation was accompanied by a marked reduction of neutral endopeptidase enzyme activity and aminopeptidase activity. From these observations it was concluded that RA induces a cholinergic neurochemical differentiation of this human teratocarcinoma cell line, and that these cells might provide a model system to investigate cholinergic properties of human origin.     

Differential regulation of keratin 8 and 18 messenger RNAs in differentiating F9 cells.

F9 embryonal carcinoma cells (F9EC) can be induced to differentiate in vitro into epithelial cells expressing keratin 8 (K8) and keratin 18 (K18). cDNAs corresponding to K8 and K18 mRNAs were cloned and used to study the change in the abundance of these mRNAs during differentiation of F9 cells into parietal endoderm-like cells by treatment with retinoic acid (RA) or with RA and dibutyryl cAMP (Bt2cAMP). Using an RNase protection assay, it was determined that K8 mRNA was induced slightly before K18 mRNA and that it accumulated to a greater extent than K18 mRNA. Furthermore, differentiation in presence of Bt2cAMP plus RA resulted in an earlier induction of the two mRNAs and a higher level of expression of K8 mRNA. These results indicate that K8 and K18 mRNAs are regulated differently in F9 cells.     

Differential regulation of keratin 8 and 18 messenger RNAs in differentiating F9 cells

F9 embryonal carcinoma cells (F9EC) can be induced to differentiate in vitro into epithelial cells expressing keratin 8 (K8) and keratin 18 (K18). cDNAs corresponding to K8 and K18 mRNAs were cloned and used to study the change in the abundance of these mRNAs during differentiation of F9 cells into parietal endoderm-like cells by treatment with retinoic acid (RA) or with RA and dibutyryl cAMP (Bt₂cAMP). Using an RNase protection assay, it was determined that K8 mRNA was induced slightly before K18 mRNA and that it accumulated to a greater extent than K18 mRNA. Furthermore, differentiation in presence of Bt₂cAMP plus RA resulted in an earlier induction of the two mRNAs and a higher level of expression of K8 mRNA. These results indicate that K8 and K18 mRNAs are regulated differently in F9 cells.     

Dexamethasone-induced changes in lung function are not prevented by concomitant treatment with retinoic acid

Alveolarization is impaired in rats treated with dexamethasone (Dex) on postnatal days 4-13, but concomitant treatment with all-trans retinoic acid (RA) increases alveolar number. To determine whether morphological changes induced by Dex and/or RA predict changes in lung function at 1 mo, we assessed resting breathing parameters, dynamic compliance, ventilation required to maintain O(2) saturation at > or = 90%, and pressure-volume curves of air-filled lungs. During resting breathing, mean tidal volume per gram was greater in Dex + RA-treated rats than in controls (P < 0.05). Dynamic compliance was also greater in Dex- and Dex + RA-treated rats than in controls or RA-treated rats (P < 0.02). In Dex- and Dex + RA-treated rats, we observed increased hysteresis ratios (P < or = 0.006), air trapping (P < 0.05), and lung volumes at 5 and 13.5 cmH(2)O pressure (P < 0.001) and decreased elastic recoil (P < 0.007). The effect of Dex on elastic recoil was greater in female than in male rats (P = 0.006). Despite impaired septation, O(2) saturation was not compromised in Dex- or Dex + RA-treated rats. Thus lung function changes induced by Dex treatment during alveolarization were not prevented by concomitant treatment with RA.     

Retinoic acid induces a specific membrane glycoprotein in human epithelial cell lines

Retinoic acid (RA) inhibits growth, increases the cytokeratin content, and alters the cytoskeleton of the human cervical cell line NHIK 3025. Using RA-treated NHIK 3025 cells as immunogen we prepared murine monoclonal antibodies (IgG1) which recognized an RA-induced cell-surface antigen which could not be detected in untreated NHIK 3025 cells. Analysis of the Triton soluble proteins by SDS-gel electrophoresis and immunoblotting revealed that the cell-surface antigen is a 140-kDa glycoprotein (gp140). gp140 was also shown to be induced by RA in HeLa S3 cells and constitutively expressed in the human trophoblast cell line BeWo. gp140 was also detected in other human epithelial cell lines, but not in human hematopoietic cells. Expression of gp140 was induced in HeLa S3 cells by nanomolar concentrations of RA, and in NHIK 3025 cells by micromolar amounts (1-10 microM). The glycoprotein was detectable 3-6 h following exposure to RA and its expression was reversible upon removal of RA from the medium. Our results indicate that gp140 is a newly identified RA-inducible epithelial membrane glycoprotein which may represent a phenotypic differentiation marker for epithelial cells.     

Effect of retinoic acid on the proliferation and phagocytic capability of murine macrophage-like cell lines

Retinoic acid (RA) exerted a variable degree of growth inhibitory activity on the macrophage-like cell lines P388D1, J774.2, WEHI-265, WEHI-3, and PU-5. Comparison of cell proliferation and clonal growth suggests that at concentrations of 10(-9)-10(-6) M the inhibitory activity stems from processes leading to elongation of cell cycle time and not from terminal differentiation processes. RA was shown to be a potent inducer of the development of high-phagocytic phenotypes (assessed by phagocytosis of heat-killed yeast cells) in the P388D1, J774.2, and WEHI-265 cell lines which differ substantially in their proliferative and adherence characteristics. The PU-5 and WEHI-3 cell lines were not induced by RA to express an enhanced phagocytic activity toward heat-killed yeast cells. The augmented phagocytic capability was dose dependent over a wide range of RA concentrations. In P388D1 cells, 2 X 10(-12) M RA already exerted significant phagocytosis augmentation effects, which progressively increased up to 2 X 10(-5) M RA, the highest concentration tested. Retinal, retinyl acetate, and retinol had similar effects to those of RA on both cell adherence and phagocytosis in P388D1 cells, albeit at concentrations four to six orders of magnitude higher. Optimal development of the high-phagocytic phenotype in P388D1, J774.2, and WEHI-265 cells required at least 96 hr of culture in the presence of RA; at 48 hr and 23 hr the effects were already substantial, whereas at 4 hr of exposure to RA no significant enhancement of phagocytosis could be detected. Thus both extended periods of culture in the presence of RA (more than two to three cell cycles) and high concentrations were needed for induction, in more than 90% of the cells, of the expression of a high-phagocytic phenotype. The reversion to a low-phagocytic phenotype upon removal of RA was also rather slow and required several cell cycles. In P388D1 cells RA also enhanced the phagocytosis of latex beads but had no effect on the phagocytosis of starch particles, or the extent of binding of immunoglobulin G-coated sheep red blood cells (SRBC). The expression of receptors for concanavalin A and for nonopsonized SRBC remarkably increased in RA-treated cells, as was the ability to perform Fc-receptor mediated erythrophagocytosis. Both P388D1 cells and WEHI-265 cells were induced by RA to express nitroblue tetrazolium reducing activity. The data suggest that RA induces profound changes in the functional capabilities of macrophage-like cell lines which are apparently not dependent on cessation of growth and terminal differentiation processes.     

Effect of retinoic acid on cell-cell adhesiveness in cloned BHK21/C13 cells which form piling-up colonies

The effect was studied of retinoic acid (RA) on cell-cell adhesiveness in Ag8-1 cells, which are piling-up colony-forming cells cloned from a Syrian hamster kidney fibroblastic cell line BHK21/C13. From the piled-up part of the colonies grown with RA (10 microM), many cells were dissociated by mere shaking or pipetting. The dissociated cells soon adhered to and spread on plastic surfaces in the presence of RA. The number of cells per colony increased almost at the same rate in the presence or absence of RA. The effect of RA on the appearance of cells dissociable from colonies was noticeable above 0.1 microM, prominent from 1 to 10 microM, greater when added in the earlier stages of colony formation and negligible when added just before the dissociation assay. Single cells from the monolayer culture grown with RA (10 microM) had less tendency to aggregate than did those from the control culture. Cells from the colonies grown with RA adhered to and spread on a plastic dish for bacterial use, but control cells seldom adhered. These results indicate that RA decreases the cell-cell adhesiveness or suppresses the development of it but increases cell-substratum adhesiveness.     

The effect of retinoic acid on protein phosphorylation in mouse melanoma cells

Vitamin A inhibits growth and increases the activity of cAMP-dependent protein kinase in B16 mouse melanoma cells. In this report we show that retinoic acid (RA) treatment of intact cells alters their subsequent in vitro protein phosphorylation, but we could not demonstrate any changes in in vivo protein phosphorylation. A 48-h treatment with RA results in a concentration-dependent decrease of protein phosphorylation of a 95K molecular weight (MW) protein in both supernatant and particulate fractions. The phosphorylation of this protein does not appear to be regulated by cAMP. Proteins at 92K and 82K MW in the supernatant fraction are increased in phosphorylation. The former (but not the latter) is regulated by cAMP. In the particulate fraction a variety of proteins 12K-68K MW are increased in phosphorylation, as the cells are treated with increasing amounts of RA. The phosphorylation of most of these proteins is regulated by cAMP. Another inhibitor of B16 cell growth, melanocyte-stimulating hormone (MSH) also alters protein phosphorylation. At short incubation periods (1 h), this hormone stimulates phosphorylation of a number of proteins (17-40K MW), while in longer incubation periods (48 h) phosphorylation is inhibited. All of these phosphorylations appear to be regulated by cAMP. We attempted to repeat these observations using intact-cell phosphorylation with 32PO4. In two experiments we saw small changes in the phosphorylation of proteins. In most experiments, however, we could find no change in the phosphoproteins. Further experiments have led us to question the in vivo phosphorylation, since treatment of the cells with MSH, cholera toxin, or db-cAMP also did not affect intact-cell protein phosphorylation. We have previously documented that under these latter conditions cAMP levels are greatly elevated and cAMP-dependent protein kinase is activated. The in vitro phosphorylation results suggests that in RA-treated cells, kinase activities and/or protein substrate levels are changing. However, the physiological significance of the particular MW phosphoproteins changes we have described must await resolution of the in vivo phosphorylation data.     

Compartmentalization of cyclic AMP elevation in neurons ofAplysia californica

We have measured by radioimmunoassay the amount of total, free, and bound forms of cyclic AMP (cAMP) within the abdominal ganglion and in five identified cell bodies of neurons from Aplysia californica. In the abdominal ganglion the unbound (free) cAMP levels comprised approximately 25-30% of the total cAMP content under the unstimulated condition, i.e., bathed in high-magnesium saline. Under pharmacological conditions that blocked endogenous phosphodiesterase and activated adenylate cyclase, ganglionic free cAMP levels were elevated more than fourfold, while bound cAMP levels more than doubled. Freeze-substitution techniques were employed to facilitate isolation of individual cell bodies either before or after pharmacological manipulation of cAMP levels. The basal, free cAMP content of cells R2, LP1, R15, L11, and L2-L6 was in the range of 10-40 pmol/mg of cell protein, which accounted for approximately one-half of the total cAMP content per cell body. Determinations of individual cell volumes indicated that the basal, free cAMP concentrations ranged from 1 to 6 microM. Under the same pharmacological conditions that elevated ganglionic cAMP in levels, no changes were measured in either the free or the bound forms of cAMP in isolated cell bodies. Our results indicate that the cAMP elevation was compartmentalized within the neuropilar region of the ganglion, most likely within the processes of the nerve cells. Previous results demonstrated that cAMP injections into the same Aplysia neurons studied here induced a cAMP-activated sodium current, INa (cAMP). In this report we discuss the possibility that pharmacological elevation of cAMP within neuronal processes may reach concentrations similar to those produced by cAMP injections into somata.