Characterization of epidermal growth factor receptor induction by retinoic acid in a chemically transformed rat liver cell line

Levels of epidermal growth factor (EGF) receptor expression vary widely among cell lines derived clonally from a chemically transformed population of rat liver epithelial cells. Retinoic acid (RA), a derivative of vitamin A that stimulates differentiation in a number of embryonal cell lines, increases the level of 125I-EGF binding in several clones of the transformed cell lines. One such cell line, GP6ac, which reverts to a less transformed phenotype when treated with RA, exhibited a 3-4-fold increase in surface EGF receptors with prolonged (2-5-day) RA exposure. The increase persisted as long as the cells were treated with RA. The increase in surface EGF receptors was due to induction of receptor biosynthesis, which occurred within 4 h at both the mRNA and protein levels and persisted until the RA was withdrawn. Paradoxically, the RA response was accompanied by an initial 40-50% decrease in 125I-EGF binding during the first 12 h of RA treatment. The decrease was due primarily to a reduction of receptor affinity. Since the phorbol ester 12-O-tetradecanoylphorbol-13-acetate also decreases 125I-EGF binding and increases EGF receptor biosynthesis in GP6ac cells, we tested the effect of RA in cells depleted of protein kinase C by prolonged treatment (18 h) with 10 microM 12-O-tetradecanoylphorbol-13-acetate. The absence of protein kinase C did not affect the induction of receptor mRNA and protein or the decrease in binding during the early period of RA exposure. This indicates that RA induction of EGF receptor synthesis in GP6ac cells involves signaling pathways distinct from those utilized by phorbol esters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Restoration of anchorage regulation in transformed cells by retinoic acid (RA) is independent of the presence of cytoplasmic RA-binding proteins

In an attempt to analyse the cause-effect relationship between anchorage-independent growth (a property which correlates best with in vivo tumorigenicity) and a set of other common transformation-related properties, the effect of retinoic acid (RA) treatment on six unrelated transformed cell lines (including DNA tumor virus, retrovirus, and spontaneously transformed cells) were studied. The data show that the changes in morphology and cellular orientation in culture, loss of cell surface fibronectin, disruption of actin microfilaments, increased hexose uptake, loss of density-dependent growth, and decreased binding of EGF, properties which are often associated with oncogenic transformation of cells, are dissociable from one another and from anchorage-independent growth. RA appears to interfere with anchorage-independent growth of all the retrovirus and spontaneously transformed cell lines (responsive cells) that we examined; however, such treatment failed to inhibit anchorage-independent growth in both of the DNA tumor virus-transformed cell lines (non-responsive cells) that we used in the present study. The presence of RA-binding proteins in both responsive and non-responsive cells suggests that the mechanism of RA action for the inhibition of anchorage-independent growth in transformed cells may be independent of the presence of such cytoplasmic proteins. Finally, the present study clearly indicates that the use of RA treatment, like partial transformation mutants of oncogenic viruses, can be a novel approach in analysing the general mechanism by which transformed cells grow without anchorage.     

Modulation of transglutaminase activity in mononuclear phagocytes and macrophage-like tumor cell lines by differentiation agents

The effect of glucocorticosteroids, retinoids, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and the tumor promoter phorbol myristate acetate (TPA) on the expression of transglutaminase activity in vitro differentiating bone marrow-derived mouse and rat mononuclear phagocytes (BMDMP) and mouse and human myeloid leukemia cell lines was assessed. Dexamethasone was found to induce an increase of about 100% in transglutaminase activity in mouse and rat BMDMP. The effect was time- and dose-dependent, and specific for steroids with glucocorticoid activity. Retinoic acid (RA) suppressed transglutaminase activity in mouse BMDMP (approximately 50%) and enhanced it in rat BMDMP (100-200%). Other retinoids were less effective. 1,25(OH)2D3 had little effect on transglutaminase expression in mouse BMDMP and suppressed it in rat BMDMP (approximately 60%). TPA exerted a suppressive effect (approximately 50%) on transglutaminase activity of both rat and mouse BMDMP. In murine (P388D1 and J774.2) and human (ML3, HL-60, KG-1, HEL, U937) myeloid leukemia cell lines, dexamethasone enhanced transglutaminase activity to a varying degree (100-1,000%), RA suppressed it in P388D1 cells (approximately 70%) and enhanced it in the other cell lines (100-1,500%), 1,25(OH)2D3 induced a rather small augmentation of enzyme expression, whereas TPA suppressed enzyme expression (70-100%). The species-specific differences previously observed by us for the effect of RA, dexamethasone and 1,25(OH)2D3 on the formation of BMDMP from mouse and rat bone marrow progenitor cells are now shown to extend also to effects on expression of transglutaminase activity. From a mechanistic point of view it is of interest that dexamethasone uniformly enhanced transglutaminase activity, whereas TPA suppressed it. RA and 1,25(OH)2D3 induced either suppression or enhancement in the various cell types, with no correlation between the direction of the effect of the two agents. The data suggest that modulation of transglutaminase activity by the four agents occurs via disparate mechanisms.     

Prevention by retinoic acid of anionic site redistribution on the surface of cultured human sarcoma cells

The distribution of cell surface negatively-charged macromolecules was determined electron microscopically on untreated and on retinoic acid (RA)-treated cultured human osteosarcoma Hs791 and chondrosarcoma Hs705 cells using cationized ferritin (CF), an electron-dense marker of anionic sites. Labeling on the surface of prefixed cells was continuous and uniform whether they were grown in the absence or presence of RA. In contrast, CF distribution on unfixed cells was markedly affected by RA; CF labeling of untreated cells occurred in patches and clusters whereas the label on RA-treated cells was continuous, as on prefixed cells. CF labeling of unfixed cells decreased considerably after incubation of the cells either with hyaluronidase or neuraminidase. There was also a reduction in patching and clustering. Changes induced by RA in the apparent membrane microviscosity, in neuraminidase-releasable sialic acid, or in transglutaminase activity could not be related to the effect of RA on CF-induced anionic site redistribution since these characteristics were modulated differently in the two cell lines. In contrast, RA increased the sialylation of specific cell surface membrane glycoproteins on both cell types. These results suggest that RA prevents redistribution of cell surface sialoglycoconjugates and glycosaminoglycans by CF. This effect may be the result of increased sialylation of specific surface components and may be related causally to the suppression of the transformed phenotype in the sarcoma cells.     

Bimodal effect of retinoic acid on melanocyte differentiation identified by time-dependent analysis

Retinoic acid (RA) is considered to control melanocytes; however, its precise mechanism remains unclear because of a bimodal effect, which promotes or inhibits melanin synthesis depending on the cell type, culture condition of melanocytes and skin conditions. In this study, we examined the effects of RA throughout each stage of differentiation of melanocytes using a mouse embryonic stem cell culture system to induce melanocytes. The results showed that RA has significantly different effects depending on the stage of differentiation of melanocytes. More specifically, RA promoted differentiation in earlier stages, wherein embryonic stem cells became melanoblasts via neural crest cells, and inhibited differentiation in later stages, wherein melanoblasts became melanocytes. It was revealed for the first time that melanocytes show markedly different reactions to RA depending on the stage of differentiation.     

Induction of F9 cell differentiation by transient exposure to retinoic acid.

The F9 cell is a mouse embryonal teratocarcinoma which can be induced to differentiate into visceral endoderm by treatment with retinoic acid (RA). Treatment with RA in conventional studies was carried out in the constant presence of RA. Here we demonstrate that treatment with RA can be as short as 3 hrs to induce differentiation of F9 cells. Morphology, alpha-fetoprotein gene activity, and temporal patterns of F9 cell differentiation are the same with both short- and long-term treatment with RA.     

Comparison of the effects of transforming growth factor beta, N,N-dimethylformamide, and retinoic acid on transformed and nontransformed fibroblasts

In order to compare the effects of transforming growth factor (TGF beta) with those of the differentiation promoters N,N-dimethylformamide (DMF) and retinoic acid (RA), the antiproliferative and fibronectin-inducing activities of the three agents were examined. AKR-2B mouse embryo fibroblasts and their chemically transformed counterpart AKR-MCA cells were used as the model system. Growth in monolayer culture of both cell lines was inhibited by TGF beta (EC50 approximately 1 ng/ml), DMF (EC50 approximately 0.5%), and RA (EC50 approximately 1 microM) in a concentration-dependent manner. Time-dependent elevation in fibronectin expression was also observed with all three agents. The EC50 for growth inhibition of both cell lines by TGF beta agreed well with that obtained for stimulation of fibronectin synthesis. A 3-h exposure to TGF beta is sufficient to obtain the maximal fibronectin level observed at 48 h in AKR-2 B cells but not in AKR-MCA cells. Our results indicate that in this system the effects of TGF beta are similar to those of the chemical differentiation inducers DMF and RA. Furthermore, our data also suggest that the TGF beta signal may be processed differently by nontransformed and transformed fibroblasts.     

Prostaglandin synthetase activity and hormone responsiveness in normal and SV40 transformed WI-38 fibroblasts.

Prostaglandin (PG) synthetase activity and selective hormone responsiveness were examined in normal and SV40 transformed WI-38 fibroblasts (VA13-2RA). The transformed VA13-2RA cells have significantly reduced rates of PGE1, PGE2, PGF1alpha and PGF2alpha synthesis as compared to the normal WI-38 fibroblast. The transformed cell in contrast to the normal cell hyperresponds to stimulation by L-epinephrine (10 muM) and PGE1 (8.5 muM) but is unresponsive to bradykinin (BK) as measured by the accumulation of intracellular cyclic AMP. Indomethacin treatment does not significantly alter the PGE1 and L-epinephrine (EPI) responsiveness of the normal WI-38 fibroblast, however it abolishes the BK response in these cells. These results provide further evidence for the dependency of cell activation by bradykinin on the PG synthetase system. No experimental data was found to support the role of PGs as negative regulators of PGE1 and EPI responsiveness in the WI-38 fibroblast. Using the VA13-2RA cells, limited attempts to recover PG synthetase activity comparable to that found in normal WI-38 cells were unsuccessful. The VA13-2RA cell and its normal counterpart represent models for investigating the role of PGs in cell function and the mechanism of BK activation and its effect on cell metabolism.     

Enhancement of melanotic expression in cultured mouse melanoma cells by retinoids

Retinoic acid (RA), which reduces the rate of cell proliferation in S91 mouse melanoma clone C2 cells, was found to stimulate the expression of their melanotic phenotype. RA treatment also induced the extension of long cellular processes. The RA effects on melanogenesis included stimulation of tyrosinase activity and augmentation of cellular melanin content to levels 3- to 4-fold higher than in untreated cultures at similar cell densities. These effects became apparent after 48 hours of exposure to 10(-5) M RA and increased thereafter. Half-maximal stimulation in cells treated for 6 days occurred at 5 X 10(-7) M RA. Although the degrees of melanogenesis enhancement by RA (10(-5) M) and by alpha-melanocyte stimulatory hormone (2 X 10(-7) M) were similar, the former did not alter the intracellular cAMP level, whereas the latter induced a transient 4-fold increase. In high-passage (p28) cells, as well as in low-passage cells (less than p10) treated with tyrosinase inhibitor phenylthiocarbamate, melanin synthesis was suppressed in the absence and presence of RA, yet the ability of RA to inhibit cell proliferation was not compromised. In the presence of the tumor promotor phorbol myristate acetate (greater than 5 X 10(-9) M) melanin synthesis in control as well as in cells exposed to RA was dramatically inhibited. Phorbol which is not active in tumor promotion had no effect on melanogenesis. In addition to RA, other retinoids, such as 13-cis-retinoic acid, retinyl acetate, the TMMP analog of RA and the phenyl analog of RA, but not the pyridyl analog of RA or retinyl palmitate, also inhibited cell growth and enhanced melanin synthesis.     

Spina bifida aperta induced by valproic acid and by all-trans-retinoic acid in the mouse: distinct differences in morphology and periods of sensitivity.

The antiepileptic drug valproic acid (VPA) has been implicated as a human teratogen causing spina bifida aperta. Recently, we developed a mouse model inducing spina bifida aperta with VPA. To elucidate the pathogenesis of VPA-induced spina bifida aperta we now investigated the anatomy and histology of this defect in the mouse. The morphology of spina bifida aperta induced by all-trans-retinoic acid (RA) was used for comparison. Various doses of VPA and RA were administered at different times to determine the periods of sensitivity for inducing spina bifida aperta with these drugs. Each administration regimen consisted of three doses applied at intervals of 6 hr. RA induced spina bifida aperta during an earlier developmental period (day 8 of gestation) than VPA (day 9 of gestation). The most effective regimens for induction of spina bifida aperta in mice were injections of 3 x 500 mg VPA-Na/kg body weight (b.w.) intraperitoneally on day 9 of gestation at 0, 6, and 12 hr; RA (12.5 mg/kg b.w.) was given orally on day 8 of gestation at 12 and 18 hr, day 9 at 0 hr. VPA did not induce spina bifida aperta on day 8 of gestation and RA did not induce this effect on day 9 of gestation. Histological studies of day 18 fetuses carrying spina bifida aperta were performed. The spina bifida aperta induced by VPA shows a disorganized and necrotic spinal cord. In the vertebral canal were observed cell debris, blood cells, capillaries, macrophages, and rests of meninges. These results indicate that the spinal cord is almost destroyed at the affected section. In contrast, the spina bifida aperta induced by RA demonstrates a spinal cord organized in the gray and white matter, the dorsal and ventral horn. But the neural canal does not exist, only a layer of ependymal cells lies on the surface of the spinal cord. Our results indicate that the morphology of spina bifida aperta induced by VPA differed distinctly from that induced by RA in the mouse fetus. Moreover VPA produced a spina bifida aperta with a specific morphology. Also the period of sensitivity for induction of this lesion differed and occurred earlier for RA than for VPA. VPA and RA may possibly induce spina bifida aperta via different mechanisms in the mouse.     

Hoxa-5 in mouse developing lung: cell-specific expression and retinoic acid regulation

Hoxa-5 is a homeobox gene that is highly expressed in the developing mouse lung. However, little is known about the molecular mechanisms controlling expression. We characterized the ontogeny of Hoxa-5 gene and protein expressions during lung development and then studied the cell-specific effects of retinoic acid (RA) on Hoxa-5 mRNA in fetal lung fibroblasts and MLE-12 mouse lung epithelial cells. Strong but constant Hoxa-5 gene and protein expressions were detected from mouse lung on embryonic day 13.5 to postnatal day 2. At baseline, the gene was strongly expressed in the fibroblasts of day 17.5 fetal mouse lungs. A very weak but reproducible expression was present in the MLE-12 cells. RA stimulated gene expression in both cell types in a time- and dose-dependent manner. Peak expression occurred much later in the MLE-12 cells compared with that in fibroblasts. Cycloheximide and actinomycin D treatment studies suggested that the differences in RA effect on each cell type may involve the presence of a repressor that can be overcome by RA.     

Modification of steroidogenesis in a mouse adrenal cell line (Y-1) transformed by simian adenovirus SA-7

Transformation of a steroidogenic mouse adrenal cell line (Y-1) by simian adenovirus SA7 produced a cell line with low apparent steroidogenic activity. The effect of ACTH and cholera toxin on cyclic AMP production was similar in both not transformed and virus-transformed cells and activity of cyclic AMP-dependent protein kinase was also similar in both cells. In transformed cells, cholesterol was metabolized to delta 5-3 beta-hydroxysteroids, mainly 20 alpha-dihydropregnenolone while in not transformed cells, the major metabolites were delta 4-3 ketosteroids (20 alpha-dihydro- and 11 beta-hydroxy-20 alpha-dihydroprogesterone). In both cell lines ACTH increased the metabolism of cholesterol. Further studies with labelled pregnenolone and progesterone revealed a loss of delta 5-3 beta-hydroxysteroid dehydrogenase/isomerase and 11 beta-hydroxylase activity in the transformed cells.     

维生素A酸和双丁酰基环腺苷单磷酸对小鼠胚...

In vitro induced differentiation of mouse embryonic stem cells (ES-5 cells), derived from 5-day 129 mouse blastocyst was studied with retinoic acid (RA) and dibutyryl cyclic adenosine monophosphate (dB-cAMP). RA only or RA with dBcAMP together can both induce monolayer ES-5 cells to differentiate into cells of two types: neuron-like cells and fibroblast-like cells. After treated with 10(-6)mol/L RA for 6 days, the differentiated cells were about 80% of all cells, among which most cells were fibroblast-like cells and others were neuron-like cells. While after 6 days of treatment with 10(-6)mol/L RA and 1 mmol/L dBcAMP, the ratio of differentiated cells can be up to 90-95%, and most cells (about 90-95% of differentiated cells) are neuron-like cells. Immunocytochemical analysis of phenotypic markers, especially GFAP and laminin, showed that the neuron-like cells were glia cells. DBcAMP affected the direction and efficiency of induction by RA. The induced differentiation by RA on attached aggregated ES-5 cells was studied as well. In this case, more cell types appeared, such as epitheloid cells, fibroblast-like cells and spindle shaped cells and so on. The exact nature of these differentiated cells was not identified. After attached culture for about 15 days, rhythmically contracting cardiac-like muscle cells were most attractive among those several differentiated cell types. The change of phenotypic markers during induced differentiation of ES-5 cells in monolayer and aggregated state was summarized in table 1. Transforming growth factor-beta 1 (TGF-beta 1) was also examined in undifferentiated and differentiated cells. Untreated ES-5 cells showed positive immunofluorescent reaction to TGF-beta 1 and various differentiated cells showed different reactions. Glia cells and cardiac-like cells displayed a much stronger TGF-beta 1 reaction. These results indicate that the exact role played by TGF-beta 1 during induced differentiation needs further investigation. The different effect of RA on monolayer and aggregated ES cells and the possible significance of cell to cell interaction in the latter case are discussed.     

Antiviral and anticellular effects of interferon on the mouse embryonic cells transformed by viruses and/or chemical carcinogen.

The antiviral and anticellular activity of partially purified mouse interferon has been tested in cell lines transformed with Simian Virus 40 (MEB-SV 40), mouse sarcoma virus (Harvey strain) (MEB-MSV), and 20-methylcholanthrene (MEB-MCH), respectively. The transformed lines were derived from C3H mouse embryonic primary cells. It has been shown that the MEB-MSV cells were 10 to 50 times less sensitive to the antiviral effect of interferon than the MEB-SV 40 or MEB-MCH cells. A 30% reduction of the number of treated cells as compared with untreated control cells was taken as basis for comparison of anticellular activity of interferon in transformed lines. While the MEB-MCH cells required 1000 units of interferon for a 30% growth inhibition, about 3000 units were necessary for a comparable suppression of MEB-SV 40 cells and/or MEB-MSV cells.     

Anti-apoptotic effect of retinoic acid on retinal progenitor cells mediated by a protein kinase A-dependent mechanism

Retinal progenitor cells （RPCs） are neural stem cells able to differentiate into any normal adult retinal cell type, except for pigment epithelial cells. Retinoic acid （RA） is a powerful growth/differentiation factor that generally causes growth inhibition, differentiation and/or apoptosis. In this study, we demonstrate that RA not only affects mouse RPC differentiation but also improves cell survival by reducing spontaneous apoptotic rate without affecting RPC proliferation. The enhanced cell survival was accompanied by a significant upregulation of the expression of protein kinase A （PKA） and several protein kinase C （PKC） isoforms. Treatment of cells grown in RA-free media with 8-bromoadenosine3＇,5＇-cyclic monophosphate, a known activator of PKA, resulted in an anti-apoptotic effect similar to that caused by RA; whereas the PKA inhibitor N-[2-（p-bromocinnamylamino）ethyl]-5-isoquinolinesul- fonamide dihydrochloride led to a significant （-32%） increase in apoptosis. In contrast, treatment of RPCs with any of two PKC selective inhibitors, 2,2＇,3,3＇,4,4＇-hexahydroxy-1,1 ＇-biphenyl-6,6＇-dimethanol dimethyl ether and bisindolylmaleimide XI, led to diminished apoptosis; while a PKC activator, phorbol 12-myristate 13-acetate, increased apoptosis. These and other data suggest that the effect of RA on RPC survival is mostly due to the increased anti-apoptotic activity elicited by PKA, which might in turn be antagonized by PKC. Such a mechanism is a new example of tight regulation of important biological processes triggered by RA. Although the detailed mechanisms remain to be elucidated, we provide evidence that the pro-survival effect of RA on RPCs is not mediated by changed expression of p53 or bcl-2, and appears to be independent of 15-amyloid, Fas ligand, TNF-α, ganglioside GM1 and ceramide C 16-induced apoptotic pathways.