Murine embryonal carcinoma cells differentiate in vitro in response to retinol

In an initial effort to determine whether circulating retinol might promote differentiation of embryonal carcinoma (EC) cells in tumor form, we have assessed the ability of retinol to stimulate differentiation of cultured EC cells. We found that retinol induces several murine EC cell lines to differentiate in vitro. Differentiated derivatives were distinguishable from parental EC cells by morphology, cell surface antigenic properties and levels of secretion of plasminogen activator. Retinol effects could be seen at concentrations as low as 8.7 × 10⁻⁸ M (0.025 (μg/ml). Only two of eight EC lines tested failed to differentiate in response to retinol: PCC4-azaIR, which dies at retinol concentrations above 3.5 × 10⁻⁷ M; and PCC4(RA)^-1, a mutant line derived from PCC4-azaIR cells, which also fails to differentiate in response to retinoic acid.     

Effects of Different Conditions of Retinoic Acid Treatment on Expression of MK Gene,which is Transiently Activated during Differentiation of Embryonal Carcinoma Cells1

MK gene was intensely expressed, when aggregates of HM-1 embryonal carcinoma (EC) cells were treated with retinoic acid for 2 days to induce the differntiation to nerve cells, myoblasts and extraembryonic endoderm cells. The conditions inhibiting nerve cell diffrentiation or extraembryonic endoderm cell differentiation affected MK gene expression only slightly. The maximum level of MK RNA was detected 2 days after initiation of retionic acid treatment, when cells were morphologically indistinguishable from undifferentiated EC cells. Thus, MK gene appears to be expressed in differentiating EC cells irrespective of the direction of differentiation. The degree of MK gene expression in sparsely cultured HM-1 cells correlated with the concentration of retinoic acid, especially between 10^-8 and 10^-7 M. When retinoic acid treatment was terminated after 1 day, the amount of MK RNA started to decrease. These two results are consistent with the view that retionic acid complexed with the receptor is directly involved in expression of MK gene.     

Retinoids and cultured human fibroblasts : Effects on cell growth and presence of cellular retinoic acid-binding protein

We have examined the effects of retinoids on growth of cultured human skin fibroblasts from four individuals. Retinoic acid and retinol both produce a dose-dependent inhibition of growth in the four strains examined; retinoic acid was more potent than retinol in this respect. The growth inhibitory effect of retinoic acid is characterized by a decrease in the exponential growth rate, which is reversible upon removal of retinoic acid from the growth medium; the final saturation density, however, is not modified by retinoic acid treatment. No alterations of cell morphology, viability, or adhesiveness to substratum are induced by the retinoid concentrations utilized. The inhibitory effect of 10⁻⁶ M retinoic acid on cell growth is not affected by the concentration of fetal calf serum (FCS) in the medium. In all four human fibroblast strains examined, specific binding of [³H]retinoic acid to cytosol is present as determined by sucrose-density gradient centrifugation. Despite the effects of retinol on fibroblast growth, no cytoplasmic binding of [³H]retinol could be demonstrated in these cells.     

Retinoic acid-induced modifications in the growth and cell surface components of a human carcinoma (HeLa) cell line

The addition of retinoic acid to cultures of HeLa-S3 cells caused a reduction in cell proliferation rate which became apparent after 72 h and was linearly dependent on retinoic acid concentration in the range 10⁻⁹–10⁻⁵ M. After 72 h of exposure to retinoic acid, the cells assumed a flattened appearance and no longer formed multilayers. These changes were reversed within 48 h after removal of retinoic acid from the medium. Structural analogs of retinoic acid with a free ---COOH group at C-15 were usually more potent in growth inhibition than compounds with an alcohol, aldehyde, ether or ester group. A cellular retinoic acid-binding protein was detected in cell homogenates, and the binding of [³H]retinoic acid to the binding protein was inhibited by most, but not all, analogs possessing a free terminal ---COOH group. For example, the 4-oxo analog of retinoic acid, while capable of inhibiting cellular proliferation, failed to bind to the retinoic acid-binding protein. Analysis of cell surface and cellular glycoproteins by lactoperoxidase-catalysed ¹²⁵I iodination and by metabolic labeling with [³H]glucosamine revealed that a 190000 D glycoprotein which was labeled by both methods and a 230000 D glycoprotein which was labeled only with [³H]glucosamine were labeled more intensely in retinoic acid-treated cells compared with untreated cells. The electrophoretic mobility of the 230000 D glycoprotein could be modified by treatment of intact cells with either neuraminidase or proteolytic enzymes, suggesting that this glycoprotein is also exposed on the cell surface. The cell surface alterations were detected much earlier than the onset of growth inhibition and appeared as early as 24 h after exposure to retinoic acid. The possible relationship between retinoic acid-induced changes in cell membrane structure, cell morphology, and cell proliferation is discussed.     

Characterization of the action of retinoids on mouse fibroblast cell lines

Retinoic acid affects 3T6 and 3T3 cells by inhibiting growth, causing a morphological change and increasing cell-to-substratum adhesiveness. Retinoic acid does not exert such effects on virus-transformed 3T3SV cells. Retinoic acid treatment of 3T6 cells causes a concentration-dependent increase in generation time and a reduction in saturation density. Analysis of cell surface proteins shows that a high molecular weight band of 230 000 D, corresponding to the position of the LETS glycoprotein, is more intensely labeled by iodination of cells treated with retinoic acid compared to control cells. Retinoic acid substantially stimulates the incorporation of ³⁵SO₄ into cell-associated glycosaminoglycans and causes a less dramatic increase in glycosaminoglycans excreted into the medium. The relationship between the increase in these cell surface components and the enhanced adhesiveness is discussed. A retinoic acid binding protein is detectable in the cytosol of 3T6 and 3T3 cells but not in 3T3SV cells, suggesting that the action of retinoids on these cells is mediated via this protein.     

Histone H10 mRNA and protein accumulate early during retinoic acid induced differentiation of synchronized embryonal carcinoma cells

The very lysine-rich replacement histone variant H1⁰ is found to be present in different murine (C1003, PC13, P19) and human (Tera-2) embryonal carcinoma cell lines. The proportion of H1⁰ increases upon induction of differentiation of the different cell lines by various treatments. In undifferentiated PC13 EC cells H1⁰ mRNA is present at a low level. During retinoic acid induced differentiation of mitotically synchronized PC13 EC cells, accumulation of H1⁰ mRNA starts in the first cell cycle. The H1⁰ protein level starts to increase in the second synchronous cycle preceding changes in the cycle parameters that become apparent in the third cycle. The results provide further support for an important role of H1⁰ in the control of cellular differentiation in early mammalian development.Abbreviations EC embryonal carcinoma - RA retinoic acid - DAPT 4-6-diamino-2-phenylindole - SDS sodium dodecylsulphate - DMSO dimethyl sulfoxide - TCA trichloro acetic acid     

Metabolism of retinoids by embryonal carcinoma cells

Several embryonal carcinoma (EC) cell lines were tested in culture for their ability to metabolize all-trans-[3H]retinol, all-trans-[3H]retinyl acetate, and all-trans-[3H]retinoic acid. There was little, if any, metabolism of all-trans-retinol to more polar compounds; we failed to detect conversion to acidic retinoids by reverse-phase high performance liquid chromatography and derivatization. We also did not observe [3H]retinoic acid when EC cells were incubated with [3H]retinyl acetate. Unlike the other retinoids, all-trans-[3H]retinoic acid, even at micromolar levels, was almost totally modified by cells from several EC lines within 24 h. Most of the labeled products were secreted into the medium. Some EC lines metabolized retinoic acid constitutively, whereas others had an inducible enzyme system. A differentiation-defective line, which contains little or no cellular retinoic acid-binding protein activity, metabolized retinoic acid poorly, even after exposure to inducers. At least eight retinoic acid metabolites were generated; many contain hydroxyl residues. Our data lead us to propose that retinol does not induce differentiation of EC cells in vitro via conversion to retinoic acid. Also, the relatively rapid metabolism of retinoic acid by EC cells suggests either that the induction of differentiation need involve only a transient exposure to this retinoid or that one or more of the retinoic acid metabolites can also promote differentiation.     

The effect of retinoic acid on the proportion of insulin cells in the developing chick pancreas

Summary We assessed the potential role of all-trans-retinoic acid on the developing chick pancreas, specifically with regard to the proportions of insulin cells. The endodermal component of the dorsal pancreatic bud of 5-d-old chick embryos was cultured on Matrigel. Retinoic acid (10⁻⁶ or 10⁻⁵ M) was added to a standard serum-free medium, Ham's F12 containing insulin, transferrin and selenium (F12.ITS). Control grafts were cultured in F12.ITS alone or in F12.ITS with DMSO (the diluent for retinoic acid). After 7 d the explants were retrieved, freeze-dried, vapor-fixed, and embedded in resin. Endocrine cell types were identified by immunocytochemistry. The numbers of insulin cells were expressed as a proportion of the sum of insulin plus glucagon cells. Retinoic acid had a dose-related effect; the proportion of insulin cells in explants treated with the lower dose of retinoic acid (10⁻⁶ M) was more than twice the proportion of insulin cells in explants treated with the higher dose (10⁻⁵ M) of retinoic acid and more than three times that of the control grafts.     

Differentiation of TERA-2 human embryonal carcinoma cells into neurons and HCMV permissive cells

Retinoic acid induces the differentiation of NTERA-2 cl. D1 human embryonal carcinoma (EC) cells into neurons, cells permissive for the replication of human cytomegalovirus (HCMV), and other cell types that cannot as yet be classified but are distinguishable from the stem cells. We tested several additional agents for their ability to induce the differentiation of these EC cells. No differentiation was induced by butyrate, cyclic AMP, cytosine arabinoside, the tumor promoter 12-0-tetradecanoylphorbol 13-acetate (TPA), or the chemotherapeutic agent cis-diaminedichloroplatinum, although morphological changes were detected at the highest concentrations of these agents that permitted cell survival. However, retinal, retinol, 5-bromouracil 2'deoxyribose (BUdR), 5-iodouracil 2'deoxyribose (IUdR), hexamethylene bisacetamide (HMBA), dimethylacetamide (DMA), and dimethylsulfoxide (DMSO) all induced some neuronal differentiation, but to a lesser extent than retinoic acid. Also, BUdR, IUdR, HMBA, and DMA induced the appearance of many cells permissive for the replication of HCMV. Differentiation was, in all cases, accompanied by the loss of SSEA-3, a globoseries glycolipid antigen characteristically expressed by human EC cells. However, another glycolipid antigen, A2B5, which appears in 60%-80% of differentiated cells 7 days following retinoic acid induction, was detected in less than 20% of the cells induced by the other agents studied. This implies that the HCMV-permissive cells induced by retinoic acid are not identical to those induced by BUdR, IUdR, and DMA.     

The Src-Family Kinase Inhibitor PP2 Rescues Inducible Differentiation Events in Emergent Retinoic Acid-Resistant Myeloblastic Leukemia Cells

Retinoic acid is an embryonic morphogen and dietary factor that demonstrates chemotherapeutic efficacy in inducing maturation in leukemia cells. Using HL60 model human myeloid leukemia cells, where all-trans retinoic acid (RA) induces granulocytic differentiation, we developed two emergent RA-resistant HL60 cell lines which are characterized by loss of RA-inducible G1/G0 arrest, CD11b expression, inducible oxidative metabolism and p47^phox expression. However, RA-treated RA-resistant HL60 continue to exhibit sustained MEK/ERK activation, and one of the two sequentially emergent resistant lines retains RA-inducible CD38 expression. Other signaling events that define the wild-type (WT) response are compromised, including c-Raf phosphorylation and increased expression of c-Cbl, Vav1, and the Src-family kinases (SFKs) Lyn and Fgr. As shown previously in WT HL60 cells, we found that the SFK inhibitor PP2 significantly increases G1/G0 cell cycle arrest, CD38 and CD11b expression, c-Raf phosphorylation and expression of the aforementioned regulators in RA-resistant HL60. The resistant cells were potentially incapable of developing inducible oxidative metabolism. These results motivate the concept that RA resistance can occur in steps, wherein growth arrest and other differentiation events may be recovered in both emergent lines. Investigating the mechanistic anomalies in resistant cell lines is of therapeutic significance and helps to mechanistically understand the response to retinoic acid’s biological effects in WT HL60 cells.     

Retinoic acid effect on cyclic AMP-dependent protein kinases in embryonal carcinoma cells: studies with differentiation-defective sublines

Retinoic acid induces the differentiation of PCC4.aza 1R and Nulli-SCC1 embryonal carcinoma (EC) cells. In response to retinoic acid treatment, the levels of cyclic AMP (cAMP)-dependent protein kinases are enhanced in the plasma membrane within 17 hours and in the cytosol fractions of these cells within 2 to 3 days, as determined by phosphotransferase activity and by 8-azido-cyclic [32P]AMP binding to the RI and RII regulatory subunits. PCC4 (RA)-1 and Nulli (RA)-1 are mutant EC lines that fail to differentiate in response to retinoic acid. The former line, but not the latter, lacks cellular retinoic acid-binding protein (cRABP). Basal levels of cAMP-dependent protein kinase activities are elevated in PCC4 (RA)-1 cells. When these cells are treated with retinoic acid, neither cAMP-dependent protein kinase activities nor cAMP binding activities are enhanced; rather, there is a decrease in cytosolic kinase activity and RI subunit. On the other hand, Nulli (RA)-1 cells exhibit increases both in cAMP-dependent protein kinase activities and cAMP binding in response to retinoic acid. These results raise the possibility that cRABP mediates the enhancement of regulatory and catalytic subunits of cAMP-dependent protein kinases in both the membrane and the cytosolic fractions of the teratocarcinoma cells. There also might be some effects of retinoic acid on the cAMP-dependent protein kinase that are unrelated to differentiation and to the presence of cRABP.     

Retinoic acid induces neuronal differentiation of a cloned human embryonal carcinoma cell line in vitro

The human embryonal carcinoma cell lines $\text{NT2}\text{D1}$ and $\text{NT2}\text{B9}$, clonally derived from Tera-2, differentiate extensively in vitro when exposed to retinoic acid. This differentiation is marked by the appearance of several morphologically distinct cell types and by changes in cell surface phenotype, particularly by the disappearance of stage-specific embryonic antigen-3 (SSEA-3), which is characteristically expressed by human EC cells. Among the differentiated cells are neurons, which form clusters interconnected by extended networks of axon bundles, and which express tetanus toxin receptors and neurofilament proteins. These observations constitute the first instance of extensive somatic differentiation of a clonal human EC cell line in vitro.     

Retinoic acid induced growth arrest of human breast carcinoma cells requires protein kinase Cα expression and activity

Retinoic acid inhibits proliferation of hormone-dependent, but not hormone-independent breast cancer cells. Retinoic acid-induced changes in cellular proliferation and differentiation are associated with disturbances in growth factor signaling and frequently with changes in protein kinase C expression. PKCδ, ϵ, and ζ are expressed in both hormone-dependent (T-47D) and hormone-independent (MDA-MB-231) cell lines. Retinoic acid arrested T-47D proliferation, induced PKCα expression and concomitantly repressed PKCζ expression. The changes in PKCα and PKCζ reflect retinoic acid-induced changes in mRNA. In contrast, retinoic acid had no effect on growth, or PKC expression in MDA-MB-231 cells. Growth arrest and the induction of PKCα, but not the reduction in PKCζ, resulted from selective activation of RARα. In total, these results support an important role for PKCα in mediating the anti-proliferative action of retinoids on human breast carcinoma cells. J. Cell. Physiol. 172:306–313, 1997. © 1997 Wiley-Liss, Inc.     

Inhibition of differentiation by leukemia inhibitory factor distinguishes two induction pathways in P19 embryonal carcinoma cells

The ability of leukemia inhibitory factor (LIF) to block differentiation of P19 embryonal carcinoma (EC) cells under a variety of induction conditions was determined. LIF inhibits differentiation under several conditions which lead to endodermal and mesodermal cell lineages including skeletal and cardiac muscle. In contrast, LIF does not block differentiation when cells are induced under conditions which lead to neuro-ectodermal cell types including neurons and astroglial cells. These studies demonstrate that P19 EC cell differentiation can be divided into LIF sensitive and insensitive pathways which correlate with differentiation of endodermal/mesodermal and neuro-ectodermal cell types, respectively. The effect of LIF on mRNA levels for several genes which have previously been implicated in mediating differentiation in P19 EC cells was determined. LIF has no effect on the mRNA levels for retinoic acid receptor (RAR) alpha, RAR beta, RAR gamma, jun A, jun D, c-fos, or fra-1. In contrast LIF stimulates jun B mRNA expression by a factor of four to six under all induction conditions.     

Differentiation induction of human leukemia cells (HL60) by a combination of 1,25-dihydroxyvitamin D3 and retinoic acid (all trans or 9-cis)

1,25(OH)₂D₃ and two stereoisomers of retinoic acid, all trans and 9-cis retinoic acid, are regulators of cell proliferation and differentiation. The aim of this study was to evaluate the effects of a combination of 1,25(OH)₂D₃ and retinoic acid (all trans or 9-cis) on proliferation and cell differentiation of the human promyelocytic leukemia cell line HL60, and to test the reversibility of the induced differentiation. Cell proliferation was inhibited as expected by 1,25(OH)₂D₃ and all trans retinoic acid alone (IC₅₀ of cell survival was 4 × 10⁻⁷ M, 9 × 10⁻⁶ M and 9 × 10⁻⁷ M for 1,25(OH)₂D₃, all trans and 9-cis retinoic acid, respectively). Combination of 1,25(OH)₂D₃ and either form of retinoic acid resulted in a partially additive decrease in cell proliferation. 1,25(OH)₂D₃ induced a monocytic differentiation (100% CD14+ cells with 10⁻⁷ M 1,25(OH)₂D₃), while retinoic acid led to a predominantly granulocytic differentiation (36 and 42% CD67+ cells with 10⁻⁶ M all trans and 9-cis retinoic acid, respectively). Additive effects on differentiation were observed upon combination of subtherapeutical doses of the drugs, achieving a mainly monocytic population, demonstrating the dominant role of 1,25(OH)₂D₃ in determining the direction of differentiation. The effects on proliferation and differentiation of the solitary drugs were reversible, while the proliferation arrest and differentiation induced by the combination persisted and even progressed after withdrawal of the drugs. We conclude that 1,25(OH)₂D₃ and retinoic acid (all trans or 9-cis) exert additive effects on inhibition of proliferation and induction of cell differentiation of HL60 cells, leading to a persistent differentiation, even after drug withdrawal.     

All-trans-retinoic acid-mediated modulation of p53 during neural differentiation in murine embryonic stem cells

All-trans-retinoic acid (RA) plays an important physiological role in embryonic development and is teratogenic in large doses in almost all species. p53, a tumor suppressor gene encodes phosphoproteins, which regulate cellular proliferation, differentiation, and apoptosis. Temporal modulation of p53 by retinoic acid was investigated in murine embryonic stem cells during differentiation and apoptosis. Undifferentiated embryonic stem cells express a high level of p53 mRNA and protein followed by a decrease in p53 levels as differentiation proceeds. The addition of retinoic acid during 8–10 days of differentiation increased the levels of p53 mRNA and protein, accompanied by accelerated neural differentiation and apoptosis. Marked increase in apoptosis was observed at 10–20 h after retinoic acid treatment when compared with untreated controls. Retinoic acid-induced morphological differentiation resulted in predominantly neural-type cells. Maximum increase in p53 mRNA in retinoic acid-treated cells occurred on day 17, whereas maximum protein synthesis occurred on days 14–17, which coincided with increased neural differentiation and proliferation. Increased p53 levels did not induce p21 transactivation, interestingly a decrease in p21 was observed on day 17 on exposure to retinoic acid. The level of p53 declined by day 21 of differentiation. The results demonstrated that retinoic acid-mediated apoptosis preceded the changes in p53 expression, suggesting that p53 induction does not initiate retinoic acid-induced apoptosis during development. However, retinoic acid accelerated neural differentiation and increased the expression of p53 in proliferating neural cells, corroborated by decreased p21 levels, indicating the importance of cell type and stage specificity of p53 function. This revised version was published online in July 2006 with corrections to the Cover Date.     

PKC delta and NADPH oxidase in retinoic acid-induced neuroblastoma cell differentiation

The role of reactive oxygen species (ROS) in the regulation of signal transduction processes has been well established in many cell types and recently the fine tuning of redox signalling in neurons received increasing attention. With regard to this, the involvement of NADPH oxidase (NOX) in neuronal pathophysiology has been proposed but deserves more investigation. In the present study, we used SH-SY5Y neuroblastoma cells to analyse the role of NADPH oxidase in retinoic acid (RA)-induced differentiation, pointing out the involvement of protein kinase C (PKC) delta in the activation of NOX. Retinoic acid induces neuronal differentiation as revealed by the increased expression of MAP2, the decreased cell doubling rate, and the gain in neuronal morphological features and these events are accompanied by the increased expression level of PKC delta and p67^phox, one of the components of NADPH oxidase. Using DPI to inhibit NOX activity we show that retinoic acid acts through this enzyme to induce morphological changes linked to the differentiation. Moreover, using rottlerin to inhibit PKC delta or transfection experiments to overexpress it, we show that retinoic acid acts through this enzyme to induce MAP2 expression and to increase p67^phox membrane translocation leading to NADPH oxidase activation. These findings identify the activation of PKC delta and NADPH oxidase as crucial steps in RA-induced neuroblastoma cell differentiation.     

Alterations in polyamine metabolism during embryonal carcinoma cell differentiation in vitro

Murine embryonal carcinoma (EC) cells can be stimulated to differentiate by several chemical inducers. Since the response of EC cells to induction is likely to occur shortly after exposure to the inducer, we report here the changes that occur in polyamine levels in a number of EC cell lines shortly after exposure to two chemical stimuli, alpha-difluoromethylornithine (DFMO) and retinoic acid (RA). Our results suggest that polyamine levels are important in determining the state of EC cell differentiation, but that reduction in these levels alone is not sufficient to induce differentiation in all the EC cell lines tested. Also, it is apparent that RA does influence levels of polyamines. However, this influence does not seem to be mediated through direct interaction with ODCase.