Induction of F9 embryonal carcinoma cell differentiation by inhibition of polyamine synthesis

alpha-Difluoromethylornithine (DFMO), a highly selective inhibitor of ornithine decarboxylase (ODC), induced terminal differentiation of F9 mouse embryonal carcinoma cells in culture. Differentiation was assessed using morphological criteria and the level of plasminogen activator activity. The observed phenotypic changes and the fact that the cells did not synthesize alpha-fetoprotein, indicate that they were parietal endoderm cells. The putrescine, spermidine and spermine content of untreated control cells increased during exponential growth and then decreased gradually with continued time in culture. The increases in putrescine and spermidine contents were prevented by DFMO treatment. In fact, the putrescine and spermidine content decreased below the limits of detection after only one day of treatment. The addition of putrescine to the culture medium at any time within 4 days of DFMO treatment, prevented the DFMO-induced differentiation, suggesting that the effects observed were indeed caused by polyamine depletion. The phenotypic changes induced by DFMO were similar to those induced by retinoic acid, a very potent inducer of embryonal carcinoma differentiation. Although retinoic acid can inhibit ODC activity and putrescine accumulation, it is unlikely that this mechanism of action is responsible for retinoic acid-induced F9 cell differentiation, inasmuch as putrescine addition did not prevent the expression of the differentiated phenotype. Undifferentiated F9 embryonal carcinoma cells exhibited a very short G1 phase, and in this respect they are similar to the cells of the preimplantation mouse embryo. In control (exponentially growing) cultures a majority of the F9 cells were in the S phase, but in DFMO-treated cultures they accumulated in the G1 phase and showed no further proliferative potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of tunicamycin on the differentiation of F9 cells induced by either retinoic acid or retinoic acid and dibutyryl cyclic AMP

Tunicamycin (0.5 micrograms/ml) inhibited differentiation of F9 cells treated either with retinoic acid or with retinoic acid and dibutyryl cyclic AMP, as monitored by the activity of alkaline phosphatase and expression of cytokeratins. On the other hand, the pattern of the polysaccharide chain synthesis changed drastically with the treatment irrespective of the presence of tunicamycin. Therefore, phenotypes induced with retinoic acid are dissociated into two categories, one that is directly induced by the drug and the other that is induced indirectly by a mechanism in which glycoproteins play a role.     

Comparative proteomic analysis of differentiation of mouse F9 embryonic carcinoma cells induced by retinoic acid

The multipotent mouse F9 embryonic carcinoma cell is an ideal model system to investigate the mechanism of retinoic acid (RA) in cell differentiation and cell growth control and the biochemical basis of early embryonic development. We reported here a proteomics approach to study protein expression changes during the differentiation of F9 cells into the visceral endoderm. F9 cells were incubated with or without RA at 0, 24, 48, and 72 h. Total proteins extracted were separated by two‐dimensional electrophoresis (2‐DE) and the protein patterns on the gels were comparatively analyzed by computer. Approximately 1,100 protein spots were detected in the F9 proteome, within the pH 3–10 range. Fourteen protein spots which the levels of expression were found to be altered dramatically during the F9 cells differentiating, and were identified by MALDI‐TOF MS or ESI‐MS/MS. These proteins included metabolism enzymes, HSP60s, RAN, hnRNP K, FUBP1, VDAC1, STI1, and prohibitin. These proteins are involved in cellar metabolism, gene expression regulation, stress response, and apoptosis, respectively. The data from proteomic analyze are consistent with the result obtained from Western blot analysis. This study increases our understanding of the proteomics changes during F9 cells differentiation induced by RA. J. Cell. Biochem. 113: 1811–1819, 2012. © 2012 Wiley Periodicals, Inc.     

Cyclic adenosine monophosphate-mediated induction of F9 teratocarcinoma differentiation in the absence of retinoic acid.

F9 teratocarcinoma stem cells differentiate into parietal endoderm-like cells when given retinoic acid (RA) and dibutyryl cyclic adenosine monophosphate (DB-cAMP). It is generally accepted that the stem cells are resistant to the action of cAMP alone and need to be primed by RA in order to respond to cAMP. In this report, we demonstrate that F9 stem cells differentiate into parietal endoderm-like cells in the absence of exogenous RA when treated with cholera toxin and 1-methyl,3-isobutyl xanthine (CT/MIX) or 8-bromo-cAMP/MIX (8B2-cAMP/MIX). Cells treated with CT/MIX or 8B2-cAMP/MIX were morphologically similar to parietal endoderm-like cells, produced high amounts of plasminogen activator, and synthesized both type IV collagen and laminin mRNA. Conversely, markers made in abundance by stem cells such as stage-specific embryonic antigen (SSEA-1) and an mRNA species of 6.8 kb (pST6-135) were markedly reduced in CT/MIX-treated cells. To prove that cAMP alone could induce differentiation Lipidex-1000, a hydrophobic gel, was used to remove 80-90% of the endogenous serum retinoids. F9 cells grown in this retinoid-depleted serum and treated with 8B2-cAMP/MIX differentiated to parietal endoderm-like cells as shown by both dramatic changes in morphology and induction of type IV collagen mRNA. Our results indicate that the differentiation of F9 to parietal endoderm-like cells can be induced by increased intracellular cAMP and is not strictly dependent on the addition of RA.     

Biphasic response to retinoic acid dose in differentiation of F9 cells into primitive endoderm-like cells

The differentiation of F9 cells, as monitored by the secretion of basement membrane proteins, was biphasic depending on the dose of retinoic acid (RA) used. Secretion of laminin A reached a plateau at about 10 nM and increased again at more than 100 mM RA. A similar biphasic response was observed in the secretion of tissue-type plasminogen activator as well. In the presence of RA alone, this biphasic pattern of differentiation was stable for a long period of time, but the addition of 1 mM dibutyryl cAMP changed it to monophasic after 12 days of exposure.     

Induction of HL-60 cell differentiation by water-soluble and nitrogen-containing conjugates of retinoic acid and retinol

Retinoids induce the promyelocytic cell line, HL-60, to differentiate along the granulocytic pathway in vitro. A number of water-soluble and nitrogen-containing retinoids were synthesized in our laboratory [retinoyl-glucose (RAGL), retinyl-glucose (ROGL), retinoyl-adenosine (RADS), retinoyl-adenine (RAD), retinoyl-beta-glucuronide (beta RAG), and retinoyl-alpha-glucuronide (alpha RAG)]. These retinoids (10(-5) to 10(-8) M), as well as retinoic acid (RA) and retinol (ROL), were tested for their ability to induce the differentiation of HL-60 cells in vitro and to affect cell growth and viability during a 24- to 72-h incubation period. Differentiation was assessed by measuring the percentage of cells expressing the Mac-1 antigen on their cell surfaces. RA and the conjugates of RA were all quite active in inducing HL-60 cell differentiation, whereas ROL and ROGL had much less activity at equimolar concentrations. beta RAG, alpha RAG, RADS, and RAD were less toxic, whereas the glucose conjugates of retinol and retinoic acid (ROGL and RAGL) were both considerably more toxic than either RA or ROL at equimolar concentrations. All retinoids affected cell growth in a dose-dependent fashion. At 24 h, free RA or ROL was not detected in the cells after incubation with any of the retinoid conjugates.     

Induction of alkaline phosphatase by retinoic acid

Treatment of mammalian cells in culture with retinoic acid causes a time- and concentration-dependent increase of the specific activity of alkaline phosphatase. The increase reaches a factor of 15 and more and begins at a concentration of 10(-8)M retinoic acid. The induction is inhibited by cycloheximide or actinomycin D. The same isoenzyme of alkaline phosphatase is expressed in control and in retinoic acid-treated cells as demonstrated by the inhibitions by amino acids and peptides. The enzyme induction occurs in rat heart, skeletal muscle, brain, lung cells and HeLa cells. No induction was found in two lines of human melanoma cells. After treatment of cells with tunicamycin, the induction of alkaline phosphatase is detectable only in the homogenate and no longer detectable by histochemical methods. This shows that the glycosylation of the protein is an important step in the insertion of this enzyme into the plasma membrane.     

Modulation of protein biosynthesis during early stages of differentiation in retinoic acid treated F9 teratocarcinoma cells

Modulation of protein biosynthesis by retinoic acid during induction of differentiation of F9 teratocarcinoma stem cells was investigated by using computerized analysis of double label autoradiography of two-dimensional polyacrylamide gels. As early as 6 h after induction increased synthesis of 5 and decreased synthesis of 2 proteins occur. By 12 h after induction, synthesis of 13 proteins is elevated and by 24 h that of 17. At 24 h the range of stimulation is from two- to fourfold, as demonstrated by a 3H:14C ratio divided by the mode ratio. Examination of the Gaussian distributions of frequency of ratio indicates that many subtle changes in protein synthesis accompany the development of the new phenotype.     

The induction of antigenic changes in a teratocarcinoma stem cell line (F9) by retinoic acid

Treatment of embryonal carcinoma cells F9 with retinoic acid results in the appearance of epithelioid cells resembling endoderm which synthesize basement membrane protein and plasminogen activator. Concomitant with the appearance of these properties of differentiated cells, the epithelial cells cease to express SSEA-1, an antigenic determinant characteristic of teratocarcinoma stem cells and early mouse embryos. Our evidence indicates that the phenotypic changes that accompany retinoic acid treatment of embryonal carcinoma cells are irreversible and a consequence of the differentiation of the cells into endoderm.     

Butyrate inhibits the retinoic acid-induced differentiation of F9 teratocarcinoma stem cells

F9 mouse teratocarcinoma stem cells differentiate into parietal endoderm cells in the presence of retinoic acid, dibutyryl cyclic AMP, and theophylline (RACT). When F9 cells are exposed to 2-5 mM sodium butyrate plus RACT, they fail to differentiate. Differentiation is assessed by induction of laminin and collagen IV mRNA, the synthesis of laminin, collagen IV and plasminogen activator proteins, and alterations in cell morphology. Butyrate inhibits differentiation only when added within 8 hr after retinoic acid addition. Thus an early event in retinoid action on F9 cells is butyrate-sensitive. The population doubling time and cell cycle distribution of F9 cells are not altered within the first 24 hr after butyrate addition, suggesting that butyrate does not inhibit differentiation by inhibition of growth or normal cycling. However, butyrate does inhibit histone deacetylation in F9 cells, and this could be the mechanism by which butyrate inhibits differentiation.     

Induction of matrix metalloproteinase MMP-9 (92-kDa gelatinase) by retinoic acid in human neuroblastoma SKNBE cells: relevance to neuronal differentiation

Retinoic acid (RA) has been shown to induce human neuroblastoma SKNBE cell differentiation into a neuronal phenotype. Whether this neuronal differentiation is associated with modulation of matrix gelatinase [matrix metalloproteinase (MMP)-2 and MMP-9] expression was investigated in SKNBE cell cultures exposed to RA for 14 days. Their differentiation into a neuronal phenotype was typified by neural cell adhesion molecule and growth-associated protein-43 expression. Gelatinase expression was assessed by gel zymography, quantitative RT-PCR, and immunocytochemistry. Neuronal markers were located in neurites and ganglion-like clusters of neuronal cells induced upon RA exposure. MMP-2 expression was constitutive and remained unchanged at both the mRNA and protein levels in response to RA, tumor necrosis factor-alpha (TNFalpha), or phorbol 12-myristate 13-acetate (PMA) treatment. In contrast, MMP-9 was inducible by RA, TNFalpha, or PMA. MMP-9 was progressively enhanced by RA as a function of time exposure until day 14. The addition of TNFalpha or PMA potentiated RA-induced MMP-9 expression with a synergic maximal effect at day 14 of RA exposure. Immunoreactive MMP-9 was located early in outgrowing neurites, but only at day 14 of RA exposure in extensive neuritic networks. Taken together, the correlation between the MMP-9 expression by SKNBE cells and the time scale of their differentiation into a neuronal phenotype allowed us to propose that MMP-9 could participate in the neurite growth process and cell migration and organization into ganglion-like clusters.     

Response of human rhabdomyosarcoma cell lines to retinoic acid: relationship with induction of differentiation and retinoic acid sensitivity

The ability of retinoids to induce growth inhibition associated with differentiation of diverse cell types makes them potent anti-cancer agents. We examined the effect of retinoic acid (RA) in cell lines derived from rhabdomyosarcoma (RMS), a malignant soft-tissue tumor committed to the myogenic lineage, but arrested prior to terminal differentiation. We showed that several RMS derived cell lines, including RD human rhabdomyosarcoma cells, are resistant to the growth-inhibitory and differentiation effects of RA. We established that this RA-resistance correlates with reduced expression and activity of RA-receptors in RD cells. We stably expressed either RARalpha, RARbeta, RARgamma, or RXRalpha expression vector into RD cells and found that only RARbeta or RARgamma induced a significant RA growth arrest without promoting differentiation indicating that changes in the amounts of RARs and RXRs are not sufficient to determine the RA myogenic response of rhabdomyosarcoma cells. Activation of RD cell differentiation by ectopic MRF4 expression enhanced RA-receptor activity and led to RA induction of differentiation. These studies demonstrate that RA-resistance of RD cells is linked to their lack of differentiation and suggest that the differentiation-promoting activity of RA requires factors other than RAR-RXR heterodimers.     

ER stress response during the differentiation of H9 cells induced by retinoic acid

Endoplasmic reticulum (ER) stress occurs during early embryonic development. The aim of this study is to determine whether ER stress occurs during human embryonic stem cell differentiation induced by retinoic acid (RA). H9 human embryonic stem cells were subjected to RA treatment for up to 29days to induce differentiation. HEK293 cells were treated with RA as a control. The results demonstrate that several ER stress-responsive genes are differentially regulated in H9 and HEK293 cells in response to 5days of RA treatment. GRP78/Bip was upregulated in H9 cells but downregulated in HEK293 cells. eIF2α was downregulated in H9 cells but not in HEK293 cells. Phosphorylation of eIF2α was downregulated in H9 cells but upregulated in HEK293 cells. XBP-1 was downregulated immediately after RA treatment in H9 cells, but its downregulation was much slower in HEK293 cells. Additionally, two ER-resident E3 ubiquitin ligases, gp78 and Hrd1, were both upregulated in H9 cells following 5 days of exposure to RA. Moreover, the protein Bcl2 was undetectable in H9 cells and H9-derived cells but was expressed in HEK293 cells, and it expression in the two types of cells was unaltered by RA treatment. In H9 cells treated with RA for 29 days, GRP78/Bip, XBP-1 and Bcl2 were all upregulated. These results suggest that ER stress is involved in H9 cell differentiation induced by RA.     

Studies on the relation of DNA synthesis to retinoic acid-induced differentiation of F9 teratocarcinoma cells

Inhibition of DNA synthesis in F9 embryonal carcinoma cells with high thymidine induces differentiation similar to that induced with retinoic acid (RA). The presence of differentiated cells is evident after 15 h of treatment with 2 mM thymidine, during which period DNA synthesis is inhibited 99%. The addition of RA during the period of high thymidine treatment does not increase the amount of differentiation seen at the end of the 15-h treatment, but does increase the amount seen after thymidine is removed. The inhibition of proliferation by low serum concentration does not induce differentiation in the absence of RA. In partially synchronized cultures of F9 cells, the addition of RA alters the pattern of DNA replication during the first third of S phase. If RA is present during this part of S phase, differentiation is evident both morphologically and biochemically during the following cell cycle. Addition of RA during the second half of S phase does not lead to obvious differentiation until after the next cell cycle. These results suggest that particular events during the early replication period of F9 cells are targets for RA action in induction of differentiation of F9 cells.     

Stimulation of differentiation of several murine embryonal carcinoma cell lines by retinoic acid

Retinoic acid stimulates several murine embryonal carcinoma (EC) cell lines, even those previously considered to be incapable of differentiating, to give rise to cell types distinguishable from the parental phenotype in morphology, production of plasminogen activator and surface protein properties. Retinoic acid promotes these changes over a range of low concentrations (10⁻⁹–10⁻⁵ M) which are generally non-toxic to the cells. The effects are clearly demonstrated when EC cells are aggregated prior to exposure to retinoic acid. It is concluded that the observed phenotypic alterations induced by retinoic acid reflect differentiation of the EC cells since non-EC cell characteristics are maintained by cloned cells several generations after retinoic acid is removed from the cultures. Our studies suggest that although retinoic acid stimulates the conversion of EC cells to differentiated derivatives, it does not influence the direction of differentiation. Furthermore, the effectiveness of retinoic acid in stimulating differentiation of EC cells from lines such as Nulli-SCC1 raises the question of whether true ‘nullipotent’ EC lines really exist.