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.     

Transforming growth factor beta 1 partially suppresses the transformed phenotype of ras-transformed hepatocytes.

The effect of transforming growth factor beta type 1 (TGF-beta 1) on DNA synthesis, anchorage-dependent and anchorage-independent proliferation, cytoskeletal organization, and gene expression in ras-transformed simian virus 40 (SV40)-immortalized hepatocyte cell lines was measured. An SV40-immortalized cell line (CWSV1), a control neo-transfected and selected cell line (N1), and neo+ras-transfected and selected cell lines (NR3 and NR4) were used for this study. CWSV1 and N1 cells do not grow in soft agarose and are not tumorigenic. The ras-transformed hepatocytes NR3 and NR4 grow in soft agar and are tumorigenic. TGF-beta 1 treatment did not inhibit DNA synthesis or anchorage-dependent growth in the SV40-immortalized hepatocyte cell line CWSV1 or in the ras-transformed hepatocytes. TGF-beta 1 treatment inhibited anchorage-independent growth, increased actin cytoskeleton organization, and altered the morphology of ras-transformed hepatocytes; that is, with regard to all three of these properties, TGF-beta 1-treated ras-transformed hepatocytes more closely resembled the immortalized parent cell line. c-Ha-ras and c-myc RNA levels were not altered in TGF-beta 1-treated NR4 cells. TGF-beta 1 treatment did alter expression of some genes in NR4 cells. The level of expression of alpha 1 integrin RNA was higher in CWSV1 cells than in NR4 cells and increased in NR4 cells when they were treated with TGF-beta 1. Similarly, the levels and profiles of integrins on the cell surface of CWSV1 cells compared to NR4 cells, as determined by cell surface protein iodination, differed and in TGF-beta 1-treated NR4 cells more closely resembled the surface integrin profile for CWSV1 cells.     

Decreased transforming growth factor-beta response and binding in insulin-independent teratoma-derived cell lines with increased tumorigenic properties.

The mouse C3H teratoma-derived cell line 1246 is an adipogenic cell line which stringently requires insulin to proliferate and differentiate in defined medium. From this cell line an insulin-independent cell line called 1246-3A was isolated. It was found that, in contrast to 1246 cells, 1246-3A cells had lost the ability to differentiate and became tumorigenic when injected at a density of 10(6) cells/mouse into syngeneic host C3H mice. In addition, they produce in their culture medium transforming growth factor alpha- and beta-like polypeptides which stimulate their proliferation. Highly tumorigenic insulin-independent cell lines able to give rise to tumor when injected at a density of 10(4) cells/mouse were isolated by using an in vitro-in vivo shuttle technique. The highly tumorigenic cell lines have lost the response to TGF-beta 1. The binding of TGF-beta 1 to the nontumorigenic parent cell line or to cells displaying increased tumorigenic properties was investigated. The data presented here indicate that the increased tumorigenicity is accompanied by a progressive decrease of specific binding of TGF-beta 1 to the cells. However, the decreased number of cell surface TGF-beta 1 binding sites in the highly tumorigenic cells did not correlate with an increase in the secretion of TGF-beta protein by the tumorigenic cells, as all of TGF-beta produced by the cells was in a latent form. Affinity cross-linking experiments indicated that the 1246 cell line displayed several TGF-beta cross-linked molecular species (MW 140, 92, and 70 kDa). Increase of tumorigenicity was accompanied by a marked decrease in the intensity of the cross-linked bands, particularly of the 92 and 70 kDa species.     

Retinoic acid stimulates the differentiation of PC12 cells that are deficient in cAMP-dependent protein kinase

Retinoic acid (RA) induced neuronal differentiation in A126-1B2 cells and 123.7 cells, two mutant lines of PC12 that are deficient in cAMP-dependent protein kinase, but not in the parental PC12 cell line. A single exposure to RA was sufficient to cause neurite formation and inhibit cell division for a period of greater than 3 wk, suggesting that RA may cause a long-term, stable change in the state of these cells. In A126-1B2 cells, RA also induced the expression of other markers of differentiation including acetylcholinesterase and the mRNAs for neurofilament (NF-M) and GAP-43 as effectively as nerve growth factor (NGF). Neither NGF nor RA stimulated an increase in the expression of smg-25A in A126-1B2 cells, suggesting that the cAMP-dependent protein kinases may be required for an increase in the expression of this marker. RA also caused a rapid increase in the expression of the early response gene, c-fos, but did not effect the expression of egr-1. RA equivalently inhibited the division of A126-1B2 cells, 123.7 cells and parental PC12 cells, so RA induced differentiation is not an indirect response to growth arrest. In contrast, the levels of retinoic acid receptors (RAR alpha and RAR beta), and retinoic acid binding protein (CRABP) mRNA were strikingly higher in both A126-1B2 cells and 123.7 cells than in the parental PC12 cells. The deficiencies in cAMP-dependent protein kinase may increase the expression of CRABP and the RARs; and, thus, cAMP may indirectly regulate the ability of RA to control neurite formation and neural differentiation. Thus, RA appears to regulate division and differentiation of PC12 cells by a biochemical mechanism that is quite distinct from those used by peptide growth factors.     

HL-60-AR白血病细胞诱导分化性状的持续性表达

HGPRT~-人早幼粒白血病细胞突变株(HL-60-AR)与RA保温一定时间后,洗去药物继续培养,细胞分化性状(NBT还原能力、细胞膜C_3补体受体及形态变化)不但继续存在,而且能持续表达。撤去RA后连续传代培养,至少在传三代后细胞分化性状仍高度表达。然而,DMSO对HL-60-AR细胞的作用特点明显不同于RA。HL-60-AR细胞分化伴随增殖能力的降低。核酸分子杂交结果表明,细胞c-myc癌基因表达受抑先于细胞分化性状的获得和增殖能力的下降。     

Differentiation of F9 embryonal carcinoma cells

We found that monolayer cultures of F9 cells induced to differentiate with trans-retinoic acid (RA) contain two major subpopulations of cells. These two cell types can be distinguished by their cellular morphology, their pattern of laminin accumulation, and their ability to undergo further differentiation in response to N6-O2-dibutyryl adenosine 3':5' cyclic monophosphoric acid (dBcAMP). Furthermore, the developmental pathway induced by RA appears to lead to two alternative pathways, and differentiation at the branch point is either directly or indirectly controlled by cAMP. Differentiation along one branch of this pathway can be induced by 5-bromodeoxyuridine, whereas differentiation along an unrelated pathway is induced by N'-N'-dimethylacetamide. In all cases, differentiation is closely paralleled by suppression of the tumorigenic phenotype, indicating that these two processes are tightly linked and probably share a common step.     

Characteristics of cyclic AMP enhancement of retinoic acid induction of increased transglutaminase activity in HL60 cells

When the human myeloid leukemia cell line (HL60) is induced to differentiate with retinoic acid (RA), there is a concentration-dependent increase in transglutaminase (TGase) activity which peaks on day 5. While dibutyryl 3',5'-cyclic adenosine monophosphate (db-cAMP) alone produced only a slight increase in TGase activity in HL60 cells, the concomitant addition of db-cAMP (100 microM) with RA (10(-12)-10(-4) M) potentiates RA induction of TGase activity. Maximal increases in TGase activity (2- to 10-fold) were observed with 10(-4)-10(-7) M RA and when db-cAMP was present from 24 to 48 h after the addition of RA. The cyclic nucleotide enhancement was dose-dependent from 10 to 100 microM of cAMP. Less marked increases were observed with 8-bromo-cAMP and with the phosphodiesterase inhibitor theophylline. Although the simultaneous addition of PGE1 or PGE2 (10(-8)-10(-6) M) produced no enhancement of RA-induced TGase activity, adding PGE1 or PGE2 24 or 48 h following RA treatments produced an enhancement of TGase activity. The phosphodiesterase inhibitor potentiated the increases produced by db-cAMP and the prostaglandins. Dibutyryl cAMP enhanced the ability of RA to induce the cells to reduce nitroblue tetrazolium (NBT), a functional measure of differentiation, at lower concentrations of RA and with shorter treatment durations. cAMP potentiates RA-induced TGase activity in HL60 cells and the combination appears to be associated with enhanced RA-induced differentiation.     

Retinoic Acid Rapidly Decreases Phosphatidylinositol Turnover During Neuroblastoma Cell Differentiation

Phosphatidylinositol (PI) turnover has recently been implicated in the regulation of cell proliferation and transformation. We have investigated its role in differentiation using LAN-1 cells, a human neuroblastoma cell line that can be induced to differentiate along the neuronal pathway by retinoic acid (RA). We have found that treatment of LAN-1 cells with RA is followed by a rapid decrease of inositol phospholipid metabolism, using myo-[1,2-3H]inositol or [1(3)-3H]glycerol. No changes were observed in both [3H]inositol and [3H]glycerol uptake within 24 h of RA treatment. Decreased incorporation of the metabolic precursor into PI 4-monophosphate and PI 4,5-bisphosphate occurred within 1 h of RA treatment. No changes were seen in the specific radioactivity of the precursor pools up to 1 h of treatment with RA. Analysis of labeled PI metabolites from prelabeled cells indicated a rapid decrease of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol content within 1 min of induction of LAN-1 cell differentiation. These findings constitute the earliest reported events in neuroblastoma cell differentiation.     

Retroviral producer cells cause sarcoma in severe combined immunodeficiency mice

Direct in situ introduction of retroviral producer cells might provide a form of treatment for localized tumors. A possible undesirable consequence of this treatment could be uncontrolled proliferation of the injected producer cells. To test this possibility, severe combined immunodeficiency (SCID) mice were reconstituted with human peripheral blood lymphocytes which were marked with a retroviral vector using a coculture method. Although specific measures were taken to remove the possible contaminating producer cells, a high percentage of mice developed fibrosarcoma 2–6 weeks after reconstitution. We hypothesized that tumors arose from a small number of contaminating producer cells in the inoculum. Tumor cells were consistently DNA tetraploid, a characteristic of the producer cell line. DNA extracted from tumor tissue was found to contain the gene (neomycin phosphotransferase) used to mark the producer cell line. Furthermore, SCID mice injected with 1 × 10⁴ producer cells developed tumors with analogous characteristics. This report indicates that the retroviral producer cell line is tumorigenic in immune-deficient animals.     

Complementation analyses of differentiation-defective embryonal carcinoma cells

We have generated cell hybrids by fusing embryonal carcinoma (EC) cells which fail to differentiate in response to retinoic acid (RA) and/or hexamethylenebisacetamide (HMBA). The first two classes of hybrids were between an RA- line (also unresponsive to HMBA) that lacks cellular RA binding protein (cRABP) activity and HMBA- lines which possess cRABP and differentiate in the presence of RA. All of the hybrid clones possessed cRABP and differentiated normally upon exposure to either RA or HMBA. When the aforementioned RA- mutant was fused with a second mutant which was refractory to RA and HMBA but possessed cRABP activity, the resultant hybrid clones were responsive to both RA and HMBA and had cRABP activity. These results suggest that all of these mutants were recessive and complementary. Tumors from these hybrid lines differentiated extensively, in some instances much more so than the mutant parental lines and even the wild-type lines from which the mutants were derived. Based upon these observations, we propose that various EC lines might differentiate poorly in tumor form for different reasons. Hybrids between two differentiation-defective, cRABP- lines appeared to be at least partially complemented for responsiveness to RA and HMBA. These hybrids contained low but detectable levels of cRABP. This is not a consequence of tetraploidy since fusions between cells from the same mutant line retained their differentiation-defective phenotype and possessed little or no cRABP activity. Unlike tumors from the other hybrids described above, tumors from these hybrid lines expressed a very restricted pattern of differentiated cell types. This might be because the mutant lines in the latter hybrids originally derived from the same wild-type EC line.     

Retinoic acid inhibits phosphatidylinositol turnover only in RA-sensitive while not in RA-resistant human neuroblastoma cells

Phosphatidylinositol (PI) turnover has recently been implicated in the regulation of cell proliferation and transformation. We have investigated its role in differentiation using LAN-1 cells, a human neuroblastoma cell line which can be induced to differentiate along the neuronal pathway by retinoic acid (RA), and a derivated RA-resistant subline of it (LAN-1-res). We have found that treatment of LAN-1 cells with RA is followed by a rapid decrease of inositol phospholipid metabolism, using myo-[1,2-3H] inositol or [1,(3)-3H] glycerol. Analysis of labelled phosphatidylinositol metabolites from prelabelled LAN-1 cells indicated a rapid decrease of inositol (1,4,5)-trisphosphate and (1,2) diacylglycerol within 1 min. of induction of differentiation by RA, while no changes were observed in RA-treated LAN-1-res cells. These findings indicate that phosphoinositides-derived metabolites may be directly implicated in the induction processes of RA-triggered NB cell differentiation.     

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.