Emergence of TPA-resistant ‘satellite’ cells during muscle histogenesis of human limb

Human satellite cells, obtained by surgical biopsies of traumatized legs of healthy individuals, were grown in culture in the presence of different concentrations of the phorbol ester tetradecanoyl-phorbol 12 acetate (TPA). Satellite cells, after an initial duplicative period, fused into large multinucleated myotubes which readily synthesized myosin and acetylcholine receptor (AChR). The presence of TPA at concentrations up to 10(-7) M did not affect the differentiation pattern, while higher concentrations were toxic. Thus human satellite cells are capable of differentiating in the presence of phorbol esters which block differentiation of embryonic myoblasts [1]. We then examined the appearance of TPA-resistant cells during human muscle histogenesis, since we had observed that differentiation of human myoblasts from a 6-week-old limb was completely and reversibly inhibited by 10(-7) M TPA. Differentiation of myoblasts from 6-, 7- and 8-week-old fetuses was completely inhibited by TPA. Myoblasts from 10-week-old limbs did not form myotubes in the presence of TPA; however, immunohistochemical staining with an antimyosin antibody revealed the presence of a few mononucleated myosin-positive cells which escaped the TPA-induced block of differentiation. At 12 weeks of development, a few oligonucleated, myosin-positive myotubes developed in cultures treated with TPA, and the level of AChR expressed (measured as [125I] alpha-bungarotoxin bound) reached 20% of controls. At 14 weeks of development, about half of the cells in culture were TPA-resistant and by 16 weeks of development no major differences could be detected between control and treated cells. We conclude from these data that a population of TPA-resistant myogenic cells emerges between the 10th and 14th week of human limb development and suggest that this population represents satellite cells.     

Induction of human malignant T-lymphoblastic cell lines MOLT-3 and jurkat by 12-O-tetradecanoylphorbol-13-acetate: Biochemical,physical, and morphological characterization

The process of induction of human malignant T-lymphoblastic cell line MOLT-3 by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined. It was found that the induction process by TPA, which included increase in cells with receptors to sheep red blood cells (E-rosette positive-E⁺) and decrease in the levels of the marker enzyme terminal deoxynucleotidyl transferase (TdT) was not affected by the presence of DNA synthesis inhibitor arabinofuranosylcytosine (Ara-C). The exposure time to TPA required to elicit these changes was found to be short, in the order of 1 hour or less. The kinetics of the increase in E⁺ cells, decrease in the levels of TdT in these cells, or decrease in the ability to proliferate as measured by colony formation were similar with exposure to TPA for 1, 6, 24, or 96 hours. We have examined the effect of antitumor promoter compounds on their ability to block induction of MOLT-3 cells by TPA. Results indicated that none of these compounds, dexamethasone, antipain, retinoic acid, and L-1-tosylamide-2-phenylethylchloromethyl ketone (TPCK), was effective in reducing the number of E⁺ cells induced by TPA. Examination of three other leukemic T-cell lines indicated that, in addition to MOLT-3, the leukemic T-cell line Jurkat also responded to TPA, whereas two other leukemic T-cells lines CCRF-CEM and CCRF-HSB-2 did not. Certain physical and morphological changes were also observed after stimulation of MOLT-3 cells and Jurkat cells by TPA. We found that, following the addition of TPA, the cell volumes of MOLT-3 cells decreased from an average of 1150 μm³ to about 500 μm³, whereas those of Jurkat were reduced to about 700 μm³ from 1100 μm³. Electron microscopic studies of these lymphoblasts also revealed that after treatment with TPA the induced cells were generally smaller in size with increase in the density of the nuclear materials and condensation of the chromatin structures.     

Role of phorbol ester receptors in the 12-0-tetradecanoyl-phorbol-13-acetate (TPA)-induced down-regulation of colony-stimulating factor (CSF-1) binding to murine peritoneal exudate macrophages

Treatment of murine peritoneal exudate macrophages (PEM) by tumor-promoting phorbol esters (TPA) results in a rapid loss of binding activity to radioactive-labeled colony-stimulating factor ([125I]-CSF-1) on the cell surface. The inhibitory effect of TPA on PEM is transient; treated cells recover full [125I]-CSF-1 binding activity in less than 6 hr at 37 degrees C either in the presence or after the removal of added TPA. The role of phorbol ester receptors in the induction of [125I]-CSF-1 binding inhibition was studied. The biologically active ligand [3H]-phorbol 12,13-dibutyrate ([3H]-PDBu) bound specifically to cultured murine PEM. At 0 degree C, stable and equilibrium binding occurred after 2-3 hr. Scatchard analysis revealed linear plots with a dissociation constant and receptor number per cell of 20.9 nM and 3.9 X 10(5)/cell, respectively. Treatment of PEM with biologically active phorbol esters at 37 degrees C rapidly inhibited the binding activity of [3H]-PDBu on cell surface (down-regulation) and rendered these cells refractory to the TPA-induced [125I]-CSF-1 binding inhibition by the subsequent TPA treatment. The inhibition of phorbol ester binding activity on TPA-treated PEM is caused by a reduction in the total number of available phorbol ester receptors rather than by a decrease in receptor affinity as judged by Scatchard analysis. The disappearance of [3H]-PDBu binding activity is reversible and transient. However, unlike CSF-1 receptors the restoration of phorbol ester receptors on TPA-treated PEM is a very slow process; a prolonged incubation of up to 72 hr after the removal of TPA was required for PEM to regain fully its [3H]-PDBu binding activity. Furthermore, the degree of TPA-induced CSF-1-receptor down-regulation is closely associated with the number of available phorbol ester receptors present on PEM at the time of treatment. Thus, the refractoriness to TPA diminished as the phorbol ester receptors on PEM recovered. A 72-hr incubation time at 37 degrees C was needed for PEM to lose their refractoriness and again become fully sensitive to TPA-induced CSF-1-receptor down-regulation. This study provides evidence that the loss of CSF-1-receptors induced by TPA treatment requires the presence of phorbol ester receptors and proceeds presumably via a co-internalization of both CSF-1 and phorbol ester receptors; the refractoriness to TPA is thereby induced by a transient loss of available phorbol ester receptors.     

Differential changes in lipid metabolism of myeloid and lymphoid cell lines induced by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA)

Treatment of the myeloid cell lines, U-937 or HL-60, with 10 nM of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), for 24 h increased the rate of incorporation of [3H]glycerol into total chloroform extracts. A proportionally greater labeling of the non-polar lipid (NL) fraction compared to the polar, phospholipid (PL), fraction was observed. Chromatographic analysis showed a 6-fold increase in the labeling of triacylglycerols (TAG), a 2-fold increase in diacylglycerols, and no changes in monoacylglycerols. PL labeling showed a 3-fold increase in phosphatidylcholine (PC). The effect of TPA on TAG labeling was selectively observed in myeloid cell lines. No such a change was found in the lymphoid cell line. MOLT-3, which did respond to TPA with increased PC labeling. Incorporation of [3H]arachidonic acid (AA) into TAG by U-937 cells was selectively increased (2.5-fold) after treatment with TPA for 24 h. Treatment of U-937 cells with TPA in serum-free medium resulted in no increased labeling of TAG. These studies suggest that changes in TAG metabolism may be characteristic of myeloid differentiation and depend on the presence of serum factor(s).     

Flow cytometric analysis of the phenotypic changes in tumour cell lines following TPA induction

Single-cell analysis by flow cytometry has enabled us to analyze the effects of a phorbol ester and known tumour promoter, TPA, on the phenotypes of four tumour lines. TPA is capable of triggering a variety of cellular alterations that can affect gene expression and the biochemical balance of intracellular events. We have investigated the effect of TPA on such properties as rate of proliferation, differentiation, expression of cell surface molecules, and susceptibility to natural killer (NK) cell-mediated cytolysis. Four human leukemia and lymphoma cell lines; K562, MOLT 4, Raji, and HL60, were studied in their response to TPA treatment. Based on measurements of the defined cellular properties, we have characterized the pleiotropic responses of each tumour cell line to the phorbol ester in relation to intensity and time of onset of each response. The effects of TPA are highly varied, ranging in time of onset from minutes to days, and in intensity from strong to weak within the four cell lines studied. However, within all the processes that are affected, the activation of protein kinase C appears to be a common initiating event of phorbol ester induction.     

12-O-tetradecanoyl phorbol 13-acetate stimulates the myristylation of an approximately 82 kDa protein in HL-60 cells

We have studied protein acylation using [3H]myristate in the two leukemia cell lines HL-60 and HL-60 Blast II. The latter is a variant which does not differentiate after treatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA). The acylation profiles of the two cell lines as examined by SDS-PAGE differed. TPA induced the myristylation of an approximately 82 kDa protein in the sensitive cells, but not in the resistant cells. Myristic acid was shown to be covalently linked to these proteins. Analysis of the cell lipids labelled with [3H]myristate showed that in contrast to observations with the proteins, the changes induced by TPA were observed in both TPA-sensitive and TPA-resistant cells. We conclude that the induction of myristylation may be an important step in the mechanism of differentiation.     

Phorbol ester causes desensitization of gonadotropin-responsive adenylate cyclase in a murine Leydig tumor cell line

The murine Leydig tumor cell line, MLTC-1, contains gonadotropin receptors and a gonadotropin-responsive adenylate cyclase system that became refractory (desensitized) when exposed to human chorionic gonadotropin (hCG). MLTC-1 cells also contain phorbol ester receptors with a Kd of 53 nM for [3H]phorbol dibutyrate. Exposing cells to 12-O-tetradecanoyl phorbol 13-acetate (TPA) also causes desensitization of the hCG response. TPA-induced desensitization was similar to hCG-induced desensitization by every criteria tested. Both TPA- and hCG-induced desensitization caused approximately 50% loss of the hormone response within 30 min. Neither TPA or hCG altered receptor affinity for hCG. The dose response of adenylate cyclase to hCG or GTP in isolated membranes was not affected by either hCG- or TPA-induced desensitization. Similarly the dose response to hCG of cAMP accumulation in intact cells was not altered by desensitization with hCG or TPA. It was determined that MLTC-1 cells have Ca2+/phospholipid-dependent protein kinase activity that displayed a dose-dependent response to TPA. The concentration of TPA required to activate the protein kinase was similar to that required for desensitization. Phorbol esters that were unable to activate protein kinase C were also unable to desensitize MLTC-1 cells. The protein kinase from MLTC-1 cells was also activated by diacylglycerol. In addition, diacylglycerols caused desensitization of the hCG response. TPA- and diacylglycerol-induced desensitization is probably mediated by protein kinase C, and the similarities between hCG- and TPA-induced refractoriness suggests a convergence of mechanisms at some point of MLTC-1 cell desensitization.     

Changes in the kinetics of inositol transport during TPA-induced differentiation of HL60 cells towards monocytes.

When exposed to the phorbol ester TPA, HL60 cells undergo growth arrest and differentiate towards monocytes. During TPA-induced differentiation there was a 2.6-fold increase in the rate of inositol transport (Vmax), a 2.1-fold increase in intracellular inositol and a 1.5-fold increase in inositol lipid. An increase in the Vmax of inositol transport did not occur when the variant cell line HL60Ast3 was exposed to TPA, which has been shown in this cell line to induce growth arrest but not differentiation. This observation suggests that the change in inositol transport during HL60 monocyte differentiation is specifically associated with the process of cell differentiation as opposed to growth arrest.     

Glucocorticoid receptors and corticosensitivity in the human monocytic cell line CM-S: changes during phorbol ester-induced differentiation

CM-S is a line of human precursor mononuclear phagocytes inducible to macrophage differentiation in response to the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Untreated CM-S cells expressed a single class of high-affinity (KD = 4.5 +/- 2.3 nM) glucocorticoid receptor sites (GCr) (27,530 +/- 3752 sites/cell) as measured by a whole-cell assay at 37 degrees C using [3H]triamcinolone acetonide as tracer, while CM-S cells induced to macrophage differentiation by 10(-7) M TPA showed reduced levels of GCr (10,729 +/- 2135 sites/cell). Kinetic studies indicated that this reduction was progressive, reaching about 34% of the original value 96 hr after TPA addition. The GCr in untreated and TPA-induced cells were similar in their specificity for corticosteroids. In the precursor cell population dexamethasone enhanced both the rate of protein synthesis and the production of autostimulatory growth factor(s), while in TPA-induced cells it inhibited the rate of protein synthesis in a dose-dependent manner. Our data with the CM-S cell line thus suggest that in the monocytic cell line lineage both the number of GCr and the cell response to glucocorticoids depend on the degree of cell maturation.     

Activation of protein kinase C by phorbol esters induces DNA synthesis and protein phosphorylations in glomerular mesangial cells

The tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) is shown to be mitogenic for quiescent glomerular mesangial cells cultured in serum-free conditions. TPA induces DNA synthesis measured by [3H]thymidine incorporation in a dose-dependent manner with an ED50 of 7 ng/ml and an optimal response for 50 ng/ml. The phorbol ester action is potentiated by insulin with an increase of the maximal effect from 232 +/- 15% for TPA alone to 393 +/- 96% for TPA plus insulin. Down-regulation of protein kinase C by prolonged exposure to TPA completely abolishes the mitogenic effect of the phorbol ester. Using a highly resolutive 2D electrophoresis, we have shown that TPA is able to stimulate the phosphorylation of 2 major proteins of Mr 80,000, pl 4.5 (termed 80K) and Mr 28,000, pI 5.7-5.9 (termed 28K). The 80K protein phosphorylation is time- and dose-dependent with an ED50 of 8 ng/ml TPA. Exposure of mesangial cells to heat-shock induces synthesis of a 28K protein among a set of other proteins suggesting that the 28K protein kinase C substrate belongs to the family of low molecular mass stress proteins. Mitogenic concentrations of TPA and phorbol 12,13-dibutyrate inhibit [125 I]epidermal growth factor binding and stimulate the 80K protein phosphorylation with the same order of potency. The inactive tumor-promoter 4 alpha-phorbol was found to be ineffective both on these 2 parameters and on DNA synthesis. These results suggest a positive role for protein kinase C on mesangial cell proliferation and indicate the existence in this cell line of 2 major protein kinase C substrates.     

Phorbol esters and mitogenesis: comparison of the proliferative response of parental and Na+K+Cl- -cotransport-defective BALB/c 3T3 cells to 12-0-tetradecanoylphorbol-13-acetate

The ability of the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) to stimulate the growth of quiescent BALB/c 3T3 cell lines lacking Na+K+Cl- cotransport activity was tested. We have previously isolated and characterized two mutant cell lines defective in this important ion transport system by mutagenesis and selection in medium containing low K+. To test our hypothesis that loss of this transport activity might abrogate the proliferative response to TPA, two kinds of mitogenesis assays were performed. First, the effect of 0.16 microM TPA on the saturation density of parental vs. mutant cell lines was determined. TPA caused a small but reproducible 30-35% increase in the saturation density of mutant cells compared to the 100-120% increase seen in parental cell lines. Second, the effect of TPA on the incorporation of 3H-thymidine into cell nuclei (labeling index) was measured. While some variability from experiment to experiment in the extent and time course of the response of mutant cells was noted, TPA either had no effect or only a small effect on the labeling index when compared to the response of parental cells. When a range of concentrations of TPA (0.016-1.6 microM) was tested, neither cell line exhibited a large response to any concentration. These results suggest that loss of Na+K+Cl- cotransport activity decreases the response of these cells to the mitogenic action of TPA.     

Response of cultured rat liver epithelial cell lines to tumour-promoting phorbol esters

The membrane effects of a potent tumour promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), were studied in a series of cultured rat liver epithelial cell lines. Treatment with TPA resulted in the formation of strand-like aggregates (ridges) of viable cells over the monolayer of IAR 6-1 cells, but not of three other cell lines tested (IAR 20, IAR 6, IAR 6-7). The morphological response of IAR 6-1 to TPA was investigated by determination of phorbol ester receptors, analysis of cellular fucoproteins, surface galactoproteins and iodinatable surface proteins, and specific immunofluorescence for components of the extracellular matrix (fibronectin, laminin-entactin, procollagen type III). A class of specific, saturable, high-affinity receptors for phorbol esters was demonstrated in all four cell lines employing a conventional [20(-3)H]phorbol-12,13-dibutyrate ([3H]PDBu)-binding assay. The dissociation constants were similar in four lines, but the number of receptors per cell in IAR 6-1 cells was about twice that in other lines. Down-regulation of receptors was demonstrated in IAR 20 and IAR 6-1 cells with similar characteristics. Iodinatable surface proteins and galactose-containing surface glycoproteins did not respond to TPA. The distribution of fibronectin, laminin-entactin and procollagen type III was not affected by TPA. A TPA-responsive cell line, IAR 6-1, contained considerably less laminin-entactin than did the other lines. TPA had no influence on metabolic labelling of [3H]fucose-containing cellular glycoprotein in IAR 6-1 cells. One specific protein, with molecular mass of 78 kD, was more heavily labelled with [3H]fucose in IAR 6-1 cells than in the other cell lines. Taken together, the results of this study show that the responsive cells (IAR 6-1) differed from non-responsive ones in having more phorbol ester receptors, increased fucosylation of a specific glycoprotein and decreased deposition of laminin-entactin in the extracellular matrix. These surface properties of IAR 6-1 cells may contribute to their ability to respond to TPA.     

A phorbol ester-nonproliferative variant of Swiss 3T3 cells is deficient in Na+K+Cl- cotransport activity

The identity of the genetic defect(s) in Swiss 3T3 TNR-2 and TNR-9 that confers nonresponsiveness to the proliferative effect of 12-0-tetradecanoylphorbol-13-acetate (TPA) is not known. In BALB/c 3T3 cells, loss (via mutation) of a specific membrane ion transport system, the furosemide-sensitive Na+K+Cl- cotransporter, is associated with decreased responsiveness to TPA. In this study, the transport properties of parental Swiss 3T3 cells and the TPA-nonresponsive lines TNR-2 and TNR-9 were determined in the presence and absence of TPA. When the rate of 86Rb+ efflux (as a tracer for K+) was measured from each of the three cell lines, a furosemide- and TPA-inhibitable component of efflux was clearly evident in parental and TNR-9 cells but was virtually absent in TNR-2 cells. 86Rb+ influx measurements indicated the presence in parental 3T3 cells and the TNR-9 line of a substantial furosemide-sensitive flux that could be inhibited by TPA. In contrast, much less furosemide-sensitive influx was present in 3T3-TNR-2 cells and it was relatively unaffected by TPA. In both parental 3T3 and 3T3-TNR-2 cells, most of the furosemide-sensitive 86Rb+ influx is dependent on extracellular Na+ and Cl-. The apparent affinities of the transporter for these two ions, as well as for K+, were similar in both cell lines. In parental cells, the inhibition of furosemide-sensitive 86Rb+ influx was quite sensitive to TPA (K1/2 approximately equal to 1 nM) and occurred very rapidly after phorbol ester exposure. As expected because of its volume-regulatory role, inhibition of Na+K+Cl- cotransport by TPA in parental cells caused a substantial reduction in cell volume (25%). In contrast, because of the reduced level of cotransport activity in TNR-2 cells, TPA had only a slight effect on cell volume. These results suggest that the genetic defect in 3T3-TNR-2 cells (but not TNR-9 cells) responsible for nonresponsiveness to phorbol esters may be the reduction of Na+K+Cl- cotransport activity. Thus this membrane transport system may be an important component of the signal transduction pathway used by phorbol esters in 3T3 cells.     

Phorbol ester-induced differentiation permits productive human cytomegalovirus infection in a monocytic cell line

The susceptibility of four different human cell lines (HUT 102, THP-1, MOLT-4, and HL-60) to infection by human CMV (HCMV) was studied. Only HUT 102 was susceptible and only immediate-early gene products were produced. However, THP-1, a monocytic cell line, could be infected by HCMV with a full cycle of replication after treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which produced differentiation of the cell line into cells with characteristics of mature macrophages. Late (structural) Ag were demonstrated, as were infectious virions as detected by electron microscopy and infectious center assay. HL-60, a promyelocytic cell line, was not susceptible to HCMV infection after treatment with TPA despite differentiation into adherent cells with properties of macrophages, suggesting that cellular lineage was important. Treatment with TPA after infection resulted in a greatly reduced frequency of infected cells, suggesting that pretreatment was essential. Furthermore, continued presence of TPA was unnecessary after differentiation was induced. This study establishes the precedent of productive HCMV infection in human monocytic cells. The potential mechanism and relevance of enhanced replication induced by TPA are discussed.     

Phorbol ester activation of phospholipase D in human monocytes but not peripheral blood lymphocytes

Previous reports have shown that 12-0-tetradecanoylphorbol-13-acetate can activate phospholipase D in human peripheral blood mononuclear cells as measured by an enzyme-catalyzed transphosphatidylation reaction (phosphatidylethanol formation). In the present study, the mononuclear cells were fractionated by two procedures to identify the responsive cells. Contrary to earlier suggestions, the results indicate that phorbol ester does not stimulate phospholipase D activity in normal lymphocytes. Thus, phosphatidylethanol was not produced by T lymphocytes (isolated by sheep erythrocyte rosette formation) or by a mixture of T and B lymphocytes (isolated by centrifugal elutriation). Under the same conditions, phorbol ester was able to activate phospholipse D in fractions that contained predominately monocytes. Preliminary experiments have further shown that phorbol ester does not induce phospholipase D activity in human T cell leukemic lines (MOLT-3, CEM, JURKAT, PEER) but can do so in some, but not all, B cell lines that have been infected with Epstein-Barr virus.     

History of immunology. Development of the concept of immunologic specificity, I

The induction of differentiation in human malignant T-lymphoblastic cell lines MOLT-3 and Jurkat by the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA) was examined using the monoclonal antibodies OKT3, OKT4, OKT6, and OKT8 which are known to react with human T-cell differentiation antigens. It was found that in the presence of nanomolar concentrations of TPA the proportion of OKT3⁺ (mature T-cell marker) cells increased while the proportion of OKT4⁺, OKT6⁺, and OKT8⁺ (relatively immature T-cell markers) cells decreased. These changes in the distribution of the OKT antigens in MOLT-3 cells were found to be more prominent with MOLT-3 cells than when the Jurkat cells were used. In studies using a double labeling approach it was found that although the OKT3⁺ and E-rosette-positive (E⁺) cells appeared to belong to the same subpopulations of MOLT-3 cells, the OKT3 antigen was probably not related to the receptor for sheep erythrocytes because adsorption of the OKT3 antibody did not block E-rosette formation. Studies using the DNA synthesis inhibitor, arabinosylcytidine (ara-C) also indicate that DNA synthesis was not required for the induction of more mature T-cell antigens in the malignant T-cell lines by TPA. These studies, taken together with our earlier reports, support the conclusion that namomolar concentrations of TPA can induce differentiation in these malignant T-cell lines. Furthermore we have shown that the T-cell hybridoma antibodies are useful markers to detect differentiation changes in human T cells.     

应用两种方法诱导SH-SY5Y细胞分化的研究

目的：观察全反式维甲酸（ATRA）处理和ATRA与十四烷酰佛波醇乙酸酯（TPA）序贯处理（ATRA/TPA）对人类神经母细胞瘤细胞系SH-SY5Y细胞增殖抑制和形态分化的影响。方法：应用10μM ATRA处理6天和10μM ATRA处理3天继以80 nMTPA处理3天这两种方法使SH-SY5Y细胞分化;用倒置光学显微镜动态观察SH-SY5Y细胞形态学变化;并用MTT比色法比较两种分化方法对SH-SY5Y细胞的体外抗增殖作用。结果：ATRA处理和ATRA与TPA序贯处理对SH-SY5Y细胞都有抗增值和诱导细胞分化作用,细胞形态发生明显的变化,分化成神经元表型,前者主要表现为两端带有长突起的纺锤体样细胞形态,而后者主要是由细胞体延伸出多个突起的多边形的细胞。ATRA分化6天的细胞的存活率下降为78.7%±2.0%。当去除ATRA后,继续培养1天的细胞存活率上升为89%±0.2%,而继续培养2天的细胞存活率为86.3%±1.4%;ATRA与TPA序贯分化6天细胞存活率下降为75.9±0.4%。当去除TPA后,继续培养一天的细胞存活率为75.5±0.7%,继续培养2天的细胞存活率为74.9±1.0%。结论：维甲酸（ATRA）处理和ATRA与十四烷酰佛波醇乙酸酯（TPA）序贯处理（ATRA/TPA）均能明显诱导SH-SY5Y细胞分化。这两种分化细胞为神经科学的研究提供了优良的体外培养模型细胞,尤其是ATRA与TPA序贯处理能获得分化完全而稳定的神经元样细胞。     

Exogenous prostaglandin E2 inhibits TPA induced matrix metalloproteinase-9 production in MCF-7 cells

Elevated levels of prostaglandin E2 (PGE2) have been reported in many high metastatic human breast cancers, but no relationship between exogenous PGE2 activity, expression of matrix metalloproteinases (MMPs) and metastasis in human tumor cells has been reported. The poorly invasive human breast cancer cell line MCF-7 was cultured for 24h in the presence of both phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 50 nM) and PGE2 (1 microM) and the activity of MMP-9, one of the MMPs involved in metastasis, was measured, in growth medium by gelatin substrate zymography. TPA induced a strong production of MMP-9 while exogenous PGE2 had no effect on the basal MMP-9 level, but inhibited the TPA induced enzyme expression and matrigel invasiveness. We showed that MCF-7 cells expressed EP2, EP3 and EP4 receptors for PGE2 and that its action was probably mediated by EP4 receptor and adenylyl cyclase activation while cAMP dependent PKA was not involved in the process of inhibition of MMP-9 production. These findings suggest a possible inhibitory role for exogenous PGE2 in the metastatic process development.     

Tetradecanoyl phorbol acetate-induced microtubule reorganization is required for sustained mitogen-activated protein kinase activation and morphological differentiation of U937 cells.

Investigation of 12-tetradecanoyl phorbol 13-acetate (TPA)-resistant U937 cell clones has demonstrated that the normal sustained p42 mitogen-activated protein kinase (p42MAPK) activation produced by TPA treatment is absent. This is shown to be due to the inability of TPA to maintain activation of MAP/extracellular signal-regulated kinase kinase (MEK) and cRaf1. A direct relationship between sustained p42MAPK activation and differentiation is provided by the demonstration that blockade of MEK activation by PD098059 prevents TPA-induced morphological differentiation of wild type U937 cells. Using TPA-resistant clones, an involvement of microtubule reorganization and granule release is demonstrated by the ability of the microtubule depolymerizing agent nocodazole, to promote sustained p42MAPK activation in the presence of TPA. This response correlates with the lack of TPA-induced microtubule reorganization in these clones and the ability of nocodazole to partially bypass resistance to TPA. The results demonstrate a causal link between protein kinase C-dependent microtubule reorganization, sustained p42MAPK activation, and the induction of differentiation in U937 cells.