Nuclear inositol lipids in Friend erythroleukemia cells. Changes related to differentiation induced by hexamethylenebisacetamide

Subcellular distribution of inositol lipids has been studied in Friend Erythroleukemia Cells following induction to erythroid differentiation with hexamethylenebisacetamide, after labelling with [3H]myo-inositol. In situ autoradiography indicated that inositol-derived molecules were present also in the nuclear compartment of uninduced and induced cells. Fractionation studies showed that the nuclear polyphosphoinositides were deeply changed after short induction times, while the whole cell inositol lipids resulted only slightly modified by the inducer. The nuclear recovery of phosphatidylinositol 4,5-bisphosphate was largely increased after 2 hrs of induction, suggesting that inositol lipid metabolism is involved in the early differentiation events occurring at the nuclear level.     

Induction of erythroid differentiation by cytoplast fusion in mouse erythroleukemia (Friend) cells

An intracellular activity, which is induced by dimethyl sulfoxide (DMSO) or hexamethylenebisacetamide (HMBA) and leads to erythroid differentiation in mouse Friend cells, was characterized by cell fusion between genetically marked intact cells and cytoplasts. For this, a procedure for rapid selection of cybrids was devised by sensitizing non-fused cells with oligomycin. We were able to demonstrate that cytoplasts derived from DMSO- (or HMBA)-treated cells trigger erythroid differentiation upon fusion with UV-irradiated cells. The activity in the cytoplasts remained only transiently and its induction was inhibited by biologically active phorbol esters or cycloheximide. The activity, however, was not induced in cytoplasts by directly treating them with DMSO (or HMBA). These results indicate that (1) the intracellular erythroid-inducing activity is located in cytoplasts, (2) it acts in trans and induces erythroid differentiation as a dominant factor and (3) its production requires de novo nuclear protein synthesis. The mechanisms of the induction of the intracellular activity and of how it triggers erythroid differentiation are discussed.     

Differentiation of murine erythroleukemia cells by hexamethylenebisacetamide involves secretion and binding to membranes of a differentiation enhancing factor

A protein factor previously shown to enhance terminal differentiation of transformed erythroid cells is synthesized by murine erythroleukemia cells and secreted in the early stages of differentiation induced by hexamethylenebisacetamide (HMBA). Secretion also occurs, constitutively, in the absence of inducer, from a murine erythroleukemia cell variant characterized by an accelerated response to HMBA. The protein factor binds to intact cells following addition of HMBA and enhances translocation of protein kinase C to the nuclear fraction. These results strongly support an important role for this natural protein factor in cell differentiation.     

HMBA诱导人肝癌SMMC-7721 细胞分化过程中 Nucleophosmin 的表达与定位变化

通过选择性抽提经环六亚甲基双乙酰胺（hexamethylene bisacetamide,HMBA）诱导处理前后的人肝癌SMMC-7721细胞核基质,并运用亚细胞蛋白质组学等分析技术,研究nucleophosmin (NPM)在核基质上的表达和定位变化,及其与相关基因产物的共定位关系,观察研究了nucleophosmin 在诱导分化前后人肝癌SMMC-7721细胞核基质中的存在、分布及其与相关基因产物的共定位关系.双向凝胶电泳和质谱鉴定结果显示,nucleophosmin 存在于 SMMC-7721 细胞核基质蛋白组分中,在 HMBA 处理后细胞核基质中表达下调.蛋白质印迹杂交实验结果确证了 nucleophosmin 在核基质中的存在及其在诱导处理后细胞核基质中表达下调的变化.免疫荧光显微镜观察显示,nucleophosmin 定位在 SMMC-7721细胞核基质上,经 HMBA处理后出现分布位置与表达水平的变化.激光扫描共聚焦显微镜观察结果显示,SMMC-7721细胞中,nucleophosmin与 c-fos、c-myc、rb、p53 等基因产物具有共定位关系,但在诱导处理后细胞内的共定位区域发生了改变.研究结果证实,nucleophosmin 是一种核基质蛋白,定位于核基质纤维上,nucleophosmin 在人肝癌 SMMC-7721 细胞诱导分化过程中的表达分布,及其与相关癌基因、抑癌基因产物的关系对 SMMC-7721 细胞分化具有重要影响.     

In vitro phosphorylation of lamin B by protein kinase C in friend erythroleukemia. Effect of chemically induced differentiation

Nuclear matrix isolated from murine erythroleukemia cells (Friend cells) has been phosphorylated with gamma 32P-ATP and purified protein kinase C in order to identify specific nuclear substrates for the enzyme. HMBA has been employed to induce the cell to differentiate and to compare the changes of phosphorylation profile after erythroid differentiation. Lamin B has been found to be hyperphosphorylated by rat brain PK-C in nuclear matrix purified from uninduced cells. This difference characterizes the cells from 14 to 72 hrs of HMBA treatment and indicates that the ability of lamin B to be phosphorylated by PK-C is linked to the differentiated state. The involvement of PK-C in lamin phosphorylation might represent an early step of the signalling pathway utilized by erythroid differentiating agents to target the cell nucleus.     

HMBA诱导人成骨肉瘤MG-63细胞分化过程中prohibitin的表达与定位变化

本文以HMBA诱导处理前后的人成骨肉瘤MG-63细胞为对象,对prohibitin在核基质中存在、分布及其与相关基因产物在HMBA处理前后MG-63细胞中的共定位关系进行观察研究.蛋白印迹杂交结果确证prohibitin存在于人成骨肉瘤MG-63细胞核基质蛋白组分中,并在HMBA处理后细胞核基质中表达下调,免疫荧光显微镜观察显示prohibitin定位在核基质上,经HMBA处理后出现分布位置与表达水平的变化.激光共聚焦显微镜观察可见prohibitin与MG-63细胞中c-fos、c-myc、p53和rb基因产物均存在共定位关系,但在HMBA处理后细胞中其共定位分布区域出现变化.研究结果证实prohibitin是一种核基质蛋白,定位于核基质上,prohibitin在人成骨肉瘤MG-63细胞诱导分化过程中的表达分布及其与相关癌基因、抑癌基因产物的共定位现象值得进一步探索和研究.     

人成骨肉瘤MG-63细胞在HMBA诱导分化后核基质蛋白表达的变化

HMBA诱导处理人成骨肉瘤MG-63细胞分化后,选择性抽提核基质蛋白,经双向凝胶电泳技术分析．共有12个蛋白点表达发生变化,经肽指纹图谱分析鉴定了9个蛋白质,其中,MHCⅡ类抗原、IFN刺激的基因因子3α蛋白、DKFZp434M2221．1蛋白、8-羟基-鸟嘌呤糖基化酶同功酶oggl和波形蛋白表达上调,hnRNP A2/B1和肌动蛋白表达下调,60S核糖体蛋白L21和ST2蛋白仅在分化的MG-63细胞中表达。研究结果表明肿瘤细胞增殖分化过程中伴随核基质蛋白表达的变化,并为深入揭示核基质蛋白与细胞癌变和逆转的关系以及阐明细胞增殖分化的基因表达调控原理提供了依据。     

Erythropoietin-induced erythroid differentiation of K562 cells is accompanied by the nuclear translocation of phosphatidylinositol 3-kinase and intranuclear generation of phosphatidylinositol (3,4,5) trisphosphate.

D-3 phosphorylated inositides are a peculiar class of lipids, synthesized by phosphatidylinositol 3-kinase (PtdIns 3-K), which are also present in the nucleus. In order to clarify a possible role for nuclear D-3 phosphorylated inositides during human erythroid differentiation, we have examined the issue of whether or not, in K562 human erythroleukemia cells, erythropoietin (EPO) may generate nuclear translocation of an active PtdIns 3-K. Immunoprecipitation with an anti-p85 regulatory subunit of PtdIns 3-K, revealed that both the intranuclear amount and the activity of the kinase increased rapidly and transiently in response to EPO. Enzyme translocation was blocked by the specific PtdIns 3-K pharmacological inhibitor, LY294002, which also inhibited erythroid differentiation. In vivo, intranuclear synthesis of phosphatidylinositol (3,4,5) trisphosphate (PtdIns (3,4,5)P(3)) was stimulated by EPO. Almost all PtdIns 3-K that translocated to the nucleus was highly phosphorylated on tyrosine residues of the p85 regulatory subunit. These findings strongly suggest that an important step in the signaling pathways that mediate EPO-induced erythroid differentiation may be represented by the intranuclear translocation of an active PtdIns 3-K.     

Synthesis of polyphosphoinositides in nuclei of Friend cells. Evidence for polyphosphoinositide metabolism inside the nucleus which changes with cell differentiation.

Previous work demonstrated the existence of phosphatidylinositol kinase and phosphatidylinositol phosphate kinase in rat liver nuclei, with the suggestion that these activities are in the nuclear membrane [Smith & Wells (1983) J. Biol. Chem. 258, 9368-9373]. Here we show that highly purified nuclei from Friend cells, washed free of nuclear membrane by Triton, can incorporate radiolabel from [gamma-32P]ATP into phosphatidic acid, phosphatidylinositol phosphate and phosphatidylinositol 4,5-bisphosphate. The degree of radiolabelling of phosphatidylinositol bisphosphate is highly dependent on the state of differentiation of the cells, being barely detectable in growing cells and much greater after dimethyl sulphoxide-induced differentiation; this difference is mostly due to different amounts of phosphatidylinositol phosphate in the isolated nuclei. We suggest that polyphosphoinositides are made inside the nucleus and that they have a role in chromatin function; either the phospholipids themselves play a role, or there is a possibility of intranuclear signalling by inositide-derived molecules.     

Synergistic induction of erythroid differentiation of mouse erythroleukemia (MEL) cells by inhibitors of topoisomerases and protein tyrosine kinases.

In vitro erythroid differentiation of mouse erythroleukemia (MEL) cells was induced by combinations of topoisomerase and protein kinase inhibitors. Neither inhibitor alone exhibited inducing activity. Although inhibitors of topoisomerases I and II were equally effective in the synergistic induction of erythroid differentiation, only inhibitors of tyrosine kinases, not of serine/threonine kinases, exhibited synergistic activity. The erythroid differentiation induced by the combination of topoisomerase and protein tyrosine kinase inhibitors was distinguished from that induced by typical erythroid inducing agents such as DMSO or HMBA by (1) earlier hemoglobin accumulation in the cells and (2) insensitivity to specific inhibitors (dexamethasone and sodium orthovanadate) of MEL cell differentiation.     

Incorporation of 2-14C-acetate into isolated nuclei and cytosolic lipids of rat thymus cells

It is generally recognized nowadays that active lipid metabolism takes place in the nucleus of a mammalian cell. Experimental data testify to the biosynthesis of polyphosphoinositides and phosphatidylcholine and reveal corresponding enzymes within nuclei of mammalian cells. These findings suggest that lipidmediated signaling pathways in nuclei operate independently of lipid-mediated regulatory mechanisms functioning in membranes and cytosol. To explore the pathways of intranuclear lipid biosynthesis, we studied incorporation of 2-¹⁴C-acetate into lipids of cytosol and isolated nuclei of rat thymus cells after separate and combined incubation with the labeled precursor. The most efficient incorporation of 2-¹⁴C-acetate into lipids (cholesterol, free fatty acids, and phospholipids) was observed in a reaction mixture containing cytosol. When the reaction mixture contained only nuclei, incorporation of the radioactive precursor into lipids also took place, but specific radioactivity of the lipids was essentially lower than in the cytosol. In both cases, 2-¹⁴C-acetate incorporated into phosphatidylethanolamine, sphingomyelin, phosphatidylserine, phosphatidylinositol, and cardiolipin. Phosphatidylcholine, the most abundant membrane phospholipid, demonstrated the lowest radioactivity, which was significantly lower than that of phosphatidylethanolamine. Incorporation of newly synthesized free fatty acids in nuclear phospholipids was inhibited, if nuclei were incubated with cytosol. As a result, radioactive free fatty acids were accumulated in nuclei, while in cytosol they were efficiently incorporated into phospholipids. The levels of phospholipids and cholesterol remained constant regardless of incubation protocol, while the overall yield of free fatty acids decreased after combined incubation of nuclear and cytosolic fractions or after incubation of cytosol without nuclei. Putative mechanisms underlying the appearance of radioactive lipids in isolated nuclei of thymus cells are discussed.     

Membrane phospholipid reorganization differentially regulates metallothionein and heme oxygenase by heme-hemopexin

Heme-hemopexin coordinately regulates genes encoding protective proteins including metallothionein-I (MT-I) and heme oxygenase 1 (HO-1). Hexamethylene-bisacetamide (HMBA), which induces differentiation and activates protein kinase C (PKC), synergistically augments the induction of both MT-I and MT-II mRNAs in response to heme-hemopexin, but attenuates the induction of HO-1. HMBA also augments the increase in MT mRNA in response to cobalt protoporphyrin-hemopexin, a hemopexin (HPX) receptor ligand that activates signaling cascades without tetrapyrrole uptake. Unlike the PKC-activating phorbol esters that induce MT-I and HO-1, HMBA has minimal effects on MT-I or HO-1. HMBA is an amphipathic molecule, and is shown here to interact physically with lipids in model membranes using differential scanning calorimetry (DSC). The data are consistent with a stabilization of the lipid bilayer and an HMBA-induced segregation of lipids into separate domains each relatively enriched in one of the lipids. HMBA also perturbs membrane-protein interactions, and causes a loss of PKC and G-protein subunits from plasma membranes in vitro. Taken together, these observations reveal an additional level of complexity in the regulation of protective proteins induced by HPX, and which may take place in vivo in response to natural compounds that reorganize membrane phospholipids. A model is proposed whereby a reorganization of lipids by HMBA alters signaling pathways and fusion events considered to be the etiology of the differential response of the MT-1 (and MT-II) and the HO-1 genes to HMBA and heme-HPX.     

Changes of nuclear matrix proteins following the differentiation of human osteosarcoma MG-63 cells

Human osteosarcoma MG-63 cells were induced into differentiation by 5 mmol/L hexamethylene bisacetamide （HMBA）. Their nuclear matrix proteins （NMPs） were selectively extracted and subjected to two-dimensional gel electrophoresis analysis. The results of protein patterns were analyzed by Melanie software. The spots of differentially expressed NMPs were excised and subjected to in situ digestion with trypsin. The maps of peptide mass fingerprinting were obtained by MALDI-TOF-MS analysis, and were submitted for NCBI database searches by Mascot tool. There were twelve spots changed remarkably during the differentiation induced by HMBA, nine of which were identified. The roles of the regulated proteins during the MG-63 differentiation were analyzed. This study suggests that the induced differentiation of cancer cells is accompanied by the changes of NMPs, and confirms the presence of some specific NMPs related to the cancer cell proliferation and differentiation. The changed NMPs are potential markers for cancer diagnosis or targets for cancer therapy.     

Structural and functional aspects of nuclear lipids in normal and tumor cells

This review summarizes available data on the structural and functional role of neutral lipids and phospholipids in normal and tumor eukaryotic cells. The role of acidic phospholipids (cardiolipin, phosphatidylinositol, and phosphatidylglycerol) in regulation of activities of DNA- and RNA-polymerases, DNA-topoisomerases I and II, DNA-methylases, and replication initiation proteins (dnaA and T-antigen) is discussed. The role of sphingolipids is emphasized considering, on one hand, the involvement of sphingosines in signal transduction, chromatin association-dissociation, and regulation of DNA and RNA synthesis and protein kinase C and, on the other hand, participation of ceramides and dihydroceramides in apoptosis. The possible role of sphingomyelin, sphingosine, cardiolipin, and diglycerides in the contacts of DNA loops with nuclear matrix is analyzed. Lipid hormones indirectly influence supercoiled DNA conformation; the effect of hormones on metabolism of phospholipids and neutral lipids in chromatin and nuclear matrix is reviewed. Characteristics of lipid composition in chromatin and nuclear matrix of the tumor cells are discussed.     

Rat liver nuclear lipids. Composition and biosynthesis

A characteristic of rat liver nuclear lipids is their high content in neutral lipids especially of tryglycerides and free fatty acids. These compounds do not arise due to hydrolysis of more complex lipids during the isolation of the nuclei. The neutral lipids fatty acid pattern is more saturated than the phospholipid one. The presence of phosphatidylinositol mono- and diphosphate in nuclei has been confirmed. Nuclei are unable to synthesize de novo phospholipids but are capable of incorporating inorganic phosphate into lipids synthesized via a kinase pathway.     

Effect of diet on the lipid composition of cell nuclei and liver mitochondria during ontogenesis of the rat

The possible influence of the type of dietary fat on quantitative changes of different lipids in microsomes, nuclei and chromatin in hepatic cells of albino rats in ontogenesis was studied. While the type of diet had no significant influence on the levels of different phospholipids and fatty acids of nuclei, the type of dietary fat exerted definite effect on the levels of whole phospholipids, cholesterol of microsomes, nuclei and fatty acids of chromatin. The age specificity was observed in nuclear structures.     

Nuclear lipids in Friend cells. Shifted profile of diacylglycerol during erythroid differentiation induced by DMSO

Friend cells were labelled for 90 min. with [3H]-Glycerol and the radioactivity in DAG# and TG was measured. The relative percentage of radiolabelled DAG differs in isolated nuclei as compared to intact cells. Moreover the level of newly synthesized DAG decreases in nuclei isolated from cells treated with DMSO for 24 hrs. as well as in nuclei from cells treated for 96 hrs. and terminally differentiated in the erythroid pathway. Since these changes are not seen in intact cells, the results are consistent with the hypothesis that nuclear lipids and namely the products of polyphosphoinositide hydrolysis, such as DAG, are involved in the regulation of the processes leading to cell differentiation.     

Synthesis of lipids in isolated nuclei from rat thymus and liver cells

Synthesis of lipids was studied in isolated nuclei from rat thymus and liver cells. On incubation of the isolated nuclei with [2-¹⁴C]acetate and [1-¹⁴C]glycerol, the label was intensively incorporated into phospholipids and with a significantly lower intensity into fatty acids and cholesterol. Only trace amounts of radioactivity were detected in the lipids of chromatin prepared from isolated thymus nuclei after their incubation, and this suggested that lipids were mainly synthesized on the nuclear membrane. On the preincubation of thymus tissue homogenate with [2-¹⁴C]acetate and the subsequent isolation of the nuclei and chromatin, the radioactivity of chromatin lipids was comparable to the radioactivity of nuclear lipids. The findings suggested that in the isolated nuclei the newly synthesized lipids were not transported into chromatin from the nuclear membrane. The specific radioactivities of individual phospholipids and fatty acids were different in the isolated nuclei and in nuclei obtained from preincubated homogenate. Mechanisms of lipid synthesis in isolated nuclei and causes of the different radioactivities of lipids in the isolated nuclei and in the nuclei obtained from the preincubated homogenate are discussed.     

Induction of differentiation in mouse neuroblastoma cells by hexamethylene bisacetamide

Hexamethylene bisacetamide (HMBA), a potent inducer of erythroid differentiation in murine erythroleukemia cells (1), induces differentiation in mouse neuroblastoma cells, as indicated by the extension of neurites and the development of an excitable membrane. HMBA is effective at concentrations 50-fold lower than dimethylsulfoxide (2), another inducer of differentiation in both mouse neuroblastoma and murine erythroleukemia cells.     

Rapid activation by erythropoietin of protein kinase C in nuclei of erythroid progenitor cells

The glycoprotein hormone erythropoietin (Ep) regulates the proliferation and differentiation of erythroid progenitor cells by a signal transduction system which is not well understood. It has recently been reported that prolactin, a mitogen and trophic hormone for liver, will activate a nuclear protein kinase C in hepatocytes. As similarities exist in the actions of Ep and prolactin in their target cells, we tested the hypothesis that Ep could activate protein kinase C in nuclei isolated from erythroid progenitor cells. In a pure population of such nuclei, Ep induced a rapid, time- and dose-dependent increase in phosphorylation of endogenous nuclear substrate which could be blocked by inhibitors of protein kinase C or by antibody to Ep. Other known activators of protein kinase C were also effective in this system. These findings show that Ep may exert its effects by a novel signalling pathway, the activation of a nuclear protein kinase C.