Induction and properties of beta-adrenergic receptors during erythroid differentiation of Friend leukemic cells.

beta-Adrenergic receptors on Friend erythroleukemic cells were identified by the use of 125I-labeled hydroxybenzylpindolol, a potent beta-adrenergic antagonist. Binding of this ligand was saturable and stereospecific. The relative orders of potency of isoproterenol, epinephrine, and norepinephrine to displace bound hydroxybenzylpindolol indicate that the Friend cells have beta 2-adrenergic receptors. After culture for 6 days in the presence of dimethyl sulfoxide or hexamethylene bisacetamide, both undifferentiated and differentiated cells have a similar number of receptors (1500 per cell), but the density of beta receptors on the cell surface increases during the process of erythroid differentiation. Incubation of the Friend cells for 24 hr with high concentrations of butyric acid, dimethyl sulfoxide, or hexamethylenebisacetamide resulted in a striking increase of th number of beta-catecholamine receptors. The induction of beta-adrenergic receptors also occurred in the presence of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate and dexamethasone.     

Globin synthesis and erythroid differentiation in a Friend cell variant deficient in heme synthesis.

Friend erythroleukemia cells of line Fw are noninducible for hemoglobin synthesis by dimethyl sulfoxide, butyric acid, and other agents. However, these agents were found to induce hemoglobin synthesis if the cells were also treated with exogenous hemin. Butyric acid (or, to a lesser extent, dimethyl sulfoxide) by itself induced accumulation of the erythroid-specific membrane protein spectrin. The basis of the control of globin gene expression by hemin was investigated. Hemin does not control globin and total protein synthesis via the action of the hemin-controlled repressor. Rather, hemin, alone or in combination with dimethyl sulfoxide, induces accumulation of globin mRNA and nuclear globin RNA sequences. Direct measurements of heme synthesis indicate that Fw cells may be significantly deficient in heme metabolism.     

Loss of chromosomal high mobility group proteins HMG1 and HMG2 when mouse neuroblastoma and Friend erythroleukemia cells become committed to differentiation.

Chromosomal high mobility group (HMG) proteins HMG1 and HMG2 from mouse neuroblastoma cells and Friend erythroleukemic cells were analyzed by acetic acid/urea/polyacrylamide gel electrophoresis. Compared to rapidly growing cells, levels of HMG1 and HMG2 were decreased in mouse neuroblastoma cells that had been induced to differentiate by serum deprivation. This comparison revealed a reciprocal relationship between these HMG proteins and H10, a histone known to be in higher concentrations in nondividing cells. When cell growth was inhibited by means of density inhibition, however, HMG1 and -2 levels were not affected in either HeLa or mouse neuroblastoma cells, even though H10 did not accumulate. This observation establishes that HMG1 and -2 contents are not correlated with mitotic rate per se. Treatment of mouse neuroblastoma by sodium butyrate, which stops cell division without commitment to differentiation, had no effect on the level of HMG1 and -2. However, the level was decreased by dibutyryl cyclic AMP and dimethyl sulfoxide treatments, which, like serum deprivation, induced irreversible morphological differentiation in the neuroblastoma cells. Moreover, induction of differentiation (hemoglobin synthesis) in Friend erythroleukemic cells by dimethyl sulfoxide showed a decrease in the contents of HMG1 and -2. These observations suggest that preferential loss of HMG1 and -2 in mouse neuroblastoma and Friend erythroleukemia cells may be related to commitment of these cells to differentiation.     

Indirect induction of erythroid differentiation in mouse Friend cells: evidence for two intracellular reactions involved in the differentiation.

The mechanism of in vitro erythroid differentiation in mouse Friend cells was studied by employing cell fusion between two genetically marked Friend cells and other nonerythroid cells, including BHK (baby hamster kidney) and FM3A (mouse mammary gland) cells. We were able to induce erythroid differentiation indirectly by fusing Friend cells that had been exposed briefly to dimethyl sulfoxide prior to fusion with nonerythroid cells that had been treated with ultraviolet light (or other DNA-damaging agents). The results suggest that two distinct reactions are involved in erythroid differentiation in Friend cells in vitro. One reaction, originating from the damaged DNA (or inhibition of DNA replication as a consequence), exhibits an inducible nature, is nonspecific to Friend cells, and is trans-acting. The other reaction is specific to Friend cells and most likely is cis-acting. We also present evidence from the cell fusion experiments that a typical tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, inhibits erythroid differentiation by affecting the latter reaction. The biological significance of these findings is discussed.     

Induction of spectrin in erythroleukemic cells transformed by Friend virus.

The presence and accumulation in murine erythroleukemic cells transformed by Friend virus of the erythrocyte membrane-associated protein spectrin has been investigated. Spectrin was present in the uninduced cells and was induced 10- to 20-fold in dimethyl sulfoxide-treated differentiating cells. The intracellular concentration of spectrin reached a peak on the third day of dimethyl sulfoxide treatment, after which it fell to levels found in mouse erythrocytes. We also found that the small subunit of spectrin was phosphorylated in the cells.     

Terminal differentiation surface antigens of myelomonocytic cells are expressed in human promyelocytic leukemia cells (HL60) treated with chemical inducers

The expression of two surface antigens present on the cell membrane of both human granulocytes and monocytes was studied during the process of myelomonocytic differentiation using two monoclonal antibodies (B9.8.1 and B13.4.1). These surface antigens are not present on immature myeloid cells nor on nonmyeloid hematopoietic cells, but can be detected when the cells are terminally differentiated. Among the bone marrow cells, B13.4.1 binds to metamyelocytes and B9.8.1 to metamyelocytes and a fraction (30%) of myelocytes. HL60 human promyelocytic leukemia cells did not react with such monoclonal antibodies. However, when such cells were induced to differentiate in vitro into mature myeloid elements by treatment with retinoic acid or dimethyl sulfoxide, 70%--90% of the differentiated cells expressed both surface antigens. Cell sorting studies on these treated HL60 cells indicated that myelocytes and metamyelocytes were the most immature cells expressing such markers. Expression of the two surface antigens was also observed when HL60 cells were induced to differentiate into monocyte/macrophage cells by treatment with the tumor promoter 12-O- tetradecanoyl-phorbol-13-acetate. Thus, human promyelocytic leukemia cells induced to differentiate in vitro by treatment with specific chemical agents express membrane antigens in the same pattern as normal bone marrow myeloid cells at the corresponding stage of differentiation.     

Lengthening of the G1 phase is not strictly correlated with differentiation in Friend erythroleukemia cells.

Friend murine erythroleukemia cells (Friend cells) undergo erythroid differentiation in vitro with an increased probability when cells are cultured in the presence of dimethyl sulfoxide (Me2SO) or other agents. Exponentially growing Friend cells, after dilution into medium containing Me2SO, underwent a transient lengthening of the G1 phase of the cell cycle before they became committed to erythroid differentiation. For nine inducing agents, a positive correlation was found between the percentage of cells that had differentiated and synthesized heme, and the percentage of progenitor cells in which a lengthened G1 phase had previously been observed. This correlation was not found, however, with two other potent inducing agents, hypoxanthine and actinomycin D. Moreover, cells that underwent a lengthened G1 phase did not always terminally differentiate. One such example was a Me2SO-resistant, variant Friend cell line (520a) grown in the presence of Me2SO. These results imply that the prolonged G1 phase, although observed with many inducers, is not a prerequisite for erythroid differentiation with all inducers.     

Nuclear protein kinase C activity is decreased in Friend erythroleukemia cells induced to differentiate.

Although it is well known that protein kinase C (PKC) is an important signaling molecule in Friend erythroleukemia cells it is not clear what role PKC may play in either regulated or unregulated erythroid cell proliferation and differentiation. The purpose of this study was to test the hypothesis that a decrease in nuclear PKC activity is associated with the induction of differentiation in Friend erythroleukemia cells. The effects of staurosporine, a selective inhibitor of PKC, and the tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate, an activator of PKC, on Friend cell proliferation and differentiation were examined. Neither the inhibitor nor the activator of PKC affected proliferation at 96 h as measured by [3H]thymidine incorporation, but both compounds inhibited cell differentiation. In addition, nuclear PKC activity was highest in untreated and in tumor promoter-treated cells that were not differentiated, and it was lowest in cells induced to differentiate with hexamethylene bisacetamide or dimethylsulfoxide. It is concluded that nuclear PKC activity is essential for Friend erythroleukemia cell proliferation, and that a decrease in enzyme activity within the nucleus is associated with differentiation.     

Degradation of cellular mRNA during infection by herpes simplex virus.

The fate of preexisting mRNA sequences was examined after infection by herpes simplex virus. Murine erythroid cells transformed by Friend leukemia virus were used as the host. Such cells, when exposed to 2% dimethyl sulfoxide, produce large amounts of globin and globin mRNA. The protein and its mRNA are easily recognized at 4 days by electrophoresis in high percentage acrylamide gels and by hybridization to cDNA, respectively. Herpes simplex virus replicates in these cells. By 2 hr after infection the rate of protein synthesis decreases to 30% of the level in mock-infected cells and only 49+/-8% (SEM) of the globin mRNA sequences present prior to infection could be detected by hybridization to cDNA. At 4 hr after infection, when the rate of protein synthesis in infected cells is at a maximum, only about 15% of the globin mRNA sequences remained. Control experiments support the hypothesis that globin mRNA sequences are degraded after infection by herpes simplex virus.     

Inducers of mammalian cell differentiation stimulate dome formation in a differentiated kidney epithelial cell line (MDCK).

Cell cultures of a differentiated kidney epithelial cell line, MDCK, spontaneously form fluid-filled domes or hemicysts composed of numbers of cells as a manifestation of specialized epithelial transport phenomena. Addition to MDCK cells of a broad spectrum of compounds that are known as potent inducers of mammalian cell differentiation in cell culture caused a striking increase in the frequency of dome formation. Polar compounds such as N,N-dimethylformamide, dimethyl sulfoxide, or hexamethylene bisacetamide stimulated increased dome formation 15--30 hr after addition. Induction of domes by these compounds was prevented either by inhibitors of protein synthesis or by ouabain, cytochalasin B, or vinblastine. Inhibition of DNA synthesis did not block chemical induction of domes. Other inducers were compounds of physiological occurrence such as n-butyrate or adenosine. Furthermore, a variety of conditions expected to elevate intracellular levels of cyclic AMP also stimulated dome formation. These findings suggest the hypothesis that domes are formed in cell culture by a form of cell differentiation that is under positive control by cyclic AMP.     

Dimethyl sulfoxide antagonizes hypotensive, metabolic, and pathologic responses induced by endotoxin

There is evidence that free radical activity may be important in the development of endotoxemia. Dimethyl sulfoxide is a hydroxyl radical scavenger that readily penetrates cell membranes. Using the conscious, instrumented rat this study tests the ability of dimethyl sulfoxide to modify the course of endotoxemia by evaluating cardiovascular, metabolic, and tissue injury parameters for 4 hr after the toxic insult. Treatment with dimethyl sulfoxide (6.5 g/kg; i.p.) evoked significant decreases in cardiac output, stroke volume, and central venous pressure and increases in heart rate, systemic vascular resistance, mean aortic pressure, respiration rate, and concentrations of blood glucose and plasma lactate. Following endotoxin (40 mg/kg, i.v. LD90- 24 hr), dimethyl sulfoxide pretreatment blocked the early hypotensive episode but all other cardiovascular and respiratory responses to endotoxin were essentially unaltered. The pH, PO2, PCO2, and hematocrit were the same for both treated and untreated groups; however, dimethyl sulfoxide prevented the endotoxin-induced hypoglycemia and significantly attenuated the hyperlacticemia at 4 hr. The severe hemorrhagic intestinal pathology characteristic of this model of endotoxemia was not present in the dimethyl-sulfoxide-treated group. From these results we conclude that dimethyl sulfoxide caused significant cardiovascular alterations conducive to impaired systemic blood flow. However, when administered prior to endotoxin, dimethyl sulfoxide induced significant beneficial modifications in the course of endotoxemia despite few improvements in cardiovascular function. The data indicate that the hydroxyl radical may be a mediator of tissue injury in this model of endotoxemia.     

Alteration of myoblast phenotype by dimethyl sulfoxide.

Application of dimethyl sulfoxide to proliferating L8 myoblasts (an established cell line of rat skeletal muscle) for 72 hr completely prevented fusion and induction of creatine phosphokinase (EC 2.7.3.2) activity (an indicator of muscle differentiation). The growth pattern changed from the usual sheets of randomly oriented cells to flattened, whorled monolayers of elongated fibroblast-like cells. By electron microscopy, rough endoplasmic reticulum increased and extracellular material appeared that had the morphologic and staining characteristics of collagen. After 120 hr in dimethyl sulfoxide-containing medium, the cells secreted about 6 times more collagen than untreated controls. Dimethyl sulfoxide was ineffective when applied to L8 cells just prior to fusion, and effects of dimethyl sulfoxide were not readily reversible unless treated cells were subcultured at low density.     

体外诱导间充质干细胞向心肌细胞方向分化的研究

目的证实来源于胎儿肝脏的间充质干细胞(FMSCs)具有向心肌细胞方向分化的潜能。方法取6~9代细胞,用不同浓度的诱导剂组合诱导细胞,分别置于不同的条件下进行培养,包括不同温度、不同氧浓度及不同培养液。结果在使用心肌分化培养液及常规培养条件下(37℃、20%O2),5-氮胞苷(50μmol/L)、维甲酸(10-3μmol/L)、二甲基亚砜(0.8%)的联合应用,诱导了FMSCs发生向心肌方向的分化。分化细胞呈小圆形细胞,具有相互聚集并形成球样细胞团结构的趋势,同时表达结蛋白及心肌肌钙蛋白Ⅰ。结论FMSCs具有向心肌细胞分化的潜能,FMSCs发生向心肌方向的分化所需条件与来源于其他动物种属的间充质干细胞的分化条件不同。     

Induction of a Ca2+, Mg2+-dependent endonuclease activity during the early stages of murine erythroleukemic cell differentiation.

A Ca2+, Mg2+-dependent endonuclease activity was detected in erythroleukemic cells undergoing differentiation in vitro in response to induction by dimethyl sulfoxide (Me2SO) or hexamethylene-bis-acetamide (HMBA). The endonuclease activity was demonstrated in isolated nuclei within 6 hr after the addition of inducer, reached maximum levels between 24 and 48 hr, and returned to control levels within 72 hr. The activity caused single strand breaks in high molecular weight native DNA, which could be labeled at exposed 3'-OH termini with Escherichia coli DNA polymerase I and radiolabeled nucleotides. Alkaline elution studies revealed DNA fragmentation that appeared coincident with the presence of the endonuclease activity. The detection and levels of single strand DNA breakage correlated with induction of terminal differentiation by Me2SO or HMBA. Induction of the endonuclease activity was reversible: depletion of Me2SO from the growth medium after treatment for 6 and 18 hr led to a rapid decrease in the level of activity. Removal of the inducer prevented terminal differentiation, a finding that strongly suggests the endonuclease activity is present during the precommitment phase of differentiation. DNA fragmentation was not observed in cells incubated with hemin, which has been shown previously to increase the cytoplasmic level of globin mRNA without causing commitment to terminal maturation. Me2SO did not induce the endonuclease activity or DNA fragmentation in an uninducible Friend cell line.     

Retinoids and phorbol esters alter release of fibronectin from enucleated cells.

Addition of 12-tetradecanoylphorbol 13-acetate (TPA) to cultures of intact Swiss mouse 3T3 fibroblasts induced a dose-dependent increase in ornithine decarboxylase (OrnDCase) activity. Over the same concentration range, 10(-9) to 10(-6) M, TPA induced the release of radioactively labeled fibronectin (FN) from the cells into the culture medium. Retinoic acid, a derivative of vitamin A, inhibited in a dose-dependent manner both the increase in OrnDCase activity and the release of FN induced by TPA. To examine the effects of TPA and retinoic acid in enucleated cells, the cells were treated with 7.5 micrograms of cytochalasin B per ml of medium during centrifugation at 10,000 X g for 35 min at 37 degrees C. In a series of five experiments, the treated cells were 94.7 +/- 4.8% (+/- SEM) enucleated as measured by [3H]thymidine incorporation and verified by Giesma staining for nuclei. In the enucleated cells, TPA did not induce increased OrnDCase activity but did induce FN release in a dose-dependent fashion over the same concentration range effective for FN release from intact cells. Moreover, addition of retinoic acid to the enucleated cells inhibited the phorbol ester-induced release of FN in a dose-dependent manner. A series of phorbol ester derivatives showed the same order of activity in causing FN release from the enucleated cells as reported for inducing OrnDcase activity in intact cells or in promoting mouse skin tumors in vivo. Similarly, several retinoids were tested for their ability to inhibit the phorbol ester-induced release of FN from enucleated cells. The efficacy of the retinoids in preventing FN release paralleled their activity in inhibiting phorbol ester-induced OrnDCase activity and skin tumor promotion, as reported in the literature. We conclude that at least one aspect of tumor promotion induced by phorbol esters--the loss of FN--does not require the cell nucleus, and further, that retinoids can inhibit this aspect of tumor promotion without nuclear involvement.     

Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds.

A human leukemic cell line (designated HL-60) has recently been established from the peripheral blood leukocytes of a patient with acute promyelocytic leukemia. This cell line displays distinct morphological and histochemical commitment towards myeloid differentiation. The cultured cells are predominantly promyelocytes, but the addition of dimethyl sulfoxide to the culture induces them to differentiate into myelocytes, metamyelocytes, and banded and segmented neutrophils. All 150 clones developed from the HL-60 culture show similar morphological differentiation in the presence of dimethyl sulfoxide. Unlike the morphologically immature promyelocytes, the dimethyl sulfoxide-induced mature cells exhibit functional maturity as exemplified by phagocytic activity. A number of other compounds previously shown to induce erythroid differentiation of mouse erythroleukemia (Friend) cells can induce analogous maturation of the myeloid HL-60 cells. The marked similarity in behavior of HL-60 cells and Friend cells in the presence of these inducing agents suggests that similar molecular mechanisms are involved in the induction of differentiation of these human myeloid and murine erythroid leukemic cells.     

Appearance of a chromatin protein during the erythroid differentiation of Friend virus-transformed cells.

Chromatin proteins from erythroleukemia cells transformed by Friend virus were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A new chromosomal protein, apparent molecular weight 25,000, was shown to appear during erythroid differentiation of the cells induced by dimethyl sulfoxide or hexamethylenebisacetamide. This protein is tightly bound to the chromatin and does not appear to be related to any of the known histones. It is also demonstrated that this protein is not induced in a dimethyl sulfoxide-resistant variant of these cells.     

Globin synthesis in mouse erythroleukemia cells in vitro: a switch in beta chains due to inducing agent.

Friend erythroleukemia cells, originally derived from DBA/2 mice, differentiate when cultured with inducing agents. Studies of the different effects of inducing agents on clone 745 have revealed that both dimethyl sulfoxide (DMSO) and hemin produce benzidine-positive cells. Butyric acid produced mature but benzidine-negative cells in this clone. All agents induced globin synthesis above the 0.1% of protein synthesis found in uninduced cells. DMSO induction stimulated globin synthesis 9%, hemin 2%, and butyric acid 3%. Total beta/alpha ratios were approximately unity with all agents. Although the inducing agents all stimulated total globin synthesis in Friend cells, the relative rates of synthesis of the two mouse beta chains were affected differently by the various agents. Hemin markedly increased the proportion of beta minor. For example, DBA/2 mouse reticulocytes synthesized 20% beta minor and 80% beta major. DMSO induction of clone 745 caused 20%-33% synthesis of beta minor. In contrast, hemin increased the proportion of beta minor to 64%-69%. Thus the Friend erythroleukemia cell system provides an in vitro approach to the study of the regulation of globin-chain switching.