Inhibition by islet-activating protein, pertussis toxin, of retinoic acid-induced differentiation of human leukemic (HL-60) cells

Human promyelocytic leukemic (HL-60) cells were induced to differentiate into neutrophil- or macrophage-like cells by incubation of the cells with retinoic acid, dibutyryl cyclic AMP (Bt2cAMP) or phorbol 12-myristate 13-acetate (PMA). Differentiation was determined by an increase in the percentage of morphologically mature cells. The retinoic acid-induced differentiation of HL-60 cells was, but the Bt2cAMP- or PMA-induced one was not, inhibited by prior exposure of the cells to islet-activating protein (IAP), pertussis toxin. The IAP-induced inhibition was correlated with the toxin-catalyzed ADP-ribosylation of a membrane GTP-binding protein with a molecular mass of 40 kDa. Thus, the IAP-substrate GTP-binding protein appears to be involved in the retinoic acid-induced differentiation of HL-60 cells.     

Retinoic acid-induced differentiation-specific, C-kinase-dependent phosphorylation of cytosolic 44 and 32 kDa proteins in HL-60 cells

The effect of various differentiation-inducers on the activity of Ca2+, phospholipid-dependent protein kinase (C-kinase) activity and endogenous protein phosphorylation by the kinase were examined in the extracts of HL-60 cells. Although all of the inducers, retinoic acid, dibutyryl cAMP, nicotinamide, dimethylsulfoxide, and 3-aminobenzamide increased the cytosolic C-kinase activity accompanied with the differentiation into mature myelocytes, only retinoic acid markedly enhanced Ca2+, phospholipid-dependent phosphorylation of 44 and 32 kDa proteins in the cytosol. These results suggest that the differentiation pathway induced by retinoic acid is different from the pathways induced by other inducers.     

Cytosolic-nuclear tumor promoter-specific binding protein (CN-TPBP) in human promyelocytic leukemia cells HL-60

12-O-Tetradecanoylphorbol 13-acetate (TPA) is a potent tumor promoter and is known to induce terminal differentiation of human promyelocytic leukemia cells HL-60 to mature monocytes. To investigate the molecular mechanism of TPA actions, TPA-specific binding proteins in HL-60 were analyzed. Anion exchange high-performance liquid chromatography revealed that HL-60 cells possess TPA-specific binding proteins other than protein kinase C (PKC). One of these TPA-specific binding proteins exists in the cytosolic fraction of HL-60 cells, but translocates into the nuclear fraction of HL-60 cells after the treatment of the cells with TPA. The results suggest that HL-60 cells take up TPA into the nuclei via the TPA-specific binding protein. The TPA-specific binding protein binds TPA, phorbol 12,13-di-butylate, teleocidin B-2, teleocidin B-3, and debromoaplysiatoxin in a mutually competitive manner. However, the protein does not bind to okadaic acid, olivoretin C, retinoic acid, or dioxin. This cytosolic-nuclear tumor promoter-specific binding protein (CN-TPBP) might play an essential role in the action of tumor promoters.     

Retinoic acid inhibits the myristoylation of a membrane protein in HL-60 cells

Incubation of nondifferentiated HL-60 cells with high specific activity myristic acid results in myristoylation of a 25 KD membrane protein. Myristoylation was inhibited by retinoic acid but not by DMSO, thus indicating that the nonmyristoylated state induced by retinoic acid is causal to rather than the result of the overall differentiation sequel induced. Similarly, incubation of HL-60 cells with high specific activity retinoic acid results in specific retinoylation of a membrane protein of similar molecular mass. The two acylated proteins could however be separated by 2-D gel electrophoresis, thus indicating that inhibition of myristoylation by retinoic acid could not be accounted for by competition between retinoic and myristic acids for the same acylated site.     

Dependence of HL-60 myeloid cell differentiation on continuous and split retinoic acid exposures: Precommitment memory associated with altered nuclear structure

The cell differentiation of HL-60 human leukemic promyelocytes along the myeloid pathway due to various continuous and distributed exposures to retinoic acid was studied. HL-60 myeloid differentiation was a continuously driven process; significant terminal cell differentiation occurred only after a minimum exposure to inducer of two division cycles. Cells so committed to differentiation retained a heritable, finite memory of differentiation commitment over a further division cycle. Prior to becoming committed, cells acquired precommitment memory of exposure to inducer. Precommitment memory abbreviated the subsequent exposure to inducer needed for commitment to differentiation. Precommitment memory was semistable. It was heritable, but was lost after four division cycles. The acquisition and loss of precommitment memory correlated with alterations in nuclear architecture detected by narrow angle light scatter using flow cytometry. The altered nuclear architecture first occurred before any overt cell differentiation or growth arrest. It was thus an early event in the induced program of terminal cell differentiation. Alterations in relative abundances of cytoplasmic proteins also occurred prior to overt cell differentiation or growth arrest. One of these was a 17 kdalton, anionic, probably Ca²⁺ binding, protein. Retinoic acid thus induced early cellular changes, including cytoplasmic and nuclear alterations, within one cell cycle when cell differentiation was not yet apparent.     

维生素A类化合物和二甲基亚砜诱导人早幼粒白血病细胞(HL-60)分化的显微与亚显微结构研究

用光镜和电镜技术研究了HL-60细胞在诱导分化过程中的显微与亚显微结构变化,10~(-6)M的维A酸处理6天,细胞按粒系途径定向分化,其核质比例降低,核浓缩、分叶,核仁减少或消失。经RA处理的细胞在电镜下出现下列明显的变化:细胞核浓缩和分叶,异染色质区域增加,约46%细胞显示出类似成熟粒细胞核的亚显微形态特征,胞质中嗜天青颗粒减少,特异颗粒显著增加,两种颗粒的比率发生明显变化;细胞质中微管、微丝的量增加;多聚和单个分散的游离核糖体减少,有些??细胞胞质空泡化;出现主要以微丝为筑架的大型钝形伪足和不规则的表面突起。上述这些变化似可作为HL-60细胞形态分化的标志。维A酸诱导HL-60细胞形态分化具有明显的时间效应关系。1.4%DMSO对HL-60细胞分化的诱导作用类似于10~(-6)MRA,而等剂量的(10~(-6)M)R Ⅰ、RⅡ其作用弱于RA。     

Human myelogenous leukemia cell line HL-60 cells resistant to differentiation induction by retinoic acid. Decreased content of glycosphingolipids and granulocytic differentiation by neolacto series gangliosides

We have recently reported that neolacto series gangliosides (NeuAc-nLc) are increased during granulocytic differentiation of human myelogenous leukemia cell line HL-60 cells induced by retinoic acid and that HL-60 cells are differentiated into mature granulocytes when the cells are cultivated with NeuAc-nLc (Nojiri, H., Kitagawa, S., Nakamura, M., Kirito, K., Enomoto, Y., and Saito, M. (1988) J. Biol. Chem. 263, 7443-7446). In contrast to these wild-type-HL-60 cells, HL-60 cells resistant to differentiation induction by retinoic acid showed a markedly decreased content of gangliosides, especially NeuAc-nLc, and did not show any increase in the content of gangliosides when cultivated with retinoic acid. Neutral glycosphingolipids, the precursors of gangliosides, were not accumulated in these resistant cells. When retinoic acid-resistant HL-60 cells were cultivated in the presence of NeuAc-nLc, the cells were found to be differentiated into mature granulocytes on morphological and functional criteria. The differentiation of cells was dependent on the concentration of gangliosides and was accompanied by inhibition of cell growth. Wild-type HL-60 cells differentiated by NeuAc-nLc showed the changes in ganglioside composition, which were similar to those in wild-type HL-60 cells differentiated by retinoic acid; among the gangliosides changed, 2----3 sialylparagloboside and 2----3 sialylnorhexaosylceramide were increased. These findings suggest (a) that the synthesis of particular NeuAc-nLe molecules is an important step for retinoic acid-induced granulocytic differentiation and this step could be bypassed or replaced by exogenous NeuAc-nLc, and (b) that the defective synthesis of particular NeuAc-nLc molecules is responsible for the failure of differentiation induction in retinoic acid-resistant HL-60 cells by retinoic acid.     

蛋白激酶C亚型在HL—60细胞诱导分化中的变化

用全反式维甲酸（ＡＴＲＡ）或佛波酯（ＰＭＡ）处理人早幼粒白血病细胞（ＨＬ－６０）３天,用形态学,ＮＢＴ还原实验,特异性和非特异性酯酶测定,证明细胞分别向粒细胞或单核／巨噬细胞分化。通过免疫组化法观察了蛋白激酶Ｃ（ＰＫＣ）α,βⅠ和βⅡ亚型在分化后的变化。结果显示,ＡＴＲＡ可引起ＨＬ－６０细胞ＰＫＣα,βⅠ和βⅡ的含量升高,分别为对照的５．０,２．８和４．２倍,并存在从胞膜向胞质转位。ＰＭＡ则使ＰＣ     

Growth inhibitory effects of 5-aza-2'-deoxycytidine in HL-60 promyelocytic leukemia cells resistant to differentiation induction

In the original HL-60 cells (HL-60-S) and an HL-60 subline (HL-60-R) respectively susceptible and resistant to induction of differentiation by retinoic acid or dimethyl sulfoxide, 5-aza-2'-deoxycytidine inhibited growth equally but induced differentiation to a greater extent in HL-60-S. Flow cytometry showed that 5-aza-2'-deoxycytidine produced in both HL-60 lines an increased proportion of cells in G2+M rather than G0/G1 as with retinoic acid. 5-aza-2'-deoxycytidine may have a differentiation-inducing effect in HL-60 provided cells have the competence to differentiate, indicating the importance of an alternate mechanism of action.     

Loss of transferrin receptors following induced differentiation of HL-60 promyelocytic leukemia cells

Human promyelocytic leukemia (HL-60) and lymphoblastoid (Daudi) cells were studied: for transferrin receptors before and after induced differentiation with dimethyl sulfoxide (DMSO), sodium butyrate or retinoic acid. None of these reagents affected the morphology or presentation of receptors in Daudi cells, but many HL-60 morphologically matured to banded neutrophils and demonstrated a concomitant loss of transferrin binding, suggesting an important role for transferrin receptors in cellular differentiation.     

Induction of cyclo-oxygenase synthesis in human promyelocytic leukaemia (HL-60) cells during monocytic or granulocytic differentiation.

Cyclo-oxygenase (COX) production in human promyelocytic leukaemia (HL-60) cells was studied during monocytic differentiation induced by 1 alpha, 25-dihydroxyvitamin D3 (24 nM; 3 days) or phorbol 12-myristate 13-acetate (100 nM; 1 day), or during granulocytic differentiation induced by retinoic acid (1 microns; 4 days). Undifferentiated or differentiated HL-60 cells were labelled with [35S]methionine, and membrane-bound COX was solubilized and quantified by SDS/PAGE. Immunoprecipitated 35S-labelled COX from cells induced to differentiate into monocytic or granulocytic lineage were clearly detected on the autoradiograms as a protein of approx. 70 kDa molecular size, whereas only a very faint COX band was detected in untreated HL-60 cells. During both monocytic and granulocytic differentiation, COX activity (measured by the conversion of exogenous arachidonic acid into prostaglandin E2) was dramatically increased. In addition, thromboxane synthesis was preferentially enhanced during monocytic differentiation. HL-60 cells, induced to differentiate into the monocytic or granulocytic lineage, provide a useful tool to investigate the cellular mechanisms involved in regulation of the synthesis of individual prostanoid-metabolizing enzymes.     

Retinoic acid effect on cyclic AMP-dependent protein kinases in embryonal carcinoma cells: studies with differentiation-defective sublines

Retinoic acid induces the differentiation of PCC4.aza 1R and Nulli-SCC1 embryonal carcinoma (EC) cells. In response to retinoic acid treatment, the levels of cyclic AMP (cAMP)-dependent protein kinases are enhanced in the plasma membrane within 17 hours and in the cytosol fractions of these cells within 2 to 3 days, as determined by phosphotransferase activity and by 8-azido-cyclic [32P]AMP binding to the RI and RII regulatory subunits. PCC4 (RA)-1 and Nulli (RA)-1 are mutant EC lines that fail to differentiate in response to retinoic acid. The former line, but not the latter, lacks cellular retinoic acid-binding protein (cRABP). Basal levels of cAMP-dependent protein kinase activities are elevated in PCC4 (RA)-1 cells. When these cells are treated with retinoic acid, neither cAMP-dependent protein kinase activities nor cAMP binding activities are enhanced; rather, there is a decrease in cytosolic kinase activity and RI subunit. On the other hand, Nulli (RA)-1 cells exhibit increases both in cAMP-dependent protein kinase activities and cAMP binding in response to retinoic acid. These results raise the possibility that cRABP mediates the enhancement of regulatory and catalytic subunits of cAMP-dependent protein kinases in both the membrane and the cytosolic fractions of the teratocarcinoma cells. There also might be some effects of retinoic acid on the cAMP-dependent protein kinase that are unrelated to differentiation and to the presence of cRABP.