Cyclic AMP-dependent and -independent protein kinases and protein phosphorylation in human promyelocytic leukemia (HL60) cells induced to differentiate by retinoic acid

The human leukemia cell line HL60 which resembles promyelocytes can be induced to differentiate to cells displaying features of the mature myeloid phenotype by a variety of agents including retinoic acid (RA) and agents that elevate intracellular adenosine 3:5 cyclic monophosphate (cyclic AMP) levels, e.g., 8-bromo-cyclic adenosine 3:5 monophosphate (8-Br-cyclic AMP), cholera toxin. Since most, if not all the effects of cyclic AMP, are mediated by adenosine 3:5 cyclic monophosphate-dependent protein kinase (cyclic AMP-dPK), we investigated the role of cyclic AMP-dPK and adenosine 3:5 cyclic monophosphate-independent protein kinase (cyclic AMP-iPK) in the induced differentiation of HL60 cells. Marked stimulation of cyclic AMP-dPK and cyclic AMP-iPK appears to be intimately involved with and specific for HL60 myeloid differentiation as evidenced by: (1) Stimulation of cyclic AMP-dPK and cyclic AMP-iPK early during HL60 myeloid differentiation and prior to phenotypic changes. (2) RA and dimethylformamide (DMF), agents that induce differentiation along the myeloid pathway, cause a marked increase in the type I cytosolic cyclic AMP-dPK and cyclic AMP-iPK (protamine kinase) while no such increases are noted in cells treated with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) which induces differentiation along the monocyte/macrophage pathway. (3) Both native polyacrylamide gel electrophoresis as well as photoaffinity labeling with 8-azido-cyclic AMP demonstrate marked increases in type I cyclic AMP-dPK in the cytosols of cells exposed to agents that induce myeloid differentiation but no increase in TPA-differentiated cells. (4) The appearance and disappearance of specific cyclic AMP-dependent and -independent protein phosphorylations are associated with the induced myeloid differentiated state.     

人畸胎瘤细胞株PA-1的体外诱导分化

PA-1细胞是一株从人卵巢性畸胎瘤衍生的细胞株,经10~(-5)mol/L视黄酸诱导后,部分细胞的形态和排列方式发生一定的改变。通过细胞免疫荧光染色,发现诱导后细胞的结蛋白和胞外基质(纤粘连蛋白、层粘连蛋白和腱粘连蛋白)表达与分布模式有较大的变化,并且这些变化与细胞形态改变相关。但大部分PA-1细胞诱导后的生长状况并没有较大的改变,仍分泌与表皮生长因子、类胰岛素生长因子-Ⅰ相关的活性物质。以上结果提示PA-1细胞经视黄酸诱导后一部分细胞可能向肌细胞方向分化。     

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.     

F9 Cells Can be Differentiated toward Two Distinct, Mutually Exclusive Pathways by Retinoic Acid and Sodium Butyrate

Both retinoic acid (RA) and sodium butyrate (NaB) induce differentiation in embryonal carcinoma F9 cells. Phenotypic changes caused by RA are irreversible, whereas those of NaB are rapid and reversible. In this study, we investigated the effects of combinations of these two agents on F9 cell differentiation and showed that RA had no effect on the cells induced to differentiate with NaB and vice versa. Thus, F9 cells are induced to differentiate along two distinct pathways which are mutually exclusive.     

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.     

PI3K/Akt pathway regulates retinoic acid‐induced Hox gene expression in F9 cells

Retinoic acid (RA), the most potent natural form of vitamin A, is a key morphogen in vertebrate development and a potent regulator of both adult and embryonic cell differentiation. Specifically, RA regulates clustered Hox gene expression during embryogenesis and is required to establish the anteroposterior body plan. The PI3K/Akt pathway was also reported to play an essential role in the process of RA‐induced cell differentiation. Therefore, we tested whether the PI3K/Akt pathway is involved in RA‐induced Hox gene expression in a F9 murine embryonic teratocarcinoma cells. To examine the effect of PI3K/Akt signaling on RA‐induced initiation of collinear expression of Hox genes, F9 cells were treated with RA in the presence or absence of PI3K inhibitor LY294002, and time‐course gene expression profiles for all 39 Hox genes located in four different clusters—Hoxa, Hoxb, Hoxc, and Hoxd—were analyzed. Collinear expression of Hoxa and ‐b cluster genes was initiated earlier than that of the ‐c and ‐d clusters upon RA treatment. When LY294002 was applied along with RA, collinear expression induced by RA was delayed, suggesting that the PI3K/Akt signaling pathway somehow regulates RA‐induced collinear expression of Hox genes in F9 cells. The initiation of Hox collinear expression by RA and the delayed expression following LY294002 in F9 cells would provide a good model system to decipher the yet to be answered de novo collinear expression of Hox genes during gastrulation, which make the gastrulating cells to remember their positional address along the AP body axis in the developing embryo.     

Anti-apoptotic effect of retinoic acid on retinal progenitor cells mediated by a protein kinase A-dependent mechanism

Retinal progenitor cells （RPCs） are neural stem cells able to differentiate into any normal adult retinal cell type, except for pigment epithelial cells. Retinoic acid （RA） is a powerful growth/differentiation factor that generally causes growth inhibition, differentiation and/or apoptosis. In this study, we demonstrate that RA not only affects mouse RPC differentiation but also improves cell survival by reducing spontaneous apoptotic rate without affecting RPC proliferation. The enhanced cell survival was accompanied by a significant upregulation of the expression of protein kinase A （PKA） and several protein kinase C （PKC） isoforms. Treatment of cells grown in RA-free media with 8-bromoadenosine3＇,5＇-cyclic monophosphate, a known activator of PKA, resulted in an anti-apoptotic effect similar to that caused by RA; whereas the PKA inhibitor N-[2-（p-bromocinnamylamino）ethyl]-5-isoquinolinesul- fonamide dihydrochloride led to a significant （-32%） increase in apoptosis. In contrast, treatment of RPCs with any of two PKC selective inhibitors, 2,2＇,3,3＇,4,4＇-hexahydroxy-1,1 ＇-biphenyl-6,6＇-dimethanol dimethyl ether and bisindolylmaleimide XI, led to diminished apoptosis; while a PKC activator, phorbol 12-myristate 13-acetate, increased apoptosis. These and other data suggest that the effect of RA on RPC survival is mostly due to the increased anti-apoptotic activity elicited by PKA, which might in turn be antagonized by PKC. Such a mechanism is a new example of tight regulation of important biological processes triggered by RA. Although the detailed mechanisms remain to be elucidated, we provide evidence that the pro-survival effect of RA on RPCs is not mediated by changed expression of p53 or bcl-2, and appears to be independent of 15-amyloid, Fas ligand, TNF-α, ganglioside GM1 and ceramide C 16-induced apoptotic pathways.     

Retinoic acid induces neuronal differentiation of embryonal carcinoma cells by reducing proteasome-dependent proteolysis of the cyclin-dependent inhibitor p27.

Retinoic acid (RA) treatment of embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) induces growth arrest and terminal differentiation along the neuronal pathway. In the present study, we provide a functional link between RA and p27 function in the control of neuronal differentiation in NT2/D1 cells. We report that RA enhances p27 expression, which results in increased association with cyclin E/cyclin-dependent kinase 2 complexes and suppression of their activity; however, antisense clones, which have greatly reduced RA-dependent p27 inducibility (NT2-p27AS), continue to synthesize DNA and are unable to differentiate properly in response to RA as determined by lack of neurite outgrowth and by the failure to modify surface antigens. As to the mechanism involved in RA-dependent p27 upregulation, our data support the concept that RA reduces p27 protein degradation through the ubiquitin/proteasome-dependent pathway. Taken together, these findings demonstrate that in embryonal carcinoma cells, p27 expression is required for growth arrest and proper neuronal differentiation.     

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。     

Differential regulation by retinoic acid of the homeobox genes of the four HOX loci in human embryonal carcinoma cells.

We studied the expression of 38 human homeobox genes belonging to the four HOX complex loci in embryonal carcinoma (EC) cells induced to differentiate by culturing them in a medium containing retinoic acid (RA). Genes located at the 3' end of each one of the four HOX loci are activated by RA in a sequential order colinear with their 3' to 5' arrangement in the cluster: 3' HOX genes respond early to the drug while upstream genes respond progressively later. Among the genes located at the 5' end of HOX loci RNase protection analysis reveals that one HOX3 gene and four HOX4 genes are weakly expressed in EC stem cells and downregulated upon treatment with 10(-5) M RA. While activation of early responding genes does not require continuous protein synthesis, the observed timing and polarity of gene activation is disrupted in the absence of protein synthesis.     

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.     

Retinoic acid prevents germ cell mitotic arrest in mouse fetal testes

During mouse fetal development, meiosis is initiated in female germ cells only, with male germ cells undergoing mitotic arrest. Retinoic acid (RA) is degraded by Cyp26b1 in the embryonic testis but not in the ovary where it initiates the mitosis/meiosis transition. However the role of RA status in fetal germ cell proliferation has not been elucidated. As expected, using organ cultures, we observed that addition of RA in 11.5 days post-conception (dpc) testes induced Stra8 expression and meiosis. Surprisingly, in 13.5 dpc testes although RA induced Stra8 expression it did not promote meiosis. On 11.5 and 13.5 dpc, RA prevented male germ cell mitotic arrest through PI3K signaling. Therefore 13.5 dpc testes appeared as an interesting model to investigate RA effects on germ cell proliferation/differentiation independently of RA effect on the meiosis induction. At this stage, RA delayed SSEA-1 extinction, p63γ expression and DNA hypermethylation which normally occur in male mitotic arrested germ cells. In vivo, in the fetal male gonad, germ cells cease their proliferation and loose SSEA-1 earlier than in female gonad and RA administration maintained male germ cell proliferation. Lastly, inhibition of endogenous Cyp26 activity in 13.5 dpc cultured testes also prevented male germ cell mitotic arrest. Our data demonstrate that the reduction of RA levels, which occurs specifically in the male fetal gonad and was known to block meiosis initiation, is also necessary to allow the establishment of the germ cell mitotic arrest and the correct further differentiation of the fetal germ cells along the male pathway.