Reversible effects of retinoic acid on glycosaminoglycan synthesis during differentiation of HL-60 leukemia cells

Glycosaminoglycans (GAGs) play an important role in cell-cell and cell-substratum interactions, and undergo specific changes during neutrophil development. Previous studies (Luikart, S.D., Maniglia, C. A., and Sartorelli, A. C. Cancer Res., 44: 2907-2912, 1984) have shown that both dimethyl sulfoxide and 4-beta-phorbol-12-beta-myristate-13-alpha-acetate decreased GAG production by a hypoxanthine-guanine phosphoribosyl transferase-deficient clone of HL-60 promyelocytic leukemia cells prior to the appearance of a mature myeloid or monocytoid phenotype. To expand these investigations further, GAGs were analyzed by cetylpyridinium chloride precipitation and DEAE-Sephacel ion-exchange chromatography after labeling of parental HL-60 cultures with [35S]sulfate and D-[3H]glucosamine for 6 h, following treatment with 1 microM all-trans retinoic acid (RA). Chondroitin sulfate represented the major GAG species produced, although endo-beta-galactosidase-sensitive undersulfated macromolecules which possibly might be keratan sulfate, were also identified. GAG production decreased over a time period of 144 h in culture. RA treatment reduced the amount of radiolabeled cell-associated GAGs by 50% after 48, 96, and 144 h of exposure. In contrast, commitment to myelocytic maturation of the majority (i.e., approximately 60%) of the cells occurred between 72 and 96 h of RA treatment. Concurrently with the appearance of mature granulocytic cells, two-thirds of the radiolabeled GAGs were recovered from the medium, compared to one-third in untreated cultures, a phenomenon that resulted in an overall alteration in the distribution of GAGs. When RA was removed by washing after either 48 h (i.e., precommitment to differentiation) or 96 h (i.e., postcommitment to differentiation), a 1.5- to 3.5-fold increase in GAG production was noted 48 h later; this increase was unrelated to the medium change or to alterations in cell cycle distribution. The amounts of endo-beta-galactosidase-sensitive macromolecules were unaltered. Thus, although 1 microM RA inhibited the synthesis of chondroitin sulfate by HL-60 leukemia cells, this inhibition was reversible by removal of the drug and appeared to be unrelated to the commitment to myelocytic maturation.     

Cholesterol starvation induces differentiation of human leukemia HL-60 cells

Cholesterol metabolism is particularly active in malignant, proliferative cells, whereas cholesterol starvation has been shown to inhibit cell proliferation. Inhibition of enzymes involved in cholesterol biosynthesis at steps before the formation of 7-dehydrocholesterol has been shown to selectively affect cell cycle progression from G(2) phase in human promyelocytic HL-60 cells. In the present work, we explored whether cholesterol starvation by culture in cholesterol-free medium and treatment with different distal cholesterol biosynthesis inhibitors induces differentiation of HL-60 cells. Treatment with SKF 104976, an inhibitor of lanosterol 14-alpha demethylase, or with zaragozic acid, which inhibits squalene synthase, caused morphologic changes alongside respiratory burst activity and expression of cluster of differentiation antigen 11c (CD11c) but not cluster of differentiation antigen 14. These effects were comparable to those produced by all-trans retinoic acid, which induces HL-60 cells to differentiate following a granulocyte lineage. In contrast, they differed from those produced by vitamin D(3), which promotes monocyte differentiation. The specificity of the response was confirmed by addition of cholesterol to the culture medium. Treatment with PD 98059, an inhibitor of extracellular signal-regulated kinase, abolished both the activation of NADPH oxidase and the expression of the CD11c marker. In sharp contrast, BM 15766, which inhibits sterol Delta(7)-reductase, failed to induce differentiation or arrest cell proliferation. These results show that changes in the sterol composition may trigger a differentiation response and highlight the potential of cholesterol pathway inhibition as a possible tool for use in cancer therapy.     

Effects of uridine on the growth and differentiation of HL-60 leukemia cells

HL-60 leukemia cells, induced to differentiate, activate a Na(+)-dependent nucleoside transport system, concomitant with a reduction in the nitrobenzylthioinosine (NBMPR)-sensitive facilitated transport of nucleosides. The consequence of these changes lead to the formation of intracellular pools of uridine. To examine the possible role of accumulated uridine in the commitment of HL-60 leukemia cells to undergo maturation, the effects of uridine on the growth and differentiation of HL-60 cells were monitored. Uridine at millimolar levels caused a concentration-dependent inhibition of cellular growth, resulting in the accumulation of cells in the G2/M phases of the cell cycle, phenomena that preceded the formation of differentiated cells. These effects of uridine were reduced by 10 microM NBMPR, an inhibitor of the facilitated transport of nucleosides. The effects of 24 mM uridine on growth and differentiation of HL-60 cells were also prevented by 5 mM inosine, and partially prevented by either 2 mM hypoxanthine or 20 microM adenosine. Pretreatment of HL-60 cells with 24 mM uridine for 6 days, followed by a 2 h exposure to TPA, resulted in the rapid attachment of cells to the tissue culture dish, and the extension of long processes. Although the concentrations of uridine required for the above effects are greater than those achieved during differentiation, these observations suggest that uridine may play a role in regulating the maturation process.     

Effects of lithium on dimethyl sulfoxide induced differentiation of HL-60 promyelocytic leukemia cells.

The human promyelocytic leukemia cell line, HL-60, was used to investigate the effects of lithium on dimethyl sulfoxide (DMSO)-induced granulocytic differentiation of these cells. Dose-response studies showed an optimal increase of cellular proliferation when cells were incubated with 5 mM lithium for 5 days (127 +/- 5% of DMSO only treated cells). This enhancement in growth was preceded by significantly increased [methyl-3H]thymidine incorporation (143 +/- 4% of DMSO only treated controls) after 2 days. However, no significant changes in the ability of cells to reduce NBT could be detected irrespective of whether the cells were incubated with 1.25% (v/v) DMSO only, or with DMSO plus non-toxic concentrations (less than or equal to 10 mM) lithium. From the results obtained it would appear as if the arrest of growth induced by DMSO and the stimulation of proliferation effected by lithium occurs along independent pathways and that lithium exerts its mitogenic effect prior to the onset of terminal differentiation initiated by DMSO.     

Changes in actin and actin-binding proteins during the differentiation of HL-60 leukemia cells.

Actin and actin-binding proteins form a peripheral network on the cytosolic side of the plasma membrane. These cytoskeleton proteins are involved in functions that require cellular movement and may also have a role in modulating signal transduction during cellular proliferation and differentiation. To measure changes in F-actin and actin-binding proteins during HL-60 differentiation, cells were induced to mature along the granulocytic pathway by exposure to 1 microM retinoic acid (RA) for 5 days and were analyzed for F-actin and actin-binding proteins by flow cytometry. The amounts of F-actin and spectrin in untreated HL-60 cells and in those undergoing differentiation by treatment with the retinoid did not differ. N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-phallacidin was used to measure F-actin content and a monoclonal antibody followed by fluorescence isothiocyanate-conjugated goat anti-mouse immunoglobulin antibody was used to measure the content of spectrin; cells were analyzed by flow cytometry. In contrast, cells exposed to RA contained larger amounts of alpha-actinin, vinculin, talin, lipocortin I, and lipocortin II, as determined with their respective antibodies followed by flow cytometric analysis as described above. An RA-supersensitive clone of HL-60, designated HL-60/S4, exhibited lower constitutive levels of alpha-actinin, vinculin, and talin but a higher constitutive level of lipocortin II than parental cells. Treatment of HL-60/S4 with RA led to increases in vinculin, talin, lipocortin I, and lipocortin II. An RA-resistant clone, designated HL-60/R3, constitutively expressed larger amounts of alpha-actinin, vinculin, lipocortin I, and lipocortin II than parental HL-60 cells. Treatment of HL-60/R3 with RA resulted in decreases in the amounts of these actin-binding proteins. Changes in actin-binding proteins that occur during the differentiation of HL-60 cells suggest that these proteins may be of importance to the expression of the mature phenotype.     

Alteration in insulin receptor expression accompanying differentiation of HL-60 leukemia cells

Differentiation of leukemic cells in vitro is characterized by the sequential appearance of morphological, functional, and biochemical markers of maturation. The interaction of insulin with its receptor may be a regulator of growth and differentiation of leukemic cells. Human promyelocytic leukemia cells (HL-60) demonstrate specific reversible insulin binding consistent with properties of human insulin receptor. HL-60 cells treated with 500 microM N6,O2-dibutyryl adenosine 3',5'-cyclic monophosphate, 1 microM 1 alpha, 25-dihydroxyvitamin D3, or 41 nM phorbol-12-myristate-13-acetate expressed monocytic markers of differentiation and an increase in insulin receptor expression. The change in insulin receptor expression with 1 microM 1 alpha, 25-dihydroxyvitamin D3 and N6,O2-dibutyryl adenosine 3',5'-cyclic monophosphate induction was further characterized by Scatchard analysis. High affinity binding (Kd) constant was not altered, and the change in binding was attributed to receptor number. Commitment to increased insulin receptor expression was demonstrated after 1-h exposure to 1 microM 1 alpha, 25-dihydroxyvitamin D3. Agents which induced granulocytic differentiation, such as 160 mM dimethyl sulfoxide and 100 nM retinoic acid, significantly decreased insulin receptor expression compared to monocytic inducing agents. This difference in insulin receptor expression correlated with binding characteristics in normal human peripheral granulocyte and monocytes. The HL-60 cell line offers a model for the study of the molecular events which lead to the contrasting insulin receptor expression during myeloid and monocytoid hematopoiesis.     

Mechanism of the induction of the differentiation of HL-60 leukemia cells by antifolates

The classic inhibitor of dihydrofolate reductase (DHFR), methotrexate (MTX), has been shown to be an effective inducer of the differentiation of HL-60 promyelocytic leukemia cells (Bodner A.J. et al.; J. Natl. Cancer Inst. 67:1025-1030; 1981). We have obtained evidence that induction of the differentiation of these cells by MTX, as well as by other folic acid antagonists, is the result of the effects of these agents on purine and thymine nucleotide biosynthesis. Thymidine (10 microM) completely blocked both the cytotoxicity and induction of differentiation produced by the specific inhibitor of thymidylate synthase (TS), N10-propargyl-5,8-dideazafolic acid (CB-3717). Thymidine also blocked the acute cytotoxicity caused by MTX and trimetrexate (TMQ); the induction of differentiation and the loss of proliferative capacity, however, were only partially prevented by thymidine. Hypoxanthine (100 microM), which completely restored antifolate-depleted purine nucleotide levels, had no effect on either the cytotoxicity or the induction of maturation produced by these agents. The growth inhibitory effects and the induction of differentiation caused by dideazatetrahydrofolic acid (DDATHF), which acts on de novo purine nucleotide biosynthesis rather than on DHFR or TS, was completely prevented by hypoxanthine. Hypoxanthine also completely prevented the inhibition of cellular replication and induction of differentiation by MTX and TMQ when combined with thymidine. The findings suggest that the depletion of intracellular thymine nucleotide levels by the antifolates, MTX, TMQ, and CB-3717 is the primary event involved in the maturation of HL-60 leukemia cells produced by these agents and that maturation occurs concomitantly with a high level of cytotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pycnogenol induces differentiation and apoptosis in human promyeloid leukemia HL-60 cells

Pycnogenol, rich of many phytochemicals of medical value, is a commercialized nutrient supplement extracted from the bark of European coastal pine. In this study, we investigated the anti-tumor effects of Pycnogenol on HL-60, U937 and K562 human leukemia cell lines. We found that Pycnogenol inhibited cell proliferation dose- and time-dependently, and the IC(50)s of Pycnogenol on HL-60, U937 and K562 cells were 150, 40 and 100 microg/ml, respectively. When HL-60 cells were incubated with low concentrations of Pycnogenol (50, 100 and 125 microg/ml) for 24 h, a prominent G0/G1 arrest was observed, followed by gradual accumulation of sub-G0/G1 nuclei. At 48 h of treatment, 50-70% of HL-60 cells differentiated, as evidenced by morphological changes, NBT reduction, induction of NSE activity, and increases of cell surface expression of CD11b. However, results from Annexin V/PI staining, DAPI staining and DNA fragmentation assay indicated that Pycnogenol induced HL-60, U937 and K562 cell apoptosis at their respective IC(50)s after 24 h of treatments. Pretreatment of z-DEVD-fmk, a caspase-3 specific inhibitor, not only decreased caspase-3 activity but also reduced the percentage of apoptotic cells induced by Pycnogenol. This indicated that caspase-3 activation was involved in Pycnogenol induced-apoptosis. In conclusion, Pycnogenol induced differentiation and apoptosis in leukemia cells. Our data suggest that Pycnogenol could serve as a potent cancer chemopreventive or chemotherapeutic agent for human leukemia.     

淫羊藿甙对人急性早幼粒白血病细胞分化的影响

目的　为寻找新的肿瘤细胞诱导分化剂 ,探讨了淫羊藿甙 ( icariin,ICA)的诱导分化作用及其机制。方法　选用从朝鲜淫羊藿中提取的单体成分 ICA,采用四唑氮蓝 ( NBT)还原试验 ,12 5I-标记的环磷酸腺苷 ( c AMP)和环磷酸鸟苷 ( c GMP)双抗体分离技术以及扫描电镜技术 ,观察了 ICA对人急性早幼粒白血病细胞 ( HL- 60 )分化的影响。结果　ICA( 0 .1g/ L)作用后的 HL- 60细胞 ,NBT还原能力明显增强 ,细胞内 c AMP/ c GMP比值升高 ,细胞膜表面发生明显变化 ,出现较多的皱褶和球状突起。结论　ICA对 HL- 60细胞有诱导分化作用 ,其机制可能与升高细胞内 c AMP/ c GMP比值有关。     

Synergistic effects of highly unsaturated fatty acid-containing phosphatidyl-ethanolamine on differentiation of human leukemia HL-60 cells by dibutyryl cyclic adenosine monophosphate.

Highly unsaturated fatty acid-containing phospholipid (HUFA-PL) has many nutritional and medical applications. We investigated the effect of HUFA-PL on differentiation of human leukemia HL-60 cells induced by dibutyryl cyclic adenosine monophosphate (dbcAMP). HUFA-containing phosphatidylethanolamine (HUFA-PE), such as salmon testis PE, significantly enhanced dbcAMP-induced cell differentiation. A combined treatment of 200 mM dbcAMP with 50 mM HUFA-PE increased the nitroblue tetrazolium (NBT)-reducing activity, which is an indicator of differentiation, to a level comparable to that in the case of 500 mM dbcAMP treatment. In contrast, HUFA-lyso PE (a monoacyl form) did not exert an enhancing effect on dbcAMP-induced differentiation. The enhancing effect of HUFA-PE was suppressed by a protein kinase C inhibitor, staurosporine, while a protein kinase A inhibitor, H-8, did not suppress the enhancing effect. These findings suggest that HUFA-PE might enhance dbcAMP-induced differentiation through modulation of the protein kinase C signaling pathway in HL-60 cells.     

突变型IκBα对HL-60细胞凋亡和分化的影响

目的：探讨缺失突变型IκBα对HL-60细胞凋亡和分化的影响。方法：通过脂质体技术,将pCLX-IκBα△N重组载体质粒DNA转移到HL-60细胞并于转染48小时和72小时后,分别分析AnnexinⅤ和PI以及CD11b的表达。结果：转染后,AnnexinⅤ＋/PI-细胞数增加。其中,转染72小时后的AnnexinⅤ＋/PI-细胞数高于转染后48小时的AnnexinⅤ＋/PI-细胞数。然而,无论是转染后48小时还是72小时,表达CD11b的阳性细胞数与未转染细胞的CD11b阳性数几乎无差别。结论：短暂表达IκBα△N cDNA能够诱导HL-60细胞发生凋亡,但似乎并不影响该细胞向粒系的分化。     

三氧化二砷对白血病HL-60细胞株分化影响的研究

目的:探讨三氧化二砷(As2O3)治疗白血病的作用机制.方法:1)用流式细胞仅检测细胞膜上的CD11b的表达和细胞凋亡率.2)用RT-PCR方法检测c-myc基因在mRNA水平上的表达.结果:2.5 μmol/LAs2O3处理HL-60细胞90 h后,细胞分化的标志性细胞膜抗原CD11b表达明显升高,由(14.5±2.1)%升高到(35.4±5.7)%,P<0.05;NBT阳性细胞由(10.0±2.1)%升高到(31.25±3.4)%(P<0.05),同时c-myc基因在mRNA水平也明显降低.结论:As2O3通过降低c-myc基因表达及上调细胞分化抗原CD11b表达,从而促进HL-60细胞分化.     

Induction of the differentiation of HL-60 promyelocytic leukemia cells by palmitoleic and myristoleic acids

Exposure of HL-60 promyelocytic leukemia cells to palmitoleic or myristoleic acids for 6 days produced both functional and morphological granulocytic maturation. Considerably less or no induction of differentiation occurred with a variety of other fatty acids. Combinations of fatty acids with the granulocytic inducer of maturation, DMSO, did not significantly increase the degree of differentiation of HL-60 cells over that produced by the fatty acids alone. A series of HL-60 cell clones were isolated which differed in sensitivity to the differentiation inducing activities of palmitoleic acid, myristoleic acid, and DMSO. These findings imply that myristoleic acid and palmitoleic acid act to initiate the maturation process by events that are distinct from those produced by DMSO. The capacity of myristoleic and palmitoleic acids to induce leukemic cell differentiation is discussed with respect to protein acylation by fatty acids.     

Attenuation of the induced differentiation of HL-60 leukemia cells by mitochondrial chaperone HSP70

The HSP70 family of heat shock proteins, which are involved in development and cellular differentiation, is elevated in various tumor cell lines. To examine the role of these proteins in neoplastic cell differentiation, four members of the HSP70 multiple gene family (i.e., HSP70, HSC70, GRP78, and mtHSP70) were examined during the induced differentiation of HL-60 promyelocytic leukemia cells. Western analyses showed that continuous exposure for 48 h of HL-60 cells to the differentiation-inducing agents, all-trans retinoic acid, 1,25-dihydroxyvitamin D3, or N-methylformamide, resulted in decreases in mitochondrial HSP70 (mtHSP70), with little change in the levels of HSP70, HSC70, and GRP78. To gain information on the role of mtHSP70 in the differentiation process, HL-60 cells were transfected with either murine mthsp70 cDNA or vector alone. Slightly greater than twofold increases in mtHSP70 protein levels occurred in cells transfected with the mthsp70 cDNA. In vector-transfected HL-60 cells, myeloid differentiation, measured as an increase in CD31 expression and nitroblue tetrazolium positivity, was observed following 3-6 days of treatment with each of the three inducing agents. In contrast, cell differentiation induced by each agent was markedly attenuated in mthsp70-transfected HL-60 cells. These findings suggest that a decrease in mtHSP70 is important for the induced differentiation of HL-60 cells.     

Regulation of transferrin receptor in myeloid and monocytic differentiation of HL-60 leukemia cells

Modulation of surface transferrin receptor activity has been associated with leukemia cell differentiation and proliferation. To examine the mechanisms involved in regulating this event, receptor protein and mRNA levels were measured in HL-60 promyelocytic leukemia cells induced to differentiate along the myelocytic and monocytic pathways. Transferrin receptor down-regulation which occurs during granulocytic differentiation by dimethyl sulfoxide, retinoic acid, or aclacinomycin A appears to be kinetically compatible with reduced biosynthesis resulting from reductions in the level of steady-state mRNA. In contrast, genetic modulation does not appear to mediate the initial receptor down-regulation seen during 12-O-tetradecanoylphorbol-13-acetate-induced monocytic differentiation. However, a reduction in levels of receptor message appears to contribute to the maintenance of diminished transferrin receptor activity in these 12-O-tetradecanoylphorbol-13-acetate-treated cells. A common feature of myelocytic and monocytic differentiating cells is the complete inhibition of cellular proliferation observed within 10 to 16 h following a four-fold reduction in surface transferrin receptor. We conclude that the early decline in transferrin receptor levels precludes its regulation as a consequence of the decrease in proliferation, but rather implicates its role in the programmed cessation of growth which is requisite for the terminal differentiation of these cells.     

Induction of differentiation of human promyelocytic leukemia cells (HL-60) by arginase

Human promyelocytic leukemia cells (HL-60) were found to be induced by dimethylulfoxide and several other compounds to phagocytize, reduce NBT dye, and change into forms that were morphologically similar to granulocytes and macrophages. Arginase also induced these differentiation-associated properties of the cells. The induction of differentiation by arginase was significantly inhibited by addition of excess arginine, but not by lysine or leucine; therefore, the effect of arginase may be due to arginase-mediated arginine depletion.     

二烯丙基三硫对人白血病HL-60细胞凋亡作用的实验研究

目的研究二烯丙基三硫对HL-60细胞凋亡的影响及其作用机制。方法将浓度为30 mg/L的二烯丙基三硫与HL-60细胞体外共培养48小时后,采用Wright染色在光学显微镜下观察细胞形态学变化;以流式细胞术（flow cytometry,FCM）检测细胞凋亡率;应用免疫组化方法检测细胞内Bcl-2、survivin蛋白的表达。结果光镜下可见二烯丙基三硫作用后细胞形态出现凋亡的形态特征,空白对照和30 mg/L的二烯丙基三硫分别与HL-60细胞作用48小时,细胞的凋亡率分别为（12.42&#177;0.76）%和（60.57&#177;3.24）%（P〈0.05）,二烯丙基三硫组HL-60细胞的Bcl-2、survivin蛋白呈弱阳性或阴性表达,而对照组均阳性表达。结论二烯丙基三硫促进HL-60细胞的凋亡的机制,可能是通过抑制Bcl-2、survivin蛋白的表达。     

Specific cytoskeleton changes during apoptosis accompanying induced differentiation of HL-60 myeloid leukemia cells

Changes in actin filaments and microtubules were studied in the human myeloid leukemia HL-60 cell line during the process of apoptotic cell death accompanying induced differentiation. These cytoskeleton changes were assessed during a 6-day cultivation in the presence of 10(-6) M all-trans retinoic acid (ATRA), a specific inductor of both differentiation into granulocytes and apoptosis, or during a 18-day cultivation in the presence of 1.6 nM phorbol myristylacetate (PMA), which induces differentiation into macrophages. The processes were studied at the morphological level by fluorescence microscopy and, quantitatively, by flow cytometry. The results showed that the actin cytoskeleton underwent specific structural changes during the apoptotic process, but microtubules were not actively involved. In the initial stages of apoptosis, a fine meshwork of actin filaments turned into actin granules that, in the final stages, were transformed into a network of long actin fibres distributed throughout the cytoplasm. These actin structures were considered to play an active role in two main morphological events of apoptosis - formation of blebs and final cell disintegration into apoptotic bodies. In addition, high proportion of cells with apoptotic nuclei and completely destroyed actin structures were found in the differentiating ATRA-treated cell population. Flow cytometric measurement of cytoskeletal proteins content confirmed all these observed changes. Alterations and rearrangements of both cytoskeletal structures are common for the apoptotic cell death of HL-60 cells and they are independent on the course of differentiation.     

Reciprocal alterations of GMP reductase and IMP dehydrogenase activities during differentiation in HL-60 leukemia cells.

The study was undertaken to elucidate the regulatory roles of GMP reductase (GMPR) and IMP dehydrogenase (IMPDH) on purine interconversion during differentiation. Treatment of HL-60 cells with retinoic acid (1 microM) induced granulocytic differentiation which was accompanied with a 2.4-fold increase in GMPR and 55% decrease in IMPDH activities. Maturation induced by 12-O-tetradecanoylphorbol 13-acetate or dimethylsulfoxide was also associated with similar reciprocal alterations. Incubation with guanosine (200 microM), which expands the guanine nucleotide pool, elevated GMPR (1.9-fold) and decreased IMPDH (73%) activities. The synchronous and opposing alterations in GMPR and IMPDH activities should amplify the metabolic response due to differentiation or guanylate pool expansion.