Cell cycle dependent regulation of IMP dehydrogenase activity and effect of tiazofurin.

The activity of IMP dehydrogenase (IMP DH), the rate-limiting enzyme of de novo GTP biosynthesis, was shown to be increased in cancer cells. Tiazofurin, an inhibitor of IMP dehydrogenase, proved to be an effective agent in the treatment of refractory granulocytic leukemia. To examine the cell cycle dependent alterations of GTP synthesis and sensitivities to tiazofurin, we measured IMP DH activities and GTP pools, as well as the effects of tiazofurin on cell cycle phase enriched HL-60 cells. We now show that IMP DH activities and GTP concentrations are increased in S-phase enriched fractions of HL-60 cells. Moreover, the depletion of GTP concentrations by tiazofurin is most effective in S-phase enriched HL-60 cells. These results may be utilized in cancer chemotherapy to combine tiazofurin with biologic response modifiers which recruit quiescent leukemic cells into the cell cycle.     

Differentiation of HL-60 myeloid leukaemia cells is associated with a transient block in the G2 phase of the cell cycle

The human promyelocytic leukaemia cell line HL-60 can be induced to differentiate towards mature granulocytes by treatment with dibutyryl cyclic adenosine-3',5'-monophosphate (dbcAMP). Differentiation begins within 16-24 h of treatment and is associated with a time- and dose-dependent accumulation of cells in the G0/G1 phase of the cell cycle with a concomitant decrease in the number of cells in the S and G2 + M phases. Using acridine orange staining, we found that the RNA content of the cells also decreased following differentiation. Stathmokinetic analysis of HL-60 cell populations following dbcAMP treatment showed no effect on the total number of cells in the G0/G1 or S phases, or the rate of progression of cells through these cell cycle compartments. In contrast, dbcAMP was found to induce a transient arrest of the cells in the G2 phase. We also found that differentiation induced by dbcAMP did not require progression of the cells through the cell cycle. Cells arrested in either G1/S by hydroxyurea or G2 + M by colcemid eventually expressed markers of mature granulocytes. These results demonstrate that dbcAMP modulates cell cycle progression. However, these cell cycle changes alone are insufficient to induce granulocytic differentiation of HL-60 cells.     

Relationships between the cell cycle and the expression of c-myc and transferrin receptor genes during induced myeloid differentiation

We examined the relationship of cellular oncogene c-myc and transferrin receptor (TfR) gene expression to cell proliferation and cell cycle progression during myeloid differentiation in the HL-60 myeloid leukemia cell line. In order to determine levels of mRNA for these genes in HL-60 cells induced to differentiate along the myeloid pathway, RNA was isolated from HL-60 cells incubated with retinoic acid for 24 h and Northern blots were probed with labeled cDNAs for c-myc and TfR. c-myc mRNA decreased within 3 h of retinoic acid addition, and TfR mRNA decreased after 9 h; both mRNAs continued to decrease over 24 h. RNA was also isolated from HL-60 cells separated by centrifugal elutriation into cell cycle phases. TfR and c-myc cDNA probes hybridized equally to RNA from uninduced cells in all phases of the cell cycle. However, after 24 h incubation with the differentiation inducer retinoic acid, TfR mRNA was expressed substantially less in the G1 stage, whereas c-myc mRNA was still expressed equally in all cell cycle phases. These data indicate that, although TfR and c-myc expression are both associated with cell proliferation in the HL-60 line, TfR is down-regulated specifically in G1 upon induction of terminal differentiation whereas c-myc expression is disassociated from cell cycle control in these cells.     

Inducers of leukemic cell differentiation cause down-regulation of RB gene expression.

Expression of the retinoblastoma (RB) tumor suppressor gene during cell differentiation induced by dimethyl sulfoxide or sodium butyrate was studied in HL-60 human promyelocytic leukemia cells. As cells progressed through the cell cycle, the amount of RB protein per cell increased with homeostasis maintained, so that the amount of RB protein relative to the total cell mass remained almost constant. Dimethyl sulfoxide was used to induce these promyelocytic leukemia cells to undergo terminal differentiation into mature myeloid cells. There was an early reduction in the RB protein expressed per cell. The reduction in expression was similar for cells in all cell cycle phases. There was also progressively reduced expression at later times as cells terminally differentiated. This was compared to the case in which sodium butyrate was used to induce the differentiation of HL-60 cells into mature monocytic cells. An early reduction in RB protein expression per cell also occurred. It occurred for cells in all cell cycle phases as well. Thus, the induced differentiation of HL-60 cells along either the myeloid or the monocytic differentiation lineage involves an early reduction in RB expression, which is common to both pathways. The reduction anteceded proliferative arrest or differentiation. In both cases, the final, resulting G0-differentiated cells had less RB protein per cell than the proliferating, immature, leukemic precursor cells.     

Cell cycle dependence of calmodulin levels during HL-60 proliferation and myeloid differentiation: No changes during pre-commitment

The putative role of Ca²⁺ and calmodulin in regulating cell proliferation and differentiation was tested in HL-60 human promyelocytic leukemia cells. The dependence of retinoic acid (RA)-induced terminal myeloid differentiation of HL-60 promyelocytic leukemia cells on calmodulin levels and calcium ion flux was ascertained. RA-treated and untreated control cells were stained for cellular DNA with a Hoechst dye. Populations of G1/0, S and G2+M phase cells were isolated by fluorescence activated cell sorting (FACS). Cytosolic calmodulin levels were then measured as a function of cell cycle phase for RA-treated and untreated cells using a radioimmunoassay. RA-treated cells were measured at early times, corresponding to the precommitment state, and late times, when significant cell differentiation had occurred. Cellular calmodulin levels increased with progression through the cell cycle. In contrast, no difference in calmodulin levels was observed between RA-untreated or -treated cells in the same cell cycle phases at early or late times. RA-induced HL-60 terminal myeloid differentiation was thus apparently not regulated by cellular cytosolic calmodulin levels. These conclusions were supported by the effects of calmodulin antagonists and calcium flux inhibitors. The calmodulin antagonists trifluoperazine and compound 48/80 both retarded cell growth in a concentration-dependent manner. But at concentrations where cellular effect was evidenced by slight growth inhibition, neither antagonist inhibited RA-induced cell differentiation or G1/0 growth arrest. The same was true of the gated calcium channel inhibitors, verapamil and nitrendipene, and the passive calcium flux inhibitor, CoC1₂. RA-induced HL-60 cell differentiation and arrest in G0 was thus apparently not strongly dependent on cellular calmodulin levels or calcium flux. This is in strong contrast to murine erythroleukemia cells. The results argue against a central regulatory role for calmodulin or calcium flux in control of HL-60 growth arrest or differentiation.     

Differentiation of HL-60 promyelocytic leukemia cells: simultaneous determination of phagocytic activity and cell cycle distribution by flow cytometry

Phagocytosis of fluorescent microspheres by HL-60 promyelocytic leukemia cells following induction of differentiation with dimethyl sulfoxide (DMSO) was monitored using flow cytometry. Initiation of phagocytic capability following initiation of differentiation with 1.5% DMSO coincided with the attainment of respiratory burst activity as measured by NBT (nitro blue tetrazolium) reduction; the degree of phagocytic activity was dependent upon parameters such as microsphere size, microsphere number, and exposure time. Ingestion of fluorescent microspheres did not interfere with the measurement of DNA content using propidium iodide; thus, simultaneous determination of phagocytic activity and the cell cycle phase was possible. Accumulation of cells in the G1/G0 phase of the cell cycle following DMSO treatment was correlated with the acquisition of the capacity to phagocytize. Analysis of two-parameter correlated data also indicated that phagocytosis is coupled with residence in the G1/G0 phase of the cell cycle, further suggesting that the ability to phagocytize fluorescent microspheres is associated with end-stage differentiation.     

P(HPMA-APMA)-ATRA的合成及其对HL-60细胞诱导分化能力的评估

以N-(2-羟丙基)甲基丙烯酰胺(HPMA),N-(3-氨基丙基)甲基丙烯酰胺(APMA)和全反式维甲酸(ATRA)为原料,采用自由基溶液聚合法设计合成P(HPMA-APMA)-ATRA,并用核磁共振氢谱对该化合物进行结构表征.相比于单体ATRA,聚合物的水溶性显著增加,同时可通过胞吞作用进入细胞.3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法评估聚合物和单体ATRA对人早幼粒白血病细胞HL-60生长的抑制作用,流式细胞术检测两者对HL-60细胞周期分布及细胞表面抗原CD11b表达的影响,进一步结合氯化硝基四氮唑蓝(NBT)还原法评估聚合物诱导HL-60细胞分化的能力.结果显示,聚合物比单体ATRA具有更强的细胞生长抑制活性,其IC50值分别为1.03和4.09μmol/L;聚合物还具有更高的G0/G1期细胞阻滞效应,1.2μmol/L时,聚合物比单体ATRA的G0/G1期细胞率高出17.7%;同样,0.4μmol/L聚合物与2.4μmol/L单体ATRA诱导HL-60的NBT还原能力相当,0.8μmol/L聚合物与2.4μmol/L单体ATRA诱导HL-60细胞表面抗原CD11b表达相当,表明聚合物比单体ATRA具有更强的诱导HL-60细胞向粒细胞分化的能力,其药效增强3~4倍.     

Mechanism of regulating human leukemia cell growth and differentiation by a lymphokine

Human myelogenous leukemia cells can be induced to differentiate in vitro along the monocyte-macrophage pathway by a T cell lymphokine maturation inducer. Maturation inducer has now been purified from a human T cell line and determined to be a single chain protein with an approximate molecular weight of 53,500. It induces the differentiation and proliferation of human leukemic HL-60 promyelocytes in a dosage-dependent fashion. Initial interaction with cells at G1 phase induced the cells to enter proliferating S phase. Subsequent differentiation from S phase was dependent on an optimal inducer quantity (18.7 pM - 18.7 nM) which mediated growth cessation and termination differentiation to monocytes-macrophages. When inducer quantity was more or less than this optimal range, the cells did not undergo differentiation but were continuously stimulated to proliferate. This may represent an important proliferation mechanism of leukemic cells.     

Synergistic down-regulation of signal transduction and cytotoxicity by tiazofurin and quercetin in human ovarian carcinoma cells.

Ovarian carcinoma is one of the most common causes of cancer death in women. Tiazofurin, a C-nucleoside, arrests the cell cycle at S phase and reduces the activities of PI (phosphatidylinositol) utilizing enzymes in signal transduction by depleting cellular GTP concentration. Quercetin (QN), a flavonoid, attacks the cell cycle at the G1 and S phase boundary and mainly inhibits PI kinase (1-phosphatidylinositol 4-kinase, EC 2.7.1.67) activity in the signal transduction pathway. Because tiazofurin and QN attack different biochemical targets and arrest different phases of the cell cycle, we tested the hypothesis that the two drugs might be synergistic against human carcinoma cells. In human ovarian carcinoma OVCAR-5 cells in growth inhibition assay, the IC50s (drug concentration that inhibits 50% of cell proliferation) for tiazofurin and QN were (mean +/- SE) 13 +/- 1.2 and 66 +/- 3.0 microM; in clonogenic assays they were 6 +/- 0.5 and 15 +/- 1.2 microM, respectively. When tiazofurin was added to cells followed 12 h later by QN, synergism was observed in both growth inhibition and clonogenic assays. The combination also yielded synergistic reduction of IP3 (inositol 1,4,5-trisphosphate) concentration in the cells which may explain, at least in part, the synergistic action of tiazofurin and QN in OVCAR-5 cells. The protocols yielding synergism may have implications in the clinical treatment of human ovarian carcinoma.     

氟苯达唑对人急性髓系白血病HL-60细胞的增殖抑制作用研究

目的:研究氟苯达唑对人急性髓系白血病HL-60细胞增殖的抑制作用,明确氟苯达唑对HL-60细胞周期,凋亡发生的作用机制。方法:噻唑蓝法(MTT)检测氟苯达唑对人急性髓系白血病HL-60细胞的生长抑制作用,流式细胞术检测氟苯达唑对HL-60细胞周期,DNA片段化的影响,免疫印迹法检测Caspase, Raf, Bcl-2家族蛋白表达。结果:氟苯达唑抑制人急性髓系白血病HL-60细胞生长,HL-60细胞G2/M期增加,与阴性对照组相比,在一定的剂量和时间内,差别具有显著统计学意义;DNA片段化上升,0.25,0.5,1μM组与对照组相比差别具有显著统计学意义,促使Cleaved PARP,Cleaved-caspase 3,Cleaved-caspase 9蛋白表达量趋势增加;Bag-1和Bcl-2蛋白表达量降低;b-raf,c-raf磷酸化蛋白表达水平逐渐降低。结论:氟苯达唑通过诱导HL-60细胞阻滞于G2/M期,增加DNA片段化水平,激活Caspase, Raf, Bcl-2家族介导的凋亡相关通路抑制人急性髓系白血病HL-60细胞增殖,诱导人急性髓系白血病HL-60细胞发生凋亡而发挥抗肿瘤作用。     

19-Nor-1alpha,25-dihydroxyvitamin D2 (paricalcitol) exerts anticancer activity against HL-60 cells in vitro at clinically achievable concentrations

19-Nor-1alpha,25-dihydroxyvitamin D2 (paricalcitol) is an analogue of 1,25(OH)2D3 with reduced calcemic effects that is approved in the United States for the suppression of parathyroid hormone in chronic renal failure. Paricalcitol has anticancer activity in prostate cancer cells. We tested the effects of paricalcitol on the HL-60 leukemia cells, studying cellular differentiation, cell cycle changes, apoptosis and cellular proliferation. Paricalcitol at 10(-8)M concentration induced the maturation of HL-60 cells in a time-dependent manner, as shown by increased expression of CD11b differentiation surface antigen. The ability of HL-60 cells to reduce nitroblue tetrazolium (NBT) was markedly increased after exposure to paricalcitol at 10(-8)M for 72 h. Paricalcitol inhibited colony formation of HL-60 cells in a soft agar semisolid media after 10-day incubation (estimated IC50 of 5 x 10(-9) M. Exposure to 10(-8)M paricalcitol for 72 h increased the number of cells in G0/G1 phase, and decreased the number of cells in S phase, and significantly increased the number of HL-60 cells undergoing apoptosis. The concentration required to achieve inhibition of growth of HL-60 cells is comparable to clinically achievable levels. These findings support the clinical evaluation of paricalcitol as an antileukemia agent.     

Caffeine induces a second wave of apoptosis after low dose-rate gamma radiation of HL-60 cells

Most cell lines that lack functional p53 protein are arrested in the G₂ phase of the cell cycle due to DNA damage. It was previously found that the human promyelocyte leukemia cells HL-60 (TP53 negative) that had been exposed to ionizing radiation at doses up to 10 Gy were arrested in the G₂ phase for a period of 24 h. The radioresistance of HL-60 cells that were exposed to low dose-rate gamma irradiation of 3.9 mGy/min, which resulted in a pronounced accumulation of the cells in the G₂ phase during the exposure period, increased compared with the radioresistance of cells that were exposed to a high dose-rate gamma irradiation of 0.6 Gy/min. The D₀ value (i.e. the radiation dose leading to 37% cell survival) for low dose-rate radiation was 3.7 Gy and for high dose-rate radiation 2.2 Gy. In this study, prevention of G₂ phase arrest by caffeine (2 mM) and irradiation of cells with low dose-rate irradiation in all phases of the cell cycle proved to cause radiosensitization (D₀=2.2 Gy). The irradiation in the presence of caffeine resulted in a second wave of apoptosis on days 5–7post-irradiation. Caffeine-induced apoptosis occurring later than day 7 post-irradiation is postulated to be a result of unscheduled DNA replication and cell cycle progress.     

Differentiation of leukemic cell lines: a review focusing on murine erythroleukemia and human HL-60 cells

Several acute leukemia cell lines respond to chemical or pharmacologic inducing agents by undergoing variable degrees of differentiation. This review focuses on the manner in which murine erythroleukemia (MEL) and HL-60 cells can be induced to differentiate into virtually fully mature erythroid cells and mature granulocytes or macrophages respectively. In this process the cells undergo irreversible "commitment" to terminal differentiation which is followed by loss of proliferative capacity and alterations in the expression of genes whose products are related to specific aspects of cell maturation in the corresponding cell pathways.     

Alteration of cell cycle progression in human leukemia cell line (KOPM-28) induced by 12-o-tetradecanoylphorbol-13-acetate

Terminal cell differentiation usually results in an irreversible arrest in the G1 phase of the cell cycle and loss of cell renewal ability. Human promyelocytic leukemia HL-60 cells induced with 12-o-tetradecanoylphorbol-13-acetate (TPA) differentiate into monocytes/macrophages and accumulate in G1. We determined the effect of TPA on the growth kinetics of a human leukemia cell line (KOPM-28), which developed several of the characteristics of megakaryocytes in response to TPA, such as the surface antigen complex IIb/IIIa, platelet peroxidase and polyploidy. Cell growth was immediately and completely inhibited by TPA. Flow cytometric analysis of cellular DNA content revealed a gradual decrease in cells in G1 and an accumulation of cells in G2. These data suggest that TPA prolonged G1 and rapidly arrested the cells in G2. Synchronized cells were utilized to further analyze the rapid G2 arrest. Cells arrested with aphidicolin at the G1/S interphase were released, and the effects of TPA (added at different intervals) on cell cycle progression were examined 14 h after release. The results showed that TPA added at the end of the S phase, as well as at the G1/S interphase incompletely but distinctly arrested cells in G2. Moreover, G2 arrest was observed when TPA was added to cells released from a colcemid-induced G2/M block, suggesting that cells already in G2 were inhibited by TPA from moving through M to G1. Since some cells became multi-nucleated in the course of incubation with TPA, this G2 accumulation may have resulted at least in part from a prolongation of the phase or a transient G2 block. These changes in cell cycle progression induced by TPA may be characteristic of and/or related to megakaryocytic differentiation of hemopoietic precursor cells.     

Down-regulation of a serine protease, myeloblastin, causes growth arrest and differentiation of promyelocytic leukemia cells

Cells from the human leukemia cell line HL-60 undergo terminal differentiation when exposed to inducing agents. Differentiation of these cells is always accompanied by withdrawal from the cell cycle. Here we describe the isolation of a cDNA encoding a novel serine protease that is present in HL-60 cells and is down-regulated during induced differentiation of these cells. We have named this protease myeloblastin. Down-regulation of myeloblastin mRNA occurs with both monocytic and granulocytic inducers. Myeloblastin mRNA is undetectable in fully differentiated HL-60 cells as well as in human peripheral blood monocytes. We found that regulation of myeloblastin mRNA in HL-60 cells is serum dependent. Inhibition of myeloblastin expression by an antisense oligodeoxynucleotide inhibits proliferation and induces differentiation of promyelocyte-like leukemia cells.     

Cell cycle regulation and induction of apoptosis by beta-carotene in U937 and HL-60 leukemia cells

In this communication, we report the efficacy of beta-carotene towards differentiation and apoptosis of leukemia cells. Dose (20 microM) and time dependence (12 h) tests of beta- carotene showed a higher magnitude of decrease (significance p < 0.05) in cell numbers and cell viability in HL-60 cells than U937 cells but not normal cell like Peripheral blood mononuclear cell (PBMC). Microscopical observation of beta-carotene treated cells showed a distinct pattern of morphological abnormalities with inclusion of apoptotic bodies in both leukemia cell lines. When cells were treated with 20 microM of beta-carotene, total genomic DNA showed a fragmentation pattern and this pattern was clear in HL-60 than U937 cells. Both the cell lines, on treatment with beta- carotene, showed a clear shift in G(1) phase of the cell cycle. In addition the study also revealed anti-oxidant properties of beta-carotene since there was reduction in relative fluorescent when treated than the control at lower concentration. Collectively this study shows the dual phenomenon of apoptosis and differentiation of leukemia cells on treatment with beta-carotene.     

Synergistic action of tiazofurin and retinoic acid on differentiation and colony formation of HL-60 leukemia cells

Tiazofurin and retinoic acid synergistically induced differentiation and inhibited colony formation in HL-60 human promyelocytic leukemia cells in cell culture. The synergism was the result of different mechanisms of action, since the effect of tiazofurin, unlike that of retinoic acid, was prevented by addition of guanosine. Since it has been shown that tiazofurin down-regulated the expression of c-Ki-ras oncogene, and retinoic acid that of the myc oncogene, the joint impact of these drugs is of clinical interest particularly in end-stage leukemia where the therapeutic usefulness of tiazofurin has recently been demonstrated.     

A growth-promoting ribosome extract (GPRE) from mouse L-929 cells stimulates growth of the HL-60 human promyelocytic leukemia cell line

An extract termed growth-promoting ribosome extract (GPRE), isolated from mouse L-929 cells stimulates growth of HL-60 human promyelocytic leukemia cells. The stimulation first becomes apparent at 72 h when the cells start to enter the quiescent state. The inhibition of protein synthesis by the addition of cycloheximide to L-929 cells before ribosomal extracts were prepared did not alter the stimulatory effect of GPRE. When GPRE was added together with 20% fetal calf serum to cultures of quiescent HL-60 cells, growth was stimulated to the extent that the generation time was reduced by approximately 9 h to 32.4 h. GPRE alone was unable to stimulate the quiescent cells. The growth stimulatory effect was not restricted to one cell generation but was a characteristic of at least the following two cell cycles. GPRE extract from L-cells synchronized by centrifugal elutriation was most efficient when isolated from cells in early G1 phase, while extract from S phase cells had virtually no effect. It is tentatively suggested that the factor belongs to the competence/progression group of growth factors.     

Induction of HL-60 monocytic cell differentiation promoted by a perturbation of DNA synthesis: hydroxyurea promotes action of TPA

Control of terminal cell differentiation was studied using the human promyelocytic leukemia cell line, HL-60. HL-60 cells are known to undergo terminal monocytic differentiation when continuously exposed to 1.6 nM tetradecanoylphorbol acetate (TPA). The dose-response relationship between TPA concentration and induced differentiation is relatively steep. TPA (1.1 nM) induces little G1/0 specific growth inhibition or phenotypic differentiation. In contrast, pretreating the cells with a pulse exposure to hydroxyurea promotes their capability to terminally differentiate in response to TPA. Initially exponentially proliferating cells exposed for 20 h, approximately one doubling time, to 0.3 mM hydroxyurea, a subcytotoxic dose, underwent rapid G1/0 specific growth arrest and cell differentiation in response to subsequent exposure to 1.1 nM TPA. The extent of terminal differentiation was comparable to that induced by 1.6 nM TPA. The results support the hypothesis that early events in induction of terminal HL-60 cell differentiation depend on an S phase-specific process which may involve gene amplification.     

Thymopentin (TP5), an immunomodulatory peptide, suppresses proliferation and induces differentiation in HL-60 cells

Thymopentin (Arg-Lys-Asp-Val-Tyr, TP5) has shown immuno-regulatory activities in humans. In the present study, we investigated the effects of TP5 on the proliferation and differentiation of a human promyelocyte leukemia cell line, HL-60. It is noteworthy that TP5 displayed concentration-dependent inhibitory effects on the proliferation and colony formation of HL-60 cells. Furthermore, the decrease or even disappearance of AgNORs from nucleoli was observed in HL-60 cells after the treatment with TP5. The suppression induced by TP5 was accompanied by an accumulation of cell cycle in the G0/G1 phase. Moreover, TP5 significantly increased the NBT-reduction activity of HL-60 cells. Cytofluorometric and morphologic analysis indicated that TP5 had induced differentiation along the granulocytes lineage in HL-60 cells. d-tubocurarine (TUB) significantly antagonized the inhibitory effects induced by TP5, whereas atropine did not exhibit such effect. All the results indicated that TP5 was able to significantly inhibit proliferation and induce differentiation in HL-60 cells. Our observations also implied that TP5 not only acted as an immunomodulatory factor in cancer chemotherapy, but is also a potential chemotherapeutic agent in the human leukemia therapy.