Nucleolin and fibrillarin expression in stimulated lymphocytes and differentiating HL-60 cells. A flow cytometric assay

Nucleolin and fibrillarin are two histone-like major proteins in the nucleolus that were found to be overexpressed in proliferating cells. Using specific antibodies to either nucleolin or fibrillarin flow cytometric, measurements were carried out to demonstrate quantitative changes of these proteins during lymphocyte mitogenic activation and differentiation of HL-60 promyelocytic leukaemia cells. The expression of nucleolin increased during lymphocyte stimulation and decreased slowly but constantly in the course of differentiation of HL-60 cells. Expression of fibrillarin reached a maximum in the first cell cycle and then dropped to a basic level in stimulated lymphocytes. Compared to nucleolin, the level of fibrillarin decreased more rapidly and more extensively in differentiating HL-60 cells. The data support other observations that nucleolin is a stabile structural protein at the ribosomal genes while fibrillarin may have a more specific functional role in nucleologenesis and ribosome production.     

Induction of the differentiation of synchronized HL-60 leukemia cells by tiazofurin

Tiazofurin is an effective inducer of the maturation of HL-60 promyelocytic leukemia cells, as monitored by increased phagocytic ability and the capacity to reduce nitroblue tetrazolium (NBT). The antimetabolite acts as a potent inhibitor of IMP dehydrogenase, which results in a profound depression in the cellular levels of guanine nucleotides. Flow cytometric analysis of DNA histograms indicated that the commitment of HL-60 cells to differentiate when exposed to tiazofurin was preceded by a transient delay in the G1 phase of the cell cycle. HL-60 leukemia cells enriched in the various phases of the cell cycle by centrifugal elutriation were utilized to further characterize the relationship between the phase of the cell cycle and the commitment to enter a pathway of differentiation. Fractions composed mainly of G1 cells demonstrated an increased capacity to mature when exposed to tiazofurin, whereas fractions containing cells from the S and G2 + M phases of the cell cycle had a lower ability to enter a differentiation pathway. The findings suggest that the commitment of HL-60 cells to mature when exposed to tiazofurin is mediated during the G1 phase of the cell cycle.     

Nucleolar silver-staining patterns related to cell cycle phase and cell generation of PHA-stimulated human lymphocytes

Silver staining (Ag-I) was used to investigate changes in the nucleolar structure of PHA-stimulated human lymphocytes through the phases of the cell cycle, G1, S and G2. Ag-I patterns and cell cycle phases of individual cells were assessed by sequential silver staining, Feulgen staining, DNA microdensitometry and 3H-thymidine autoradiography. The morphology and number of Ag-I nucleoli in a particular cell depended upon the phase of the cell cycle reached and on the number of generations the cell had passed through in culture. Resting, unstimulated cells usually had one small silver positive nucleolus. During blast transformation, the silver stained nucleoli increased in number and size, and then fused to form one very large, rounded or irregular-shaped nucleolus which was present through all cell cycle phases of the first reproductive cycle. Many lymphocytes developed a band-shaped nucleolus during their first S phase in culture. Lymphocytes at all cell cycle stages of the second and third generations after PHA-stimulation had multiple nucleoli whose combined areas approximated that of the single large nucleolus observed in first generation 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.     

Decrease in CuZn-superoxide dismutase mRNA level during differentiation of human monocytic and promyelotic leukemia cells

Change in the level of CuZn-superoxide dismutase (SOD) mRNA was examined using a molecular probe during differentiation of human monocytic leukemia U937 cells or promyelotic leukemia HL-60 cells induced by either 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or dimethylsulfoxide (DMSO). CuZn-SOD mRNA levels were found to decrease during the course of differentiation, and this response is specific for differentiation, since the treatment of human B cell leukemia cells or normal diploid fibroblasts with TPA failed to have any effect on the level of CuZn-SOD mRNA. The activity of CuZn-SOD in U937 cells also decreased during differentiation, but following that of the CuZn-SOD mRNA level. The expression of the CuZn-SOD gene is thus concluded to diminish during the differentiation of HL-60 and U937 cells.     

LncRNA UCA1 elevates the resistance of human leukemia cells to daunorubicin by the PI3K/AKT pathway via sponging miR-613

Acute leukemia is a hematological malignant tumor. Long non-coding RNA urothelial cancer-associated 1 (UCA1) is involved in the chemo-resistance of diverse cancers, but it is unclear whether UCA1 is associated with the sensitivity of acute leukemia cells to daunorubicin (DNR). DNR (100 nM) was selected for functional analysis. The viability, cell cycle progression, apoptosis, and invasion of treated acute leukemia cells (HL-60 and U-937) were evaluated by cell counting kit-8 (CCK-8) assay, flow cytometry assay, or transwell assay. Protein levels were detected with Western blot analysis. Expression patterns of UCA1 and miR-613 were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between UCA1 and microRNA-613 (miR-613) was verified by dual-luciferase reporter assay. We observed that UCA1 expression was elevated in HL-60 and U-937cells. DNR constrained viability, cell cycle progression, invasion, and facilitated apoptosis of HL-60 and U-937 cells in a dose-dependent manner, but these impacts mediated by DNR were reverted after UCA1 overexpression. MiR-613 was down-regulated in HL-60 and U-937 cells, and UCA1 was verified as a miR-613 sponge. MiR-613 inhibitor reversed DNR treatment-mediated effects on viability, cell cycle progression, apoptosis, and invasion of HL-60 and U-937 cells, but these impacts mediated by miR-613 inhibitor were counteracted after UCA1 inhibition. Notably, the inactivation of the PI3K/AKT pathway caused by DNR treatment was reversed after miR-613 inhibitor introduction, but this influence mediated by miR-613 inhibitor was offset after UCA1 knockdown. In conclusion, UCA1 up-regulation facilitated the resistance of acute leukemia cells to DNR via the PI3K/AKT pathway by sponging miR-613.     

Imaging of Nucleolar Dynamics During the Cell Cycle of Cancer Cells in Live Mice

The synthesis and assembly of ribosomal subunits take place in the nucleolus. The nucleolus forms in the nucleus around the repeated ribosomal gene clusters and undergoes cyclic changes during the cell cycle. Although the nucleolus is easily visualized by light microscopy of cells in vitro, the nucleolus has not been imaged in cells in vivo. We report here development of a mouse model to visualize the nucleolus cycle of cancer cells in live mice. HT-1080 human fibrosarcoma cells were labeled in the nucleus with histone H2B-GFP and with retroviral RFP in the cytoplasm. The nucleolus was visualized by contrast to the fluorescence of GFP expressed in the nucleus. HT-1080 dual-color cells were seeded on the surface of a skin-flap of nude mice. The inside surface of the skin-flap was directly imaged with a laser scanning microscope 24 hours after seeding. The nucleoli of the cancer cells were clearly imaged in real-time. The appearance of the nucleoli changed dramatically during the cell cycle. During mitosis, the nucleolus disappeared. After mitosis, the nucleoli decreased in number and increased in size. The nucleolus appears to have a major role in cell cycle regulation effected at least in part by sequestering proteins which affect cell cycle progression. Nucleolar imaging could be used for more precise determination of cancer-cell position in the cell cycle in vivo.     

NF-kappaB pathway is involved in griseofulvin-induced G2/M arrest and apoptosis in HL-60 cells

Griseofulvin (GF), an oral antifungal agent, has been shown to exert antitumorigenesis effect through G2/M cell cycle arrest in colon cancer cells. But the underlying mechanisms remained obscure. The purpose of this study is to test the cytotoxic effect of GF on HL-60 and HT-29 cells and elucidate its underlying molecular pathways. Dose-dependent and time-course studies by flow cytometry demonstrated that 30 to 60 microM GF significantly induced G2/M arrest and to a less extend, apoptosis, in HL-60 cells. In contrast, only G2/M arrest was observed in HT-29 cells under similar condition. Pretreatment of 30 microM TPCK, a serine protease inhibitor, completely reversed GF-induced G2/M cell cycle arrest and apoptosis in HL-60 cells but not in HT-29 cells. The GF-induced G2/M arrest in HL-60 cells is reversible. Using EMSA and super-shift analysis, we demonstrated that GF stimulated NF-kappaB binding activity in HL-60 cells, which was completely inhibited by pretreatment of TPCK. Treatment of HL-60 with 30 microM GF activated JNK but not ERK or p38 MAPK and subsequently resulted in phosphorylation of Bcl-2. Pretreatment of TPCK to HL-60 cells blocked the GF-induced Bcl-2 phosphorylation but not JNK activation. Time course study demonstrated that activation of cdc-2 kinase activity by GF correlated with Bcl-2 phosphorylation. Taken together, our results suggest that activation of NF-kappaB pathway with cdc-2 activation and phosphorylation of Bcl-2 might be involved in G2/M cell cycle arrest in HL-60 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.     

Observations on prostaglandins in normal and leukemic human lymphocytes

Prostaglandins E (PGE) and F₂ (PGF₂) were measured in lymphocytes of normal subjects, children with acute lymphocytic leukemia (ALL), and adults with chronic lymphocytic leukemia (CLL). In ALL lymphocytes PGE increased from a normal value of 25 pgrams to 270 pgrams/10⁶ cells, and PGF₂ increased from a normal value of 31 pgrams to 482 pgrams/10⁶ cells. In CLL lymphocytes, levels of PGE and PGF₂ were normal or low. When normal lymphocytes were stimulated with phytohemagglutinin (PHA), the level of PGE and PGF₂ fluctuated, followed by corresponding changes in the level of cyclic nucleotides. In cultured ALL lymphocytes, the level of PGE remained high, while cyclic 3′:5′-adenosine monophosphate (c-AMP) level was constantly low, and the initial high level of PGF₂ fluctuated in relation to similar oscillations of cyclic 3′:5′-guanosine monophosphate (c-GMP). These values were lower, although not significantly, when ALL lymphocytes were stimulated with PHA. When CLL lymphocytes were stimulated with PHA, the level of PGE remained low (20 pgrams), as did that of c-AMP. The level of PGF₂, after a brief initial increase (130 pgrams), returned to and remained at a lower level (60 pgrams) while the level of c-GMP was persistently high. These results suggest: (1) prostaglandins may indirectly influence the cell cycle, possibly through modulation of cyclase activity and levels of cyclic nucleotides; and (2) some derangement of this regulatory mechanism may be present in leukemic lymphocytes.     

Early response to tumor necrosis factor of human promyelocytic leukemia cell lines sensitive and resistant to the factor

Early cellular events with respect to protein synthesis and the steady-state level of cellular myc (c-myc) mRNA were analyzed in the tumor necrosis factor (TNF)-sensitive human promyelocytic leukemia cell line HL-60 and in its TNF-resistant variant HL-60R after their exposure to TNF. Addition of TNF at 100 units (U)/ml induced de novo synthesis of two proteins with apparent molecular masses of 100 kDa and 40 kDa in HL-60 cells. The induced synthesis of the 100 kDa protein continued for 6 h, while that of the 40 kDa protein was transient. The 100 kDa protein was detectable in HL-60R cells which were maintained in medium containing 1,000 U/ml TNF, whereas the synthesis of the 40 kDa protein could be transiently induced by TNF at 10(5) U/ml. Dot blot hybridization revealed that the steady-state level of c-myc mRNA in HL-60 cells was transiently reduced by TNF at 100 U/ml but remained at a reduced level for 6 h when 10(5) U/ml TNF was present. In HL-60R cells, TNF at 10(5) U/ml could transiently reduce the c-myc mRNA level. These results showed that induction of the synthesis of a 40 kDa protein and a reduction in the steady-state level of c-myc mRNA were concomitant with cellular sensitivity to the cytostatic action of TNF in HL-60 cells.     

Evidence of multifactorial mechanisms in an adriamycin-resistant HL-60 promyelocytic leukemia cell line

HL-60/AR leukemia cells, which were 60-fold resistant to the growth inhibitory activity of adriamycin, remained sensitive to the antiproliferative and differentiation-inducing activities of aclacinomycin A. The replication of HL-60/AR and of adriamycin sensitive parental HL-60 cells was inhibited by greater than 80% by 30 nM aclacinomycin A and the majority of cells (about 60 to 70%) of each line underwent granulocytic differentiation when treated with this agent, as assessed by the reduction of nitroblue tetrazolium. Measurement of the initial rates of uptake of daunorubicin and steady-state levels of adriamycin in sensitive and resistant lines indicated that transport differences do not fully account for the insensitivity of HL-60/AR cells to these anthracyclines. Furthermore, 30-fold greater levels of cell-associated adriamycin were required in HL-60/AR cells for toxic effects equivalent to those occurring in parental HL-60 cells. Analysis of DNA histograms of adriamycin treated HL-60 cells indicated that cell-cycle progression was blocked in G2-M, while this antibiotic blocked progression of resistant HL-60/AR cells in the S phase. These results suggest that, in addition to alterations in membrane permeability, differential sensitivity of multiple biochemical targets may be important in the toxicity and the development of resistance to anthracyclines. Furthermore, the finding that HL-60/AR cells do not exhibit cross-resistance to aclacinomycin A indicates that this oligosaccharide-containing anthracycline may have utility in the treatment of adriamycin resistant neoplasms.     

Expression pattern of a hematopoietic proteoglycan core protein gene during human hematopoiesis

Expression of the hematopoietic proteoglycan core protein (HpPG) gene was examined in normal peripheral blood, normal bone marrow, and leukemic peripheral blood leukocytes samples to assess the expression pattern of the HpPG gene in these cells and to ascertain points of regulation of this gene during hematopoiesis. In situ hybridization to normal bone marrow and peripheral blood leukocytes demonstrated that the gene was expressed in the promyelocytes at a approximately two fold greater level than in the segmented neutrophils and the expression decreased as the granulocytes matured. The ratio of expression in the other leukocytes to expression in the segmented neutrophils were as follows: eosinophils/basophils approximately 7; monocytes approximately 2; lymphocytes less than 1. Expression of the HpPG gene during myeloblast differentiation was assessed by Northern blot analysis of acute myelogenous leukemia (AML) RNA samples. The expression of this gene, when compared to the levels in HL-60 cells, was approximately ten fold lower in the poorly differentiated blast cells obtained from three AML patients classified M"0". Conversely, the expression in the more differentiated blast cells obtained from 10 of 11 AML patients classified as M1 and M2 were at levels similar to the levels in HL-60 cells. The expression level found in eight lymphoid leukemias was approximately ten fold or more lower than in HL-60 cells. Gene copy number determination confirmed that the HpPG gene is present in one copy per haploid genome. Thus the HpPG gene's expression pattern denotes a single copy gene being differentially expressed during hematopoiesis with initial regulation occurring very early in this developmental process and an additional up-regulatory event occurring during granule genesis.     

Formation of the N'-methylnicotinamide adenine dinucleotide derivative of NAD in intact rat pituitary tumor GH3 and human promyelocytic leukemia HL-60 cells

The NAD analog N'-methylnicotinamide adenine dinucleotide (N'AD) is formed in intact human promyelocytic leukemia HL-60 and in rat pituitary tumor GH3 cells during treatment of the cultured cells with the nicotinamide derivative N'-methylnicotinamide (N'CH3NAm). N'AD formation is associated with the induced maturation of HL-60 cells and increased hormone production by GH3 cells during treatment with the nicotinamide derivative. N'AD is detected by HPLC analysis of cytoplasmic extracts as a peak which elutes near NAD. Four facts indicate that this compound is N'AD. First, a compound which elutes with identical time retention is produced by transglycosylation during reaction of NAD with pig brain NAD glycohydrolase in the presence of excess N'CH3NAm. Second, the putative N'AD is degraded by prolonged digestion with the NAD glycohydrolase to ADP-ribose. Third, N'AD formation is prevented by addition of nicotinamide along with N'CH3NAm to compete with binding of N'CH3NAm to the NAD glycohydrolase. Fourth, radioactive precursor labeling demonstrates that it contains adenosine, but it is not labeled by radioactive nicotinamide. The biological relevance of N'AD formation was evaluated. The appearance of N'AD precedes development of HL-60 maturation, and NAD levels increase, not decrease, as observed in other cell types, during treatment with N'CH3NAm. Therefore, we propose that N'AD, not the pyridine base itself, is the active species in inducing maturation. The results provide support of a role for NAD metabolism, probably ADP-ribosylation, in the regulation of HL-60 maturation and in hormone production by pituitary cells.     

Studies on changes in nucleolar organizer region of human promyelocytic leukemia cells (HL-60) treated with retinoic acid

Changes of nucleolar organizer region in HL-60 cells after treated with retinoic acid (RA) were studied with techniques of silver-staining nucleolar organizer region (Ag-NOR) in metaphase karyotypes, Brachet's reaction and with our improved TEM techniques for studying silver-stained active nucleolar organizer region (Ag-aNOR) in interphase nucleoli. Number of Ag-NOR in HL-60 cells is 4.5/cell on average. The Ag-NOR number of cells treated with RA showed no remarkable difference from that of control group. Ag-aNOR number treated with RA was reduced obviously as compared with that of control group. Meanwhile, the changes of nucleolus number showed by Brachet's reaction were in accordance with those of Ag-aNOR. Therefore, it may be concluded: (1). Though the number of active rRNA genes did not changed after the differentiation of HL-60 cells induced by RA, their expression was clearly inhibited: (2). The relationship between the changes of Brachet-No and Ag-aNOR is in positive correlation (r = 0.98, p less than 0.01). EM examination of Ag-aNOR of HL-60 cells reveals that Ag-protein (RNA polymerase I) only presented in fibrillar centers (FC) and the dense fibrillar components (DFC) of nucleolus. In addition, in control group, large amount of Ag-protein, FC, DFC and granular components (GC) were observed, and there were many large nucleoli in a nucleus, meanwhile, the cells of the treated group tended to be mature, with a decrease in the amount of Ag-protein, FC, DFC and GC accordingly, and the nucleoli reduced both in size and number significantly.(ABSTRACT TRUNCATED AT 250 WORDS)