Acid-induced Differentiation Of HL-60 Cells Research Articles

Ring finger protein 138 inhibits transcription factor C/EBPα protein turnover leading to differentiation arrest in acute myeloid leukemia.

E3 ubiquitin ligase, ring finger protein 138 (RNF138) is involved in several biological processes; however, its role in myeloid differentiation or tumorigenesis remains unclear. RNAseq data from TNMplot showed that RNF138 mRNA levels are highly elevated in acute myeloid leukemia (AML) bone marrow samples as compared with bone marrow of normal volunteers. Here, we show that RNF138 serves as an E3 ligase for the tumor suppressor CCAAT/enhancer binding protein (C/EBPα) and promotes its degradation leading to myeloid differentiation arrest in AML. Wild-type RNF138 physically interacts with C/EBPα and promotes its ubiquitin-dependent proteasome degradation while a mutant RNF-138 deficient in ligase activity though interacts with C/EBPα, fails to down-regulate it. We show that RNF138 depletion enhances endogenous C/EBPα levels in peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers. Our data further shows that RNF138-mediated degradation of C/EBPα negatively affects its transactivation potential on its target genes. Furthermore, RNF138 overexpression inhibits all-trans-retinoic acid-induced differentiation of HL-60 cells whereas RNF138 RNAi enhances. In line with RNF138 inhibiting C/EBPα protein turnover, we also observed that RNF138 overexpression inhibited β-estradiol (E2)-induced C/EBPα driven granulocytic differentiation in C/EBPα inducible K562-p42C/EBPα-estrogen receptor cells. Furthermore, we also recapitulated these findings in PBMCs isolated from AML patients where depletion of RNF138 increased the expression of myeloid differentiation marker CD11b. These results suggest that RNF138 inhibits myeloid differentiation by targeting C/EBPα for proteasomal degradation and may provide a plausible mechanism for loss of C/EBPα expression often observed in myeloid leukemia. Also, targeting RNF138 may resolve differentiation arrest by restoring C/EBPα expression in AML.

Retinoic Acid-Induced HL-60 Cell Differentiation Is Augmented by Copper Supplementation1–4

The objective of this study was to determine how copper influences the ability of HL-60 cells to differentiate into cells of the granulocytic lineage. We hypothesized that granulopoiesis requires copper because copper-deficient humans become neutropenic. Differentiation of HL-60 cells along the granulocytic lineage with retinoic acid was enhanced by copper. The results showed a greater number of cells were more differentiated when copper was added to the medium for 96 h. The respiratory burst activity of retinoic acid-induced cells was increased by copper supplementation, but intracellular superoxide anion generation was not affected. Supplementation with copper resulted in more cell-associated copper in both noninduced and induced cells; however, the induced cells accumulated three times more copper than the noninduced cells. Even though the amount of copper associated with retinoic acid-treated cells was greater than in untreated cells, the activity of a copper-requiring enzyme, copper/zinc superoxide dismutase, was significantly lower. Copper supplementation increased the activity of this enzyme in both retinoic acid-treated and untreated cells. Cytochrome c oxidase activity was not affected by retinoic acid treatment or by copper supplementation. Copper seems to play a specific role during the early stages of granulocyte differentiation.

Probing the requirement for CD38 in retinoic acid-induced HL-60 cell differentiation with a small molecule dimerizer and genetic knockout

CD38 is an ectoenzyme and receptor with key physiological roles. It metabolizes NAD+ to adenosine diphosphate ribose (ADPR) and cyclic ADPR, regulating several processes including calcium signalling. CD38 is both a positive and negative prognostic indicator in leukaemia. In all-trans retinoic acid (RA)-induced differentiation of acute promyelocytic leukaemia and HL-60 cells, CD38 is one of the earliest and most prominently upregulated proteins known. CD38 overexpression enhances differentiation, while morpholino- and siRNA-induced knockdown diminishes it. CD38, via Src family kinases and adapters, interacts with a MAPK signalling axis that propels differentiation. Motivated by evidence suggesting the importance of CD38, we sought to determine whether it functions via dimerization. We created a linker based on the suicide substrate arabinosyl-2′-fluoro-2′-deoxy NAD+ (F-araNAD+), dimeric F-araNAD+, to induce homodimerization. CD38 homodimerization did not affect RA-induced differentiation. Probing the importance of CD38 further, we created HL-60 cell lines with CRISPR/Cas9-mediated CD38 truncations. Deletion of its enzymatic domain did not affect differentiation. Apart from increased RA-induced CD11b expression, ablation of all but the first six amino acids of CD38 affected neither RA-induced differentiation nor associated signalling. Although we cannot discount the importance of this peptide, our study indicates that CD38 is not necessary for RA-induced differentiation.

Application of selected reaction monitoring and parallel reaction monitoring for investigation of HL-60 cell line differentiation.

Targeted mass spectrometry represents a powerful tool for investigation of biological processes. The convenient approach of selected reaction monitoring using stable isotope-labeled peptide standard (SIS) is widely applied for protein quantification. Along with this method, high-resolution parallel reaction monitoring has been increasingly used for protein targeted analysis. Here we applied two targeted approaches (selected reaction monitoring with SIS and label-free parallel reaction monitoring) to investigate expression of 11 proteins during all-trans retinoic acid-induced differentiation of HL-60 cells. In our experiments, we have determined the proteins expression ratio at 3, 24, 48, and 96 h after all-trans retinoic acid treatment in comparison with 0 h, respectively. Expression profiles of four proteins (VAV1, PRAM1, LYN, and CEBPB) were highly correlated ( r > 0.75) and FGR expression was detected on proteome level starting from 24 h by both techniques. For prominent differences (fold change ≥ 2) label-free parallel reaction monitoring is not inferior to selected reaction monitoring with isotopically labeled peptide standards. Differentially expressed proteins, that have been determined in our study, can be considered as potential drug targets for acute myeloid leukemia (AML) treatment.

Arsenic trioxide augments all-trans retinoic acid-induced differentiation of HL-60 cells

AimsAll-trans retinoic acid (ATRA) is used to treat patients with acute promyelocytic leukemia (APL) due to its ability to resume the differentiation of APL cells. Recently, clinical trials have been started to evaluate ATRA plus arsenic trioxide (ATO) as a combination treatment for APL patients. However, little is known about the detailed mechanisms underlying its efficacy. We therefore investigated the effects of this combination on the differentiation and differentiation-related gene expression. Main methodsHuman leukemia HL-60 cells differentiation was examined using nitro blue tetrazolium and CD11b. The levels of mRNA and protein were determined by RT-qPCR, microarray, western blot and ELISA, respectively. The promoter activity was assessed by luciferase activity. The arsenic concentration was determined by ICP-MS. Key findingsATRA-induced HL-60 differentiation was augmented by co-treatment with ATO. A microarray analysis showed that ATRA plus ATO treatment markedly down-regulated the expression of proteinase 3 (PRTN3), which is involved in the differentiation arrest of leukemia cells, compared with treatment with ATRA alone. The PRTN3 mRNA level was suppressed by treatment with ATRA alone, and then further suppressed by co-treatment with ATO, accompanied by a concomitant increase in Sp1 protein, which is known to facilitate differentiation. The expression levels of azurocidin, telomerase reverse transcriptase, ferritin, and interleukin-1β were also altered by co-treatment with ATO. SignificanceCo-treatment with ATO enhances ATRA-induced HL-60 differentiation by altering the expression of genes involved in cell differentiation, providing the molecular basis for a combination therapy using ATO plus ATRA to treat leukemia patients.

Analysis of gene profiles involved in the enhancement of all-trans retinoic acid-induced HL-60 cell differentiation by sesquiterpene lactones identifies asparagine synthetase as a novel target for differentiation-inducing therapy

All-trans retinoic acid (ATRA) is one of the most useful drugs in the treatment for acute promyelocytic leukemia (APL), but its adverse effects, which include drug resistance and hypercalcemia are obstacles to achieving complete remission. Our previous study showed that some sesquiterpene lactones (STLs), i.e., helenalin (HE) and parthenolide (PA) but not sclareolide (SC), enhance ATRA-induced differentiation of HL-60 APL cells with no unexpected effects, but the precise mechanism on underlying this synergism is not yet fully understood. In this study, we investigated the distinctive transcriptional profile of cells treated with effective STL compounds, which were identified by comparing the profile with that of cells treated with SC. Genome-wide approaches using cDNA microarrays showed that co-treatment with the differentiation-enhancing STLs HE and PA maximized the transcriptional variation regulated by the suboptimal concentration of ATRA in HL-60 cells. Of the genes of interest, asparagine synthetase was remarkably downregulated by ATRA co-treated with either HE or PA, but not with SC. In an additional analysis for the role of asparagine synthetase, ATRA-mediated HL-60 cell differentiation was enhanced when asparagine in the culture media was depleted by an addition of L-asparaginase, indicating that downregulation of asparagine synthetase gene expression may be involved in the enhanced cell differentiation by STL compounds. These results provide useful insight into differentiation-inducing therapy in the treatment of leukemia.

Alteration of the expression ratio between WT1 gene and its isomers during all-trans retinoic acid-induced differentiation of HL-60 cells

To explore expression ratio alteration between WT1 gene and its isomers during differentiation of leukemia cell line HL-60 induced by all-trans retinoic acid (ATRA) and the relationship existed between them. The degree of cellular maturation was verified by NBT reduction test and immunophenotyping. Expression of unspliced WT1, WT1 (17AA+) and WT1 (KTS+) were determined by real-time fluorescence quantitative RT-PCR during the induced differentiation of HL-60 cells. The relative ratio of four splicing variants WT1 (+/+), WT1 (+/-), WT1 (-/+), WT1 (-/-) were analyzed. During the induced differentiation of HL-60 cells, NBT reduction rate and CD11b positive rate increased significantly (P < 0.05 and P < 0.001, respectively). The expression of WT1 gene decreased from (4.17 +/- 2.21) x 10(-3) at 0 hour to (7.53 +/- 2.30) x 10(-4) at the 96th hour. The ratio of 17AA+decreased from 0.60 +/- 0.05 at 0 hour to 0.42 +/- 0.08 at the 96th hour. The ratio of KTS+ decreased from 0.53 +/- 0.08 at 0 hour to 0.41 +/- 0.04 at the 96th hour. The ratio of WT1 (+/+) decreased gradually from 0.32 +/- 0.06 at 0 hour to 0.17 +/- 0.03 at the 96th hour. Change of ratios of other two isomers not significant. During the induced differentiation of HL-60 cells, WT1 gene expression decreases gradually. The phenotype of the majority of uninduced HL-60 cells is WT1 (+/+), in contrast to WT1 (-/-) phenotype after the induction of cell differentiation, indicating that WT1 gene may participate in the regulation of hematopoietic cell differentiation through modulation of the expression ratios of its four spliced variants.

Ribose enhances retinoic acid–induced differentiation of HL-60 cells

Ribose, a critical building block for nucleotides, plays an important role in energy metabolism, transcription, translation, and second messenger systems. This 5-carbon sugar, synthesized from glucose via the pentose phosphate pathway, has a rate-limiting step at glucose-6-phosphate dehydrogenase. Therefore, we hypothesized that when cells are required to proliferate or differentiate, as in an immune response, the requirement for D-ribose may be greater than what could be supplied by the synthetic pathway. We hypothesized that providing an exogenous source of D-ribose during cell differentiation will enhance the process of differentiation. We used a retinoic acid-induced HL-60 cell differentiation culture as a model of neutrophil maturation. The addition of 10 to 25 mmol/L D-ribose was shown to reduce cell proliferation and move the cell population toward apoptosis in a dose-dependent manner. The expression of a cell surface marker representing maturity (CD11b) significantly increased and a cell surface marker indicative of immaturity (CD117) significantly decreased. Functionally, the cells had a greater oxidative burst function dependent on time and dose. The mechanism by which ribose enhances HL-60 cell differentiation is not known; however, as adenosine triphosphate levels did not change, adenosine triphosphate is not thought to be involved. We conclude that in this cell culture model, ribose supplementation enhanced cellular differentiation and function. Thus, ribose might be conditionally essential during time of higher need as in an immune response.

Effect of all trans retinoic acid on lysosomal α-d-mannosidase activity in Hl-60 cell: correlation with Hl-60 cells differentiation

Human promyelocytic leukemia HL-60 cells represent an in vitro model of acute promyelocytic leukemia (APL), and are inducible to terminally differentiate into morphologically mature granulocytes by incubation with all trans retinoic acid (ATRA). Lysosomal glycohydrolases are involved in the changes of the membrane surface proteins' glycosylation, linked to the metastatic progression potential of neoplastic cells. In particular, it has been demonstrated that the Asn-linked glucidic residues were directly responsible for the metastatic potential, and it is known that the glycohydrolase alpha-D-mannosidase specifically hydrolyze the Asn-linked oligosaccharides. In this report, we present an in vitro study on the ATRA effects on lysosomal glycohydrolases expression and the eventual relationship with the retinoic acid-induced differentiation of HL-60 cells. We have investigated two highly expressed lysosomal glycohydrolases, namely beta-D-hexosaminidase and alpha-D-mannosidase, and showed that they were differently affected by ATRA differentiating action. In particular, due to the specific action on Asn-linked oligosaccharides, we tested alpha-D: -mannosidase enzymatic activity and observed that it was dramatically decreased after ATRA incubation, indicating a relationship with the differentiation state of the cells. These observations may directly be linked with the loss of metastatic progession of differentiated HL-60.

Enhancement of All-trans Retinoic Acid-Induced HL-60 Cell Differentiation by Thalidomide and Its Metabolites

Thalidomide (Thal: 1) and its two metabolites, 5-hydroxythalidomide (5-HT: 2) and N-hydroxythalidomide (N-HT: 3), showed an enhancing effect on all-trans retinoic acid (ATRA)-induced HL-60 cell differentiation. 5-HT and N-HT showed tubulin polymerization-inhibiting activity, but thalidomide did not.

Alternative Splicing of Human Telomerase Reverse Transcriptase May Not Be Involved in Telomerase Regulation During all-trans-Retinoic Acid-Induced HL-60 Cell Differentiation

Alternative splicing of the human telomerase reverse transcriptase subunit (hTERT) suppresses telomerase activity during the development of human fetal kidney cells into mature cells. Tumor cell differentiation is the process of turning abnormal tumor cells into ‘normal’ cells accompanied by down-regulation of telomerase activity. However, the precise mechanism of the regulation of telomerase activity in differentiated cells is not fully understood. In this study, we observed the role of alternative splicing of hTERT in the regulation of telomerase activity in all-trans-retinoic acid (ATRA)-induced, differentiated HL-60 cells. ATRA-induced down-regulation of telomerase activity in differentiated HL-60 cells was associated with a decrease in hTERT and an increase in human telomerase-associated protein-1 (hTP1) transcription. Expression of full length variant hTERT α+β+ mRNA decreased in a dose- and time-dependent manner. The drop of hTERT β- mRNA was time-dependent. hTERT α- and hTERT α-b- mRNA were reduced dramatically after ATRA treatment. In the dose-effect study, hTERT α+β+ and hTERT β- maintained a relatively stable ratio when telomerase activity decreased largely from treatment with 1 to 5 μM ATRA. Although the splicing pattern of hTERT mRNA was altered in time-effect research, the change was not related to the ATRA-treated decline of telomerase activity. The expression of alternative splicing variants of hTERT also decreased at the protein level. All these results suggested that alternative splicing of hTERT mRNA may not contribute to the suppression of telomerase activity during ATRA-induced HL-60 leukemia cell differentiation.

Capsaicin potentiates 1,25-dihydoxyvitamin D 3- and all- trans retinoic acid-induced differentiation of human promyelocytic leukemia HL-60 cells

Human promyelocytic leukemia HL-60 cells are differentiated into monocytic or granulocytic lineage when treated with 1,25-dihydroxyvitamin D 3 [1,25-(OH) 2D 3] or all- trans retinoic acid, respectively. In this study, the effect of capsaicin, an active component of the red pepper of the genus Capsocum, on cell differentiation was investigated in a HL-60 cell culture system. Treatment of HL-60 cells with 5–30 μg/ml capsaicin for 72 h inhibited cell proliferation and induced a small increase in cell differentiation. Interestingly, synergistic induction of HL-60 cell differentiation was observed when capsaicin was combined with either 5 nM 1,25-(OH) 2D 3 or 50 nM all- trans retinoic acid. Flow cytometric analysis indicated that combinations of 1,25-(OH) 2D 3 and capsaicin stimulated differentiation predominantly to monocytes whereas combinations of all- trans retinoic acid and capsaicin stimulated differentiation predominantly to granulocytes. Capsaicin enhanced protein kinase C activity in 1,25-(OH) 2D 3- and all- trans retinoic acid-treated HL-60 cells. In addition, inhibitors for protein kinase C [bisindolylmaleimide (GF-109203X), chelerythrine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7)] and an inhibitor for extracellular signal-regulated kinase [2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one (PD-098059)] significantly inhibited HL-60 cell differentiation induced by capsaicin in combination with either 1,25-(OH) 2D 3 or all- trans retinoic acid. These results indicate that capsaicin potentiates 1,25-(OH) 2D 3- or all- trans retinoic acid-induced HL-60 cell differentiation and that both protein kinase C and extracellular signal-regulated kinase are involved in the cell differentiation synergistically enhanced by capsaicin.

Retinoic acid increases amount of phosphorylated RAF; ectopic expression of cFMS reveals that retinoic acid-induced differentiation is more strongly dependent on ERK2 signaling than induced GO arrest is.

Retinoic acid is known to cause the myeloid differentiation and G1/0 cell cycle arrest of HL-60 cells in a process that requires mitogen-activated protein/extracellular signal regulated kinase (MEK)-dependent extracellular signal regulated kinase (ERK)2 activation. It has also been shown that ectopic expression of cFMS, a platelet-derived growth factor (PDGF)-family transmembrane tyrosine kinase receptor, enhances retinoic acid-induced differentiation and G1/0 arrest. The mechanism of how the retinoic acid and cFMS signaling pathways intersect is not known. The present data show that the ectopic expression of cFMS results in the differential loss of sensitivity of retinoic acid-induced differentiation or G1/0 arrest to inhibition of ERK2 activation. PD98059 was used to inhibit MEK and consequently ERK2. In wildtype HL-60 cells, PD98059 blocked retinoic acid-induced differentiation; but in cFMS stable transfectants, PD98059 only attenuated the induced differentiation, with the resulting response resembling that of retinoic acid-treated wild-type HL-60. In wild-type HL-60, PD98059 greatly attenuated the retinoic acid-induced G1/0 arrest allied with retinoblastoma (RB) hypophosphorylation; but in cFMS stable transfectants, PD98059 had no inhibitory effect on RB hypophosphorylation and G1/0 arrest. This differential sensitivity to PD98059 and uncoupling of retinoic acid-induced differentiation and G1/0 arrest in cFMS transfectants is associated with changes in mitogen-activated protein kinase signaling molecules. The cFMS transfectants had more activated ERK2 than did the wild-type cells, which surprisingly was not attributable to enhanced mitogen-activated protein-kinase-kinase-kinase (RAF) phosphorylation. Retinoic acid increased the amount of activated ERK2 and phosphorylated RAF in both cell lines. But PD98059 eliminated detectable ERK2 activation, as well as inhibited RAF phosphorylation, in untreated and retinoic acid-treated wild-type HL-60 and cFMS transfectants, consistent with MEK or ERK feedback-regulation of RAF, in all four cases. Since PD98059 blocks the cFMS-conferred enhancement of the retinoic acid-induced differentiation, but not growth arrest, the data indicate that cFMS-enhanced differentiation acts primarily through MEK and ERK2, but cFMS-enhanced G1/0 arrest allied with RB hypophosphorylation depends on another cFMS signal route, which by itself can effect G1/0 arrest without activated ERK2. Ectopic expression of cFMS and differential sensitivity to ERK2 inhibition thus reveal that retinoic acid-induced HL-60 cell differentiation and G1/0 arrest are differentially dependent on ERK2 and can be uncoupled. A significant unanticipated finding was that retinoic acid caused a MEK-dependent increase in the amount of phosphorylated RAF. This increase may help sustain prolonged ERK2 activation.

Changes in Magnesium Content and Subcellular Distribution during Retinoic Acid-Induced Differentiation of HL60 Cells

Magnesium (Mg) is required for cellular proliferation; however, the differences in subcellular regulation of Mg between proliferating and differentiated cells has not been determined. We used electron probe microanalysis (EPMA) to investigate the subcellular distribution of Mg in HL60 cells (a promyelocytic leukemia cell line) before and after retinoic acid (RA)-induced differentiation. Most intracellular Mg is bound to ATP and the Mg–ATP complex regulates several metabolic enzymes. We also compared alterations in Mg content following differentiation with the changes in ATP and ADP levels. Using atomic absorption spectrophotometry, we observed a significant decrease (−20%) in cellular Mg content in RA-differentiated HL60 cells. To investigate which intracellular compartments were involved in these changes, we analyzed subcellular elemental composition in freeze-dried cryosections of rapidly frozen undifferentiated and differentiated HL60 cells by EPMA. Following differentiation of HL60 cells, we observed an 18% decrease in Mg content in both the cytoplasm (regions of the cell excluding mitochondria and nuclei) and mitochondria. There was also a significant (40%) decrease in cytoplasmic Ca content after RA-induced differentiation. Nuclear Mg concentration was not significantly different between differentiated and undifferentiated HL60 cells, although differentiation was accompanied by a 30% decrease in the nuclear K/Na ratio. After differentiation, cellular ATP and ADP content decreased by 31 and 40%, respectively. We conclude that during exit from the cell cycle, Mg redistributes within cells and that the decrease in cytoplasmic and mitochondrial Mg is accompanied by a decrease in ATP and ADP content.

Altered Expression and Activity of Topoisomerases During All-Trans Retinoic Acid-Induced Differentiation of HL-60 Cells

Regulation of topoisomerase II (TOPO II) isozymes α and β is influenced by the growth and transformation state of cells. Using HL-60 cells induced to differentiate by all-trans retinoic acid (RA), we have investigated the expression and regulation of TOPO II isozymes as well as the levels of topoisomerase I (TOPO I). During RA-induced differentiation of human leukemia HL-60 cells, levels of TOPO I remained unchanged, whereas the levels and phosphorylation of TOPO IIα and TOPO IIβ proteins were increased twofold to fourfold and fourfold to eightfold, respectively. The elevation of TOPO II (α and β) protein levels and phosphorylation was apparent at 48 hours of treatment with RA and persisted through 96 hours. The increased level of TOPO IIβ protein was also detected in differentiated cells subsequently cultured for 96 hours in RA-free medium. Pulse chase experiments in cells labeled with 35S-methionine showed that the rate of degradation of TOPO IIβ protein in control cells was about twofold faster than that in the differentiated RA-treated cells. The level of decatenation activity of kDNA was comparable in nuclear extracts from control or RA-treated cells. Whereas etoposide (1 to 10 μmol/L) -induced DNA cleavage was not significantly different, apoptosis was significantly lower (P = .012) in RA-treated versus control cells after exposure to 10 μmol/L etoposide. Consistent with unaltered levels of TOPO I, camptothecin (CPT) -induced DNA cleavage was similar in control or RA-treated cells. However, apoptosis after exposure to 1 to 10 μmol/L CPT was significantly lower (P = .003 to P < .001) in RA-treated versus control cells. Results suggest that TOPO IIβ protein levels are posttranscriptionally regulated and that degradation of TOPO IIβ is decreased during RA-induced differentiation. Furthermore, whereas the total level of TOPO II (α + β) is increased with RA, the level of TOPO II catalytic activity and etoposide-stabilized DNA cleavage activity remains unaltered. Thus, TOPO IIβ may have a specific role in transcription of genes involved in differentiation with RA treatment.© 1998 by The American Society of Hematology.

Altered Expression and Activity of Topoisomerases During All-Trans Retinoic Acid-Induced Differentiation of HL-60 Cells

Regulation of topoisomerase II (TOPO II) isozymes  and β is influenced by the growth and transformation state of cells. Using HL-60 cells induced to differentiate by all-trans retinoic acid (RA), we have investigated the expression and regulation of TOPO II isozymes as well as the levels of topoisomerase I (TOPO I). During RA-induced differentiation of human leukemia HL-60 cells, levels of TOPO I remained unchanged, whereas the levels and phosphorylation of TOPO II and TOPO IIβ proteins were increased twofold to fourfold and fourfold to eightfold, respectively. The elevation of TOPO II ( and β) protein levels and phosphorylation was apparent at 48 hours of treatment with RA and persisted through 96 hours. The increased level of TOPO IIβ protein was also detected in differentiated cells subsequently cultured for 96 hours in RA-free medium. Pulse chase experiments in cells labeled with 35S-methionine showed that the rate of degradation of TOPO IIβ protein in control cells was about twofold faster than that in the differentiated RA-treated cells. The level of decatenation activity of kDNA was comparable in nuclear extracts from control or RA-treated cells. Whereas etoposide (1 to 10 μmol/L) -induced DNA cleavage was not significantly different, apoptosis was significantly lower (P = .012) in RA-treated versus control cells after exposure to 10 μmol/L etoposide. Consistent with unaltered levels of TOPO I, camptothecin (CPT) -induced DNA cleavage was similar in control or RA-treated cells. However, apoptosis after exposure to 1 to 10 μmol/L CPT was significantly lower (P = .003 to P &lt; .001) in RA-treated versus control cells. Results suggest that TOPO IIβ protein levels are posttranscriptionally regulated and that degradation of TOPO IIβ is decreased during RA-induced differentiation. Furthermore, whereas the total level of TOPO II ( + β) is increased with RA, the level of TOPO II catalytic activity and etoposide-stabilized DNA cleavage activity remains unaltered. Thus, TOPO IIβ may have a specific role in transcription of genes involved in differentiation with RA treatment.© 1998 by The American Society of Hematology.

Potentiation of retinoic acid-induced differentiation of HL-60 cells by prostaglandin EP2 receptor

Human promyeloid HL-60 cells are differentiated by all-trans retinoic acid (RA) to granulocytes, and prostaglandin (PG) E 2 potentiates the RA-induced differentiation. Here we examined which subtype of PGE receptors was involved in this potentiating activity of PGE 2. Northern blot analysis demonstrated that HL-60 cells expressed three subtypes of PGE receptor, EP2, EP3, and EP4. Among various EP agonists, and EP2-selective agonist, butaprost, preferentially potentiated the RA-induced differentiation of HL-60 cells. Butaprost not only decreased the half-maximal concentration of RA but also increased the maximal level of the differentiation. Butaprost concentration-dependently stimulated the cAMP formation, and 8-Br-cAMP strongly potentiated the RA-induced differentiation. These results demonstrate that the EP2 receptor enhances the RA-induced differentiation of HL-60 cells via stimulation of adenylate cyclase.

The Interconversion of Protein Phosphatase 2A between PP2A 1and PP2A 0during Retinoic Acid-Induced Granulocytic Differentiation and a Modification on the Catalytic Subunit in S Phase of HL-60 Cells

Alterations in protein phosphatase 2A (PP2A) during retinoic acid-induced differentiation of HL-60 cells have been investigated. PP2A activity of HL-60 cells for phosphorylated myelin basic protein showed a sharp and transient increase after 18-h treatment with 1 μ mretinoic acid, which corresponded to G1/S boundary of the cell cycle. This PP2A of the 18-h treated cells was eluted from a DEAE-Sepharose column with 0.13 mNaCl, while PP2A from control cells was eluted with 0.23 mNaCl. The phosphorylase phosphatase activity of PP2A in the 0.13 meluate was greatly enhanced in the presence of protamine compared with that of the later eluting PP2A. Immunoblot analyses with antisera against B′ and Bα subunits showed that the PP2A in the 0.13 mNaCl eluate from 18-h retinoic acid-treated cells was PP2A 0(AC-B′), whereas the PP2A eluted with 0.23 mNaCl from 24-h retinoic acid-treated cells and 0-, 18-, and 24-h control cells was PP2A 1(AC-Bα). These results strongly suggest that PP2A undergoes a transient and reversible interconversion of holoenzyme forms during the initial stage of retinoic acid-induced granulocytic differentiation. PP2A activity assayed after dissociation of the catalytic subunit, for phosphorylase as substrate, showed a sharp and transient decrease in S phase of HL-60 cells irrespective of the presence or absence of retinoic acid. Immunoblot analyses with antisera against C-terminus and N-terminus of the catalytic subunit of PP2A suggested that a modification at the C-terminus is responsible for the decrease in PP2A activity. Immunoreactivity to the C-terminal antibody was restored after treatments of the S-phase extract with alkali or ethanol, the conditions which remove the methyl group from the C-terminus. These results suggest that the C-terminus of PP2A catalytic subunit is transiently methylated in S phase of HL-60 cells.

Transient accumulation, phosphorylation and changes in the oligomerization of Hsp27 during retinoic acid-induced differentiation of HL-60 cells: possible role in the control of cellular growth and differentiation.

Expression of the mammalian small stress protein Hsp27 has been increasingly linked to cell growth regulation and differentiation. Hsp27 is a phosphoprotein which forms oligomers with native sizes ranging between 100 and 800 kDa. We have examined the fate of Hsp27 transiently expressed during the retinoic acid (tRA)-induced granulocytic differentiation of human leukemic HL-60 cells. We show that tRA, in addition to its effects on Hsp27 accumulation and phosphorylation, transiently increased the oligomerization state of this protein. While Hsp27 phosphorylation by tRA is an early phenomenon that takes place before cellular growth is altered, the redistribution of Hsp27 oligomers occurred later and concomitantly with the maximal accumulation of this protein. Hence, complex regulations of Hsp27 are induced by tRA which suggest that this protein plays a role in the pathway through which retinoids exert their effects. To approach Hsp27 function during HL-60 cell differentiation, experiments aimed at reducing the cellular content of this protein were performed by transiently inhibiting Hsp27 mRNA translation by a specific anti-sense oligonucleotide. This process, which decreased the basal level of Hsp27 by about 40%, did not interfere with the growth of undifferentiated HL-60 cells. In contrast, a decreased level of Hsp27 diminished the differentiation-mediated down-regulation of cell growth and altered some morphological changes induced by this retinoid. These results suggest that Hsp27 is a mediator of granulocytic differentiation.

Reduced Level of Octamer Binding Transcription Factor (Oct-1) Is Correlated with H2B Histone Gene Repression During Differentiation of HL-60 Cells by All-trans-Retinoic Acid

To gain insight on the role of transacting factors in the regulatory mechanism of H2B histone gene expression during the differentiation of HL-60 cells by all-trans retinoic acid (retinoic acid), the binding pattern of the nuclear proteins to various elements in the human H2B histone upstream region has been investigated with DNase I footprinting and DNA mobility shift assay. The level of H2B histone mRNA was markedly reduced at 48 hr in retinoic acid-treated HL-60 cells. The H2B histone mRNA was repressed in proportion to the concentration of retinoic acid. In DNase I footprinting analysis, a nuclear factor (octamer binding transcription factor, Oct-1) bound at −42 bp (ATTTGCAT) both before and after retinoic- acid-induced differentiation of HL-60 cells. One DNA-protein complex was formed by DNA mobility shift assay, and the level of Oct-1 decreased during retinoic- acid-induced differentiation. In the cycloheximide-treated HL-60 cells, the level of Oct-1 also reduced. These results suggest that the transcriptional repression of H2B histone gene during retinoic-acid-induced differentiation in HL-60 cells may be mediated by reduced level of Oct-1.