Retinoic acid induces expression of SLP-76: expression with c-FMS enhances ERK activation and retinoic acid-induced differentiation/G0 arrest of HL-60 cells

Retinoic acid (RA) is known to cause MAPK signaling which propels G0 arrest and myeloid differentiation of HL-60 human myeloblastic leukemia cells. The present studies show that RA up-regulated expression of SLP-76 (Src-homology 2 domain-containing leukocyte-specific phospho-protein of 76 kDa), which became a prominent tyrosine-phosphorylated protein in RA-treated cells. SLP-76 is a known adaptor molecule associated with T-cell receptor and MAPK signaling. To characterize functional effects of SLP-76 expression in RA-induced differentiation and G0 arrest, HL-60 cells were stably transfected with SLP-76. Expression of SLP-76 had no discernable effect on RA-induced ERK activation, subsequent functional differentiation, or the rate of RA-induced G0 arrest. To determine the effects of SLP-76 in the presence of a RA-regulated receptor, SLP-76 was stably transfected into HL-60 cells already overexpressing the colony stimulating factor-1 (CSF-1) receptor, c-FMS, from a previous stable transfection. SLP-76 now enhanced RA-induced ERK activation, compared to parental c-FMS transfectants. It also enhanced RA-induced differentiation, evidenced by enhanced paxillin expression, inducible oxidative metabolism and superoxide production. RA-induced RB tumor suppressor protein hypophosphorylation was also enhanced, as was RA-induced G0 cell cycle arrest. A triple Y to F mutant SLP-76 known to be a dominant negative in T-cell receptor signaling failed to enhance RA-induced paxillin expression, but enhanced RA-induced ERK activation, differentiation and G0 arrest essentially as well as wild-type SLP-76. Thus, SLP-76 overexpression in the presence of c-FMS, a RA-induced receptor, had the effect of enhancing RA-induced cell differentiation. This is the first indication to our knowledge that RA induces the expression of an adapter molecule to facilitate induced differentiation via co-operation between c-FMS and SLP-76.     

Low-dose ionizing radiation effects on differentiation of HL-60 cells

The biological effects of low-dose radiation have attracted attention, but data are currently insufficient to fully understand the beneficial role of the phenomenon. In the present study, we have investigated the effects of low doses of gamma-irradiation alone and in combination with all-trans-retinoic acid (RA) on proliferation, apoptosis and differentiation of the human promyelocytic leukemia HL-60 cells. Changes in cell behavior and protein expression were determined with the use of light and fluorescent microscopy, immunocytochemical and Western blot analysis. Low-dose irradiation with 1–100 cGy caused a dose-dependent inhibition of HL-60 cell proliferation, and induced apoptosis and differentiation to granulocytes with an increase in the number of CD15-positive cells. Pre-irradiation with 1–100 cGy for 24 h before treatment with RA promoted apoptosis but did not impair RA-induced differentiation. Both processes were associated with a decrease in the expression of the proliferating cell nuclear antigen (PCNA), BCL-2, c-MYC, and changes in both cytosolic and nuclear levels of protein tyrosine-phosphorylation as well as protein kinase C alpha or beta isoforms. These results demonstrate the beneficial role of low-dose irradiation in modulating leukemia cell proliferation, differentiation and apoptosis.     

Retinoic acid differentiation of HL-60 cells promotes cytoskeletal polarization

Retinoic acid (RA) treatment of HL-60 cells in vitro induces granulocytic differentiation, involving reorganization of the nucleus and cytoplasm, development of chemoattractant-directed migration, and eventual apoptosis. The present studies with HL-60/S4 cells document that major elements of the cytoskeleton are changed: actin increases by 50%; vimentin decreases by more than 95%. The cellular content of alpha-tubulin does not significantly change; but the centrosomal-microtubule (MT) array moves away from the lobulating nucleus. Cytoskeletal-modifying chemicals modulate this polarized reorganization: Taxol and cytochalasin D enhance centrosome movement; nocodazole reverses it. Cytoskeletal-modifying chemicals do not appear to affect nuclear lobulation or the integrity of envelope-limited chromatin sheets (ELCS). Employing bcl-2-overexpressing HL-60 cells permitted demonstration of nuclear lobulation, ELCS formation, and centrosome-MT movement concomitantly during RA-induced differentiation, implying independence between the cellular reorganization and apoptotic programs. RA appears to promote an inherent potential in HL-60 cells for cytoskeletal polarization, likely to be important for chemoattractant-directed cell migration, an established characteristic of mature granulocytes.     

Polyomavirus small t antigen prevents retinoic acid-induced retinoblastoma protein hypophosphorylation and redirects retinoic acid-induced G0 arrest and differentiation to apoptosis

Polyomavirus small t antigen (ST) impedes late features of retinoic acid (RA)-induced HL-60 myeloid differentiation as well as growth arrest, causing apoptosis instead. HL-60 cells were stably transfected with ST. ST slowed the cell cycle, retarding G2/M in particular. Treated with RA, the ST transfectants continued to proliferate and underwent apoptosis. ST also impeded the normally RA-induced hypophosphorylation of the retinoblastoma tumor suppressor protein consistent with failure of the cells to arrest growth. The RA-treated transfectants expressed CD11b, an early cell surface differentiation marker, but inducible oxidative metabolism, a later and more mature functional differentiation marker, was largely inhibited. Instead, the cells underwent apoptosis. ST affected significant known components of RA signaling that result in G0 growth arrest and differentiation in wild-type HL-60. ST increased the basal amount of activated ERK2, which normally increases when wild-type cells are treated with RA. ST caused increased RARalpha expression, which is normally down regulated in RA-treated wild-type cells. The effects of ST on RA-induced myeloid differentiation did not extend to monocytic differentiation and G0 arrest induced by 1,25-dihydroxy vitamin D3, whose receptor is also a member of the steroid-thyroid hormone superfamily. In this case, ST abolished the usually induced G0 arrest and retarded, but did not block, differentiation without inducing apoptosis, thus uncoupling growth arrest and differentiation. In sum, the data show that ST disrupted the normal RA-induced program of G0 arrest and differentiation, causing the cells to abort differentiation and undergo apoptosis.     

Nuclear CD38 in retinoic acid-induced HL-60 cells

The cell surface antigen, CD38, is a 45-kDa transmembrane protein which is predominantly expressed on hematopoietic cells during differentiation. As a bifunctional ectoenzyme, it catalyzes the synthesis of cyclic ADP-ribose (cADPR) from NAD(+) and hydrolysis of either NAD(+) or cADPR to ADP-ribose. All-trans-retinoic acid (RA) is a potent and specific inducer of CD38 in myeloid cells. In this report, we demonstrate that the nuclei of RA-treated human HL-60 myeloblastic cells reveal enzymatic activities inherent to CD38. Thus, GDP-ribosyl cyclase and NAD(+) glycohydrolase activities in the nuclear fraction increased very significantly in response to incubation with RA. With Western blotting, we detected in the nuclear protein fraction from RA-treated cells a approximately 43-kDa protein band which was reactive with the CD38-specific monoclonal antibody OKT10. The expression of CD38 in HL-60 nuclei was also shown with FACScan analysis. RA treatment gave rise to an increase in in vitro ADP ribosylation of the approximately 43-kDa nuclear protein. Moreover, nuclei isolated from RA-treated HL-60 cells revealed calcium release in response to cADPR, whereas a similar response was not observed in control nuclei. These results suggest that CD38 is expressed in HL-60 cell nuclei during RA-induced differentiation.     

A splicing factor phosphorylated by protein kinase A is increased in HL60 cells treated with retinoic acid

Retinoic acid (RA) induces the differentiation of human promyelocytic leukemia HL60 cells into granulocytic cells and inhibits proliferation. Certain of actions of RA are mediated by RA nuclear receptors that regulate gene expression. However, it is also known that direct protein modification by RA (retinoylation) can occur. One such retinoylated protein in HL60 cells is a regulatory subunit of protein kinase A (PKA), which is increased in the nucleus following RA treatment and which then increases phosphorylation of other nuclear proteins. However, a complete understanding of which nuclear proteins are phosphorylated is lacking. In the current study, we employed mass spectrometry to identify one of the PKA-phosphorylated proteins as a serine/arginine-rich splicing factor 1 (SF2, SRSF1). We found that RA treatment increased the level of PKA-phosphorylated SF2 but decreased the level of SF2. While SF2 regulates myelogenous cell leukemia-1 (Mcl-1, anti-apoptotic factor), RA treatment reduced the level of Mcl-1L (full-length Mcl-1 long) and increased the level of Mcl-1S (Mcl-1 short; a short splicing variant of the Mcl-1). Furthermore, treatment with a PKA inhibitor reversed these effects on Mcl-1 and inhibited RA-induced cell differentiation. In contrast, treatment with a Mcl-1L inhibitor enhanced RA-induced cell differentiation. These results indicate that RA activates PKA in the nucleus, increases phosphorylation of SF2, raises levels of Mcl-1S and lowers levels of Mcl-1L, resulting in the induction of differentiation. RA-modified PKA may play an important role in inducing cell differentiation and suppressing cell proliferation.     

Proteins in human myeloid leukemia cell line HL60 reacting with retinoic acid monoclonal antibodies

The vitamin A derivative, retinoic acid (RA) has various biological effects in mammalian cells and tissues. It is well known that RA induces differentiation of leukemia cells and inhibits cell growth. There are two pathways for RA action; one via RA nuclear receptors (RARs), and one via acylation of proteins by RA (retinoylation). However, an understanding of which actions of RA occur via RARs and which occur via retinoylation is lacking. Thus, we undertook the examination of HL60 proteins using anti-RA monoclonal antibodies (ARMAs). These ARMAs showed specific binding to proteins in a saturable manner depending on protein and antibody concentration. Proteins eluted by Mono Q anion exchange chromatography and separated using two-dimensional polyacrylamide gel electrophoresis were detected by ARMAs. One of these ARMA-bound proteins in HL60 cells was identified as alpha-actinin. These results indicate that retinoylated proteins in HL60 cells can be recognized by ARMAs and that alpha-actinin modified by RA may play a significant role in RA-induced differentiation, including the promotion of cytomorphology changes.     

ASB-2 inhibits growth and promotes commitment in myeloid leukemia cells.

In acute promyelocytic leukemia (APL) cells harboring the promyelocytic leukemia retinoic acid receptor alpha (PML-RARalpha) chimeric protein, retinoic acid (RA)-induced differentiation is triggered through a PML-RARalpha signaling resulting in activation of critical target genes. Induced differentiation of APL cells is always preceded by withdrawal from the cell cycle and commitment events leading to terminal differentiation. Here we have identified the human ankyrin repeat-containing protein with a suppressor of cytokine signaling box-2 (ASB-2) cDNA, as a novel RA-induced gene in APL cells. PML-RARalpha strongly enhanced RA-induced ASB-2 mRNA expression. In myeloid leukemia cells, ASB-2 expression induced growth inhibition and chromatin condensation recapitulating early events critical to RA-induced differentiation of APL cells.     

维生素A类化合物和二甲基亚砜诱导人早幼粒白血病细胞(HL-60)分化的显微与亚显微结构研究

用光镜和电镜技术研究了HL-60细胞在诱导分化过程中的显微与亚显微结构变化,10~(-6)M的维A酸处理6天,细胞按粒系途径定向分化,其核质比例降低,核浓缩、分叶,核仁减少或消失。经RA处理的细胞在电镜下出现下列明显的变化:细胞核浓缩和分叶,异染色质区域增加,约46%细胞显示出类似成熟粒细胞核的亚显微形态特征,胞质中嗜天青颗粒减少,特异颗粒显著增加,两种颗粒的比率发生明显变化;细胞质中微管、微丝的量增加;多聚和单个分散的游离核糖体减少,有些??细胞胞质空泡化;出现主要以微丝为筑架的大型钝形伪足和不规则的表面突起。上述这些变化似可作为HL-60细胞形态分化的标志。维A酸诱导HL-60细胞形态分化具有明显的时间效应关系。1.4%DMSO对HL-60细胞分化的诱导作用类似于10~(-6)MRA,而等剂量的(10~(-6)M)R Ⅰ、RⅡ其作用弱于RA。     

Protein kinase C activity during sphinganine potentiation of retinoic acid-induced differentiation in a human leukemia cell line (HL-60).

The differentiation of HL-60 promyelocytic cells toward mature granulocytic cells induced by retinoic acid (RA) was accompanied by a decrease in protein kinase C (PKC) activity. The enhancement of RA-induced differentiation and the potentiation of the decrease of PKC activity by sphinganine (SP) seemed to correlate with each other. Kinetically, PKC activity during RA-induced differentiation without SP decreased to its lowest (75% of the control) after 48h; about 50% of the reduction was observed at 24h. In the presence of SP, PKC activity decreased more rapidly to its lowest (60% of the control) within 24h of incubation of RA. SP, added 24h before or concomitantly with the addition of RA, could potentiate the RA-induced differentiation and the reduction of PKC activity. Our results indicate that the effect of SP and the role of PKC during RA-induced differentiation may be critical at the early stages of induction of differentiation (within 24h of RA exposure).     

Nuclear retinoic-acid-binding proteins and receptors in retinoic-acid-responsive cell lines

Nuclear retinoic-acid-binding activity and the expression of retinoic acid receptor mRNA (RAR-alpha and RAR-beta) were assayed in the F9 embryonal carcinoma, HeLa, HL-60 promyelocytic leukaemia and S91 melanoma cell lines. A 4-svedberg nuclear retinoic-acid-binding activity was detected in all 4 cell lines, but the levels in the HeLa and HL-60 cells were lower than in the F9 and S91 lines. RAR-alpha mRNA was expressed in all 4 cell lines, although at a very low level in S91 cells. Conversely, RAR-beta mRNA was expressed in S91 cells and, at a lower level, in F9 cells but was undetectable in HeLa and HL-60 cells. RAR-beta, transcribed and translated in vitro from the cloned cDNA coding region, sedimented at 4 S and this suggests that the 4-svedberg nuclear retinoic-acid-binding activity may represent the retinoic acid receptors.     

Retinoic acid causes MEK-dependent RAF phosphorylation through RARα plus RXR activation in HL-60 cells

Retinoic acid (RA) is known to cause the myeloid differentiation of HL-60 human myeloblastic leukemia cells in a process requiring MEK-dependent ERK2 activation. This RA-induced ERK2 activation appears after approximately 4 h and persists until the cells are differentiated and G0 arrested (Yen et al, 1998). This motivates the question of whether RA also activated RAF as part of a typical RAF/MEK/MAPK cascade. Retinoic acid is shown here to also increase the phosphorylation of RAF, but in an unusual way. Surprisingly, increased RAF phosphorylation is first detectable after 12 to 24 hours by phosphorylation-induced retardation of polyacrylamide gel electrophoretic mobility. The RA-induced increased RAF phosphorylation is still apparent after 72 hours of treatment when most cells are differentiated and G0 arrested. There is a progressive dose-response relationship with 10(-8), 10(-7), and 10(-6) M RA. The RA-induced RAF phosphorylation corresponds to increased in vitro kinase activity. Inhibition of MEK with a PD98059 dose which inhibits ERK2 phosphorylation and subsequent cell differentiation also inhibits RAF phosphorylation. RA-induced MEK-dependent RAF phosphorylation is not due to changes in the amount of cellular MEK. The induced RAF phosphorylation, as well as anteceding ERK2 activation, depends on ligand-induced activation of both an RARalpha receptor and an RXR receptor. This and the slow kinetics of activation suggest a need for prior RA-induced gene expression. In summary, RA induces a MEK-dependent prolonged RAF activation, whose slow onset occurs after ERK2 activation but still well before cell cycle arrest and cell differentiation. The RA-induced increased RAF phosphorylation thus differs from typical mitogenic growth factor signaling, features that may contribute to cell cycle arrest and differentiation instead of division as the cellular outcome.     

Retinoic-acid-induced differentiation of HL-60 cells is associated with biphasic activation of the Na+− K+ pump

The human promyelocytic leukemia cell line, HL-60, can be induced to differentiate into granulocyte-like cells when cultured in the presence of 10(-6) M retinoic acid (RA) for several days. Following the addition of RA two kinds of changes occur. First, there are early changes that comprise an increase in the intracellular concentration of sodium ions [Na]i, which reaches its maximum after 6 h, and an increase in the activity of the Na+-pump, which is reflected by an ouabain-sensitive K+ influx that peaks at 8 h (170% of the control value) and that occurs without any change in the number of pump molecules, as measured by the binding of 3H-ouabain. Second, beginning after 12 h of culture with RA, a decrease in the number of ouabain-binding sites occurs, this being accompanied by an increase in the number of K+ ions actively transported by each site. The effect of modulation of Na+-pump activity on the RA-induced differentiation of HL-60 cells was studied using low, noncytotoxic concentrations of ouabain which, although alone having no differentiating effect, accelerated and potentiated the effect of RA on differentiation. When added in combination, these drugs induced rapid stimulation of the Na+-pump, which reached its peak after 2 h. These results indicate that a concomitant increase in the level of [Na+]i and in the activity of the Na+-pump constitute primary events in the interaction between RA and HL-60 cells, and that cation fluxes may play a role in the initiation of the process of differentiation.