Phosphorylation of c-Cbl and p85 PI3K driven by all-trans retinoic acid and CD38 depends on Lyn kinase activity

The leukocyte antigen CD38 is expressed after all-trans retinoic acid (ATRA) treatment in HL-60 myelogenous leukemia cells and promotes induced myeloid differentiation when overexpressed. We found that Vav1 and SLP-76 associate with CD38 in two cell lines, and that these proteins complex with Lyn, a Src family kinase (SFK) upregulated by ATRA. SFK inhibitors PP2 and dasatinib, which enhance ATRA-induced differentiation, were used to evaluate the involvement of Lyn kinase activity in CD38-driven signaling. Cells treated with ATRA for 48 h followed by one hour of PP2 incubation show SFK/Lyn kinase inhibition. We observed that Lyn inhibition blocked c-Cbl and p85/p55 PI3K phosphorylation driven by the anti-CD38 agonistic mAb IB4 in ATRA-treated HL-60 cells and untreated CD38 + transfectants. In contrast, cells cultured for 48 h following concurrent ATRA and PP2 treatment did not show Lyn inhibition, suggesting ATRA regulates the effects on Lyn. 48 h of co-treatment preserved CD38-stimulated c-Cbl and p85/p55 PI3K phosphorylation indicating Lyn kinase activity is necessary for these events. In contrast another SFK inhibitor (dasatinib) which blocks Lyn activity with ATRA co-treatment prevented ATRA-induced c-Cbl phosphorylation and crippled p85 PI3K phosphorylation, indicating Lyn kinase activity is important for ATRA-propelled events potentially regulated by CD38. We found that loss of Lyn activity coincided with a decrease in Vav1/Lyn/CD38 and SLP-76/Lyn/CD38 interaction, suggesting these molecules form a complex that regulates CD38 signaling. Lyn inhibition also reduced Lyn and CD38 binding to p85 PI3K, indicating CD38 facilitates a complex responsible for PI3K phosphorylation. Therefore, Lyn kinase activity is important for CD38-associated signaling that may drive ATRA-induced differentiation.     

A PU.1 Suppressive Target Gene,Metallothionein 1G,Inhibits Retinoic Acid-Induced NB4 Cell Differentiation

We recently revealed that myeloid master regulator SPI1/PU.1 directly represses metallothionein (MT) 1G through its epigenetic activity of PU.1, but the functions of MT1G in myeloid differentiation remain unknown. To clarify this, we established MT1G-overexpressing acute promyelocytic leukemia NB4 (NB4MTOE) cells, and investigated whether MT1G functionally contributes to all-trans retinoic acid (ATRA)-induced NB4 cell differentiation. Real-time PCR analyses demonstrated that the inductions of CD11b and CD11c and reductions in myeloperoxidase and c-myc by ATRA were significantly attenuated in NB4MTOE cells. Morphological examination revealed that the percentages of differentiated cells induced by ATRA were reduced in NB4MTOE cells. Since G1 arrest is a hallmark of ATRA-induced NB4 cell differentiation, we observed a decrease in G1 accumulation, as well as decreases in p21^WAF1/CIP1 and cyclin D1 inductions, by ATRA in NB4MTOE cells. Nitroblue tetrazolium (NBT) reduction assays revealed that the proportions of NBT-positive cells were decreased in NB4MTOE cells in the presence of ATRA. Microarray analyses showed that the changes in expression of several myeloid differentiation-related genes (GATA2, azurocidin 1, pyrroline-5-carboxylate reductase 1, matrix metallopeptidase -8, S100 calcium-binding protein A12, neutrophil cytosolic factor 2 and oncostatin M) induced by ATRA were disturbed in NB4MTOE cells. Collectively, overexpression of MT1G inhibits the proper differentiation of myeloid cells.     

Retinoic acid-induced CD38 expression in HL-60 myeloblastic leukemia cells regulates cell differentiation or viability depending on expression levels

Retinoic acid-induced expression of the CD38 ectoenzyme receptor in HL-60 human myeloblastic leukemia cells is regulated by RARalpha and RXR, and enhanced or prevented cell differentiation depending on the level of expression per cell. RARalpha activation caused CD38 expression, as did RXR activation but not as effectively. Inhibition of MAPK signaling through MEK inhibition diminished the induced expression by both RARs and RXRs. Expression of CD38 enhanced retinoic acid-induced myeloid differentiation and G0 cell cycle arrest, but at higher expression levels, induced differentiation was blocked and retinoic acid induced a loss of cell viability instead. In the case of 1,25-dihydroxyvitamin D3, induced monocytic differentiation was also enhanced by CD38 and not enhanced by higher expression levels, but without induced loss of viability. Expression levels of CD38 thus regulated the cellular response to retinoic acid, either propelling cell differentiation or loss of viability. The cellular effects of CD38 thus depend on its expression level.     

Roscovitine enhances All-trans retinoic acid (ATRA)-induced leukemia cell differentiation: Novel effects on signaling molecules for a putative Cdk2 inhibitor

All-trans retinoic acid (ATRA)-based differentiation therapy has been unsuccessful in treating t(15;17) negative acute myeloid leukemia (AML) patients, motivating interest in combination therapies using ATRA plus other agents. Using the t (15, 17) negative HL-60 human myeloblastic leukemia model, we find that the cyclin-dependent kinase (CDK) inhibitor, roscovitine, augments signaling by an ATRA-induced macromolecular signalsome that propels differentiation and enhances ATRA-induced differentiation. Roscovitine co-treatment enhanced ATRA-induced expression of pS259- pS289/296/301- pS621-c-Raf, pS217/221-Mek, Src Family Kinases (SFKs) Lyn and Fgr and SFK Y416 phosphorylation, adaptor proteins c-Cbl and SLP-76, Vav, and acetylated 14–3-3 in the signalsome. Roscovitine enhanced ATRA-induced c-Raf interaction with Lyn, Vav, and c-Cbl. Consistent with signalsome hyper-activation, roscovitine co-treatment enhanced ATRA-induced G1/0 arrest and expression of differentiation markers, CD11b, ROS and p47 Phox. Because roscovitine regulated Lyn expression, activation and partnering, a stably transfected Lyn knockdown was generated from wt-parental cells to investigate its function in ATRA-induced differentiation. Lyn-knockdown enhanced ATRA-induced up-regulation of key signalsome molecules, c-Raf, pS259-c-Raf, pS289/296/301-c-Raf, Vav1, SLP-76, and Fgr, but with essentially total loss of pY416-SFK. Compared to ATRA-treated wt-parental cells, differentiation markers p47 phox, CD11b, G1/G0 arrest and ROS production were enhanced in ATRA-treated Lyn-knockdown stable transfectants, and addition of roscovitine further enhanced these ATRA-inducible markers. The Lyn-knockdown cells expressed slightly higher c-Raf, pS259-c-Raf, pS289/296/301-c-Raf, and SLP-76 than wt-parental cells, and this was associated with enhanced ATRA-induced upregulation of Fgr and cell differentiation, consistent with heightened signaling, suggesting that enhanced Fgr may have compensated for loss of Lyn to enhance differentiation in the Lyn-knockdown cells.     

Cytosolic CD38 protein forms intact disulfides and is active in elevating intracellular cyclic ADP-ribose

CD38 catalyzes the synthesis of cyclic ADP-ribose (cADPR), a Ca(2+) messenger responsible for regulating a wide range of physiological functions. It is generally regarded as an ectoenzyme, but its intracellular localization has also been well documented. It is not known if internal CD38 is enzymatically active and contributes to the Ca(2+) signaling function. In this study, we engineered a novel soluble form of CD38 that can be efficiently expressed in the cytosol and use cytosolic NAD as a substrate to produce cADPR intracellularly. The activity of the engineered CD38 could be decreased by mutating the catalytic residue Glu-226 and increased by the double mutation E146A/T221F, which increased its cADPR synthesis activity by >11-fold. Remarkably, the engineered CD38 exhibited the ability to form the critical disulfide linkages required for its enzymatic activity. This was verified by using a monoclonal antibody generated against a critical disulfide, Cys-254-Cys-275. The specificity of the antibody was established by x-ray crystallography and site-directed mutagenesis. The engineered CD38 is thus a novel example challenging the general belief that cytosolic proteins do not possess disulfides. As a further refinement of this approach, the engineered CD38 was placed under the control of tetracycline using an autoregulated construct. This study has set the stage for in vivo manipulation of cADPR metabolism.     

ATRA-Induced Cellular Differentiation and CD38 Expression Inhibits Acquisition of BCR-ABL Mutations for CML Acquired Resistance

Acquired resistance through genetic mutations is a major obstacle in targeted cancer therapy, but the underlying mechanisms are poorly understood. Here we studied mechanisms of acquired resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors (TKIs) by examining genome-wide gene expression changes in KCL-22 CML cells versus their resistant KCL-22M cells that acquire T315I BCR-ABL mutation following TKI exposure. Although T315I BCR-ABL is sufficient to confer resistance to TKIs in CML cells, surprisingly we found that multiple drug resistance pathways were activated in KCL-22M cells along with reduced expression of a set of myeloid differentiation genes. Forced myeloid differentiation by all-trans-retinoic acid (ATRA) effectively blocked acquisition of BCR-ABL mutations and resistance to the TKIs imatinib, nilotinib or dasatinib in our previously described in vitro models of acquired TKI resistance. ATRA induced robust expression of CD38, a cell surface marker and cellular NADase. High levels of CD38 reduced intracellular nicotinamide adenine dinucleotide (NAD⁺) levels and blocked acquired resistance by inhibiting the activity of the NAD⁺-dependent SIRT1 deacetylase that we have previously shown to promote resistance in CML cells by facilitating error-prone DNA damage repair. Consequently, ATRA treatment decreased DNA damage repair and suppressed acquisition of BCR-ABL mutations. This study sheds novel insight into mechanisms underlying acquired resistance in CML, and suggests potential benefit of combining ATRA with TKIs in treating CML, particularly in advanced phases.     

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.     

CD38 is expressed as noncovalently associated homodimers on the surface of murine B lymphocytes.

CD38 is a transmembrane glycoprotein that functions as an ectoenzyme and as a receptor. Based on the structural similarity between CD38 and ADP-ribosyl cyclase from Aplysia californica, it was hypothesized that CD38 is expressed as a homodimer on the surface of cells. Indeed, CD38 dimers have been reported, however, the structural requirements for their stabilization on the plasma membrane are unknown. We demonstrate that the majority of CD38 is assembled as noncovalently associated homodimers on the surface of B cells. Analysis of CD38 mutants, expressed in Ba/F3 cells, revealed that truncation of the cytoplasmic region or mutation of a single amino acid within the alpha1-helix of CD38 decreased the stability of the CD38 homodimers when solubilized in detergent. Cells expressing the unstable CD38 homodimers had diminished expression of CD38 on the plasma membrane and the half-lives of these CD38 mutant proteins on the plasma membrane were significantly reduced. Together, these results show that CD38 is expressed as noncovalently associated homodimers on the surface of murine B cells and suggest that appropriate assembly of CD38 homodimers may play an important role in stabilizing CD38 on the plasma membrane of B cells.     

Magnolol and honokiol enhance HL-60 human leukemia cell differentiation induced by 1,25-dihydroxyvitamin D3 and retinoic acid

Magnolol (MG) and honokiol (HK), two lignans showing anti-inflammatory and anti-oxidant properties and abundantly available in the medicinal plants Magnolia officinalis and M. obovata, were found to enhance HL-60 cell differentiation initiated by low doses of 1,25-dihydroxyvitamin D3 (VD3) and all-trans-retinoic acid (ATRA). Cells expressing membrane differentiation markers CD11b and CD14 were increased from 4% in non-treated control to 8-16% after being treated with 10-30 microM MG or HK. When added to 1 nM VD3, MG or HK increased markers expressing cells from approximately 30% to 50-80%. When either MG or HK was added to 20 nM ATRA, only CD11b, but not CD14, expressing cells were increased from 9% to 24-70%. Under the same conditions, adding MG or HK to VD3 or ATRA treatment further enlarged the G0/G1 cell population and increased the expression of p27(Kip1), a cyclin-dependent kinase inhibitor. Pharmacological studies using PD098059 (a MEK inhibitor), SB203580 (a p38 MAPK inhibitor) and SP600125 (a JNK inhibitor) suggested that the MEK pathway was important for VD3 and ATRA-induced differentiation and also its enhancement by MG or HK, the p38 MAPK pathway had a inhibitory effect and the JNK pathway had little influence. It is evident that MG and HK are potential differentiation enhancing agents which may allow the use of low doses of VD3 and ATRA in the treatment for acute promyelocytic leukemia.     

Anti-tumor effects of all-trans retinoic acid are enhanced by genistein

The effects of all-trans retinoic acid (ATRA) on cancer are complex. ATRA has anti-cancer effects as it promotes cancer cell differentiation. However, ATRA also up-regulates expression of vascular endothelial growth factor (VEGF) in cancer cells, which leads to angiogenesis and can, thus, facilitate cancer growth. Genistein, a crucial non-nutrient component in soybean, exhibits anti-cancer effects by inhibiting protein tyrosine kinase that is involved in up-regulation of VEGF. We hypothesized that genistein, applied simultaneously with ATRA, would counter its undesired angiogenic effects and, thus, enhance the anti-cancer effects of ATRA. The purpose of this study was to document potential synergistic effects of genistein and ATRA in A549 lung adenocarcinoma cells. We further explored the role of genistein on countering the ATRA-induced VEGF expression. We demonstrate that genistein enhances the ATRA-induced growth inhibition of A549 cells by promoting apoptosis. Further, the combined use of ATRA and genistein leads to cancer cell arrest in G0/G1 and G2/M cell cycle phases. Finally, expression of VEGF (both mRNA and protein) was diminished in A549 cells exposed to both ATRA and genistein. In conclusion, our results demonstrate that genistein effectively enhances anti-cancer effects of ATRA, particularly, by countering the ATRA-induced up-regulation of VEGF. Our study provides an experimental basis for combined use of ATRA and genistein in the treatment of lung cancer.     

Radotinib Induces Apoptosis of CD11b+ Cells Differentiated from Acute Myeloid Leukemia Cells

Radotinib, developed as a BCR/ABL tyrosine kinase inhibitor (TKI), is approved for the second-line treatment of chronic myeloid leukemia (CML) in South Korea. However, therapeutic effects of radotinib in acute myeloid leukemia (AML) are unknown. In the present study, we demonstrate that radotinib significantly decreases the viability of AML cells in a dose-dependent manner. Kasumi-1 cells were more sensitive to radotinib than NB4, HL60, or THP-1 cell lines. Furthermore, radotinib induced CD11b expression in NB4, THP-1, and Kasumi-1 cells either in presence or absence of all trans-retinoic acid (ATRA). We found that radotinib promoted differentiation and induced CD11b expression in AML cells by downregulating LYN. However, CD11b expression induced by ATRA in HL60 cells was decreased by radotinib through upregulation of LYN. Furthermore, radotinib mainly induced apoptosis of CD11b⁺ cells in the total population of AML cells. Radotinib also increased apoptosis of CD11b⁺ HL60 cells when they were differentiated by ATRA/dasatinib treatment. We show that radotinib induced apoptosis via caspase-3 activation and the loss of mitochondrial membrane potential (ΔΨ_m) in CD11b⁺ cells differentiated from AML cells. Our results suggest that radotinib may be used as a candidate drug in AML or a chemosensitizer for treatment of AML by other therapeutics.     

CD38 is constitutively expressed in the nucleus of human hematopoietic cells

CD38 is a type II glycoprotein that acts both as a bifunctional enzyme, responsible for the synthesis and hydrolysis of cyclic ADP-ribose, and as a signal-transducing surface receptor. Although CD38 was originally described as a plasma membrane molecule, several reports indicate that CD38 is expressed in the nucleus, even in cells known to be CD38 surface-negative. In this study, firstly we investigated the presence of nuclear CD38 by immunofluorescence and confocal microscopy using a panel of hematopoietic cell lines that exhibit different levels of CD38 plasma membrane expression. Our second aim was to explore the relationship between the nuclear and plasma membrane forms of CD38 in human cell lines which represent discrete early maturation stages of the human lymphoid and myeloid compartments. Our results indicate that CD38 is constitutively present in the nucleus of cells belonging to distinct lineages. Furthermore, nuclear CD38 appears to be independent of the plasma membrane pool. The presence of nuclear CD38 during different stages of hematopoietic differentiation suggests that it may play a role in the control of nuclear Ca(2+) homeostasis and NAD levels.     

C/EBPbeta: a major PML-RARA-responsive gene in retinoic acid-induced differentiation of APL cells

In acute promyelocytic leukemia (APL), the translocation t(15;17) induces a block at the promyelocytic stage of differentiation in an all-trans-retinoic acid (ATRA)-responsive manner. Here we report that upon treatment with ATRA, t(15;17) cells (NB4) reveal a very rapid increase in protein level and binding activity of C/EBPbeta, a C/EBP family member, which was not observed in an ATRA-resistant NB4 cell line. We further provide evidence that ATRA mediates a direct increase of C/EBPbeta, only in PML-RARA (promyelocytic leukemia-retinoic acid receptor alpha)-expressing cells. In addition, transactivation experiments indicate that the PML-RARA fusion protein, but not PML-RARA mutants defective in transactivation, strongly transactivates the C/EBPbeta promoter. These results suggest that PML-RARA mediates ATRA-induced C/EBPbeta expression. Finally, we demonstrate the importance of C/EBPbeta in granulocytic differentiation. We show that not only does C/EBPbeta induce granulocytic differentiation of non-APL myeloid cell lines independent of addition of ATRA or other cytokines, but also that C/EBPbeta induction is required during ATRA-induced differentiation of APL cells. Taken together, C/EBPbeta is an ATRA-dependent PML-RARA target gene involved in ATRA-induced differentiation of APL cells.     

Human CD38, a surface receptor, an enzyme, an adhesion molecule and not a simple marker.

Human CD38 is a 45 kDa type II trans-membrane glycoprotein with a peculiar discontinuous pattern of expression in leukocytes, although evidence is accumulating of its quite widespread expression outside of the hematopoietic system. CD38 is a member of an emerging family of cytosolic and membrane-bound enzymes whose substrate is nicotinamide adenine dinucleotide (NAD), a coenzyme ubiquitously distributed in nature. CD38 is a multifunctional molecule able to exert not only an enzymatic activity but also to mobilize calcium, to transduce signals, to adhere to hyaluronan and to other ligands. Interaction with CD38 on various leukocyte subpopulation has profound though diverse effects on their life-span, however, the immunoregulatory activities seem to be independent of the enzymatic functions of the molecule.     

CD38/ADP-ribosyl cyclase: A new role in the regulation of osteoclastic bone resorption.

The multifunctional ADP-ribosyl cyclase, CD38, catalyzes the cyclization of NAD(+) to cyclic ADP-ribose (cADPr). The latter gates Ca(2+) release through microsomal membrane-resident ryanodine receptors (RyRs). We first cloned and sequenced full-length CD38 cDNA from a rabbit osteoclast cDNA library. The predicted amino acid sequence displayed 59, 59, and 50% similarity, respectively, to the mouse, rat, and human CD38. In situ RT-PCR revealed intense cytoplasmic staining of osteoclasts, confirming CD38 mRNA expression. Both confocal microscopy and Western blotting confirmed the plasma membrane localization of the CD38 protein. The ADP-ribosyl cyclase activity of osteoclastic CD38 was next demonstrated by its ability to cyclize the NAD(+) surrogate, NGD(+), to its fluorescent derivative cGDP-ribose. We then examined the effects of CD38 on osteoclast function. CD38 activation by an agonist antibody (A10) in the presence of substrate (NAD(+)) triggered a cytosolic Ca(2+) signal. Both ryanodine receptor modulators, ryanodine, and caffeine, markedly attenuated this cytosolic Ca(2+) change. Furthermore, the anti-CD38 agonist antibody expectedly inhibited bone resorption in the pit assay and elevated interleukin-6 (IL-6) secretion. IL-6, in turn, enhanced CD38 mRNA expression. Taken together, the results provide compelling evidence for a new role for CD38/ADP-ribosyl cyclase in the control of bone resorption, most likely exerted via cADPr.     

EGFR inhibitors exacerbate differentiation and cell cycle arrest induced by retinoic acid and vitamin D3 in acute myeloid leukemia cells

By means of an unbiased, automated fluorescence microscopy-based screen, we identified the epidermal growth factor receptor (EGFR) inhibitors erlotinib and gefitinib as potent enhancers of the differentiation of HL-60 acute myeloid leukemia (AML) cells exposed to suboptimal concentrations of vitamin A (all-trans retinoic acid, ATRA) or vitamin D (1α,25-hydroxycholecalciferol, VD). Erlotinib and gefitinib alone did not promote differentiation, yet stimulated the acquisition of morphological and biochemical maturation markers (including the expression of CD11b and CD14 as well as increased NADPH oxidase activity) when combined with either ATRA or VD. Moreover, the combination of erlotinib and ATRA or VD synergistically induced all the processes that are normally linked to terminal hematopoietic differentiation, namely, a delayed proliferation arrest in the G₀/G₁ phase of the cell cycle, cellular senescence, and apoptosis. Erlotinib potently inhibited the (auto)phosphorylation of mitogen-activated protein kinase 14 (MAPK14, best known as p38^MAPK) and SRC family kinases (SFKs). If combined with the administration of ATRA or VD, the inhibition of p38^MAPK or SFKs with specific pharmacological agents mimicked the pro-differentiation activity of erlotinib. These data were obtained with 2 distinct AML cell lines (HL-60 and MOLM-13 cells) and could be confirmed on primary leukemic blasts isolated from the circulation of AML patients. Altogether, these findings point to a new regimen for the treatment of AML, in which naturally occurring pro-differentiation agents (ATRA or VD) may be combined with EGFR inhibitors.