Retinoic acid receptors and tissue-transglutaminase mediate short-term effect of retinoic acid on migration and invasion of neuroblastoma SH-SY5Y cells

Long-term treatment with all trans-retinoic acid (RA) induces neuronal differentiation and apoptosis. However, the effect of short-term RA treatment on cell proliferation, migration and invasion of neuroblastoma cell lines (SH-SY5Y and IMR-32) remains unclear. RA induces expression of tissue-transglutaminase (TGase) and promotes migration and invasion after 24 h of treatment in SH-SY5Y cells, but not in IMR-32 cells. RA receptor (RAR) agonist (4-(E-2-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl) benzoic acid) and RAR/retinoid X receptor (RXR) agonist (9-cis-RA) promote expression of TGase, migration and invasion of SH-SY5Y cells, while RXR agonist has no significant effect. RAR antagonist blocks RA effect on migration and invasion, indicating that RAR receptors are required. Retinoid receptors are expressed and activated by RA in both cell lines. However, only transient activation of RAR is observed in IMR-32 cells. These findings suggest that different responses observed in SH-SY5Y and IMR-32 cells could be due to differential activation of retinoid receptors. Overexpression of TGase has no effect on migration or invasion, while overexpression of antisense TGase blocks RA-induced migration and invasion, indicating that other molecules along with TGase mediate RA effects. In addition to the long-term effects of RA that are coupled with cell differentiation, short-term effects involve migration and invasion of neuroblastoma SH-SY5Y cells.     

Co-resistance to retinoic acid and TRAIL by insertion mutagenesis into RAM

Retinoic acid (RA), used as first-line therapy for acute promyelocytic leukemia (APL), exerts its antileukemic activity by inducing blast differentiation and activating tumor-selective TNF-related apoptosis-inducing ligand (TRAIL) signaling. To identify downstream mediators of RA signaling, we used retrovirus-mediated insertion mutagenesis in PLB985 leukemia cells and established the RA-resistant cell line WY-1. In PLB985, but not WY-1 cells, RA induced TRAIL and its DR4 and DR5 receptors. Knocking down TRAIL expression by RNA interference blocked RA-induced apoptosis. WY-1 cells are defective for RA-induced differentiation, G1 arrest and exhibit co-resistance to TRAIL. In WY-1 cells, a single virus copy is integrated into a novel RA-regulated gene termed RAM (retinoic acid modulator). RAM is expressed in the myelomonocytic lineage and extinguished by RA in PLB985, but not WY-1 cells. Whereas knocking down RAM expression by RNA interference promoted RA-induced differentiation and TRAIL-triggered apoptosis of PLB985 and WY-1 cells, overexpression of the predicted 109 amino-acid RAM open reading frame did not alter RA signaling in PLB985 cells. This indicates that, apart from encoding the putative RAM protein, RAM RNA may exert additional functions that are impaired by the retrovirus insertion. Our study demonstrates that RA induction of the TRAIL pathway is also operative in leukemia cells lacking an RARalpha oncofusion protein and identifies RAM as a novel RA-dependent modulator of myeloid differentiation and death.     

Functional retinoid and thyroid hormone receptors in human thyroid-carcinoma cell lines and tissues

Thyroid carcinomas no longer accessible to radio-iodide or TSH-suppressive T4 therapy, due to loss of thyroid-specific functions, might be sufficiently re-differentiated by retinoic acid (RA) to be treated by conventional methods again. To help evaluate the feasibility of RA re-differentiation therapy in thyroid carcinomas, we examined the functionality of RA receptors (RARs/RXRs), central RA signal mediators, in human thyroid-carcinoma cell lines as model systems. [³H]-RA binding assays with nuclear extracts from follicular thyroid-carcinoma cell lines FTC-133 and -238 revealed high-affinity binding sites for RA. Electrophoretic mobility shift and supershift assays using a DR2 (“direct repeat” 2) RA response element demonstrated DNA-binding of RARα, RARγ, RXRα and RXRβ in nuclear extracts of FTC-133 and anaplastic HTh74 cells. Use of a DR5 RA response element revealed no difference in DNA binding. In supershift assays with a DR4 T3 response element, we found DNA-binding by TRα1, TRα2, and TRβ. Northern-blot analysis showed low expression of RXRβ mRNA in FTC-133 and of TRα1 mRNA in FTC-133 and FTC-238 cells. Using RT-PCR, we detected mRNA for RARα, RARβ, RARγ, RXRα, and RXRβ in the 4 cell lines and in human thyroid-carcinoma samples. RARβ mRNA was reduced in FTC-238 cells and RXRβ mRNA was decreased in anaplastic C643 cells and 9 of 12 tumor samples. Differential RA regulation of RA-receptor-mRNA expression was observed in the various cell lines. Thus, RA and T3 nuclear receptors are present in thyroid-carcinoma cell lines or tissues, albeit with cell-line and tumor-dependent variations; in the cell lines, they were shown to be functional with respect to DNA and/or ligand binding. Int. J. Cancer 76:368–376, 1998.© 1998 Wiley-Liss, Inc.     

Granulocyte-colony Stimulating Factor and Retinoic Acid Cooperatively Induce Granulocytic Differentiation of Acute Promyelocytic Leukemia Cells in vitro

The interaction of granulocyte-colony stimulating factor (G-CSF) and retinoic acid (RA) in proliferation and differentiation of acute promyelocytic leukemia (APL) cells was examined. G-CSF stimulated proliferation of APL cells at concentrations of 0.1 to 50 ng/ml in a dose dependent manner. More than 10⁻⁸ M RA induced granulocytic differentiation of APL cells. Although G-CSF induced lysozyme activities in APL cells, it alone did not induce terminal differentiation of APL cells. G-CSF significantly enhanced the RA-induced granulocytic differentiation of APL cells in vitro. Enhancement by G-CSF was not due to the prolongation of survival of RA-induced differentiated cells, but the differentiation-inducing effects of G-CSF might be evident only in the presence of RA. Since G-CSF has a potential to induce the granulocytic differentiation of myeloid leukemia cells, G-CSF in combination with RA may be applicable in differentiation induction therapy for some types of myeloid leukemia.     

The Notch ligand, Delta-1, alters retinoic acid (RA)-induced neutrophilic differentiation into monocytic and reduces RA-induced apoptosis in NB4 cells

Effects of Notch activation on retinoic acid (RA)-induced differentiation and apoptosis were investigated. NB4, an acute promyelocytic leukemia (APL) cell line, undergoes neutrophilic differentiation and apoptosis by RA. Notch activation induced by a recombinant Notch ligand, Delta-1, did not affect the growth by itself. Treatment with RA plus Delta-1 made part of NB4 cells monocyte-like shaped and reduced the apoptosis. Similar phenomenon was also observed in primary APL cells. RA treatment induced cleavage of caspase-8 and PARP in NB4. Delta-1 suppressed the RA-induced cleavage of them, which may be a possible mechanism through which Delta-1 suppressed the RA-induced apoptosis.     

Influence of exogenous RAR alpha gene on MDR1 expression and P-glycoprotein function in human and rodent cell lines.

The goal of our study was to obtain direct evidence of co-ordinated regulation of P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) and differentiation in tumour cells and to study some signalling pathways involved in joint regulation of these two cell phenotypes. The sublines of human melanoma (mS) and hepatoma (human HepG2 and rat McA RH 7777) cell lines were obtained by retroviral infection of the wild-type cells with the cDNA of the human retinoic acid receptor alpha (RAR alpha). The resulting sublines stably overexpressed exogenous RAR alpha gene. The infectants became more differentiated than the parental cells as determined by a decrease in the synthesis of the embryo-specific alpha-fetoprotein in HepG2 and McA RH 7777 hepatoma cells and by an increase in melanin synthesis in mS cells. The differentiation of human cells was accompanied by an increase in the amounts of MDR1 mRNA but not by an increase in P-gp activity as a drug transporter, in contrast, in the rat RAR alpha overexpressing cells P-gp functional activity was elevated. Treatment with cytotoxic drug (colchicine) or retinoic acid (RA) resulted in a slight increase in P-gp activity in the parental and RAR alpha-infected melanoma cells, whereas the increase in P-gp function in the infected hepatoma cells (both human and rat) was very prominent. Thus, we provide new evidence that cell differentiation caused by the overexpression of the gene participating in the differentiation programme leads to overexpression of MDR1 gene and drug resistance and that this effect is tissue and species specific. These data imply that the activation of the RA-controlled signalling pathway up-regulates MDR1 gene expression.     

Characterization of two novel retinoic acid-resistant cell lines derived from HL-60 cells following long-term culture with all-trans-retinoic acid.

Either all-trans-retinoic acid (RA) or vitamin D3 (VD) induces differentiation of the myeloid leukemia cell line HL-60. RA is available for the treatment of acute promyeloleukemia, although the development of resistance to the agent is a serious problem for differentiation-inducing therapy. To approach the mechanisms of resistance to RA, we developed two novel cell lines, HL-60-R2 and R9, which were subcloned after exposure to increasing concentrations of RA. The growth rate of HL-60-R2 cells was significantly increased by RA treatment, whereas the growth rate of HL-60-R9 was not affected. RA induces apoptosis in the parental HL-60 cells. The number of apoptotic cells, however, was not increased and nitroblue tetrazolium (NBT) reduction was not altered by 1 microM RA in either of the cloned cell lines. Treatment with VD induced monocytic differentiation and increased the expression of CD11b in HL-60 and HL-60-R9 cells, but not in HL-60-R2 cells. Flow cytometric and G-banding analysis demonstrated that R2 cells were near-triploid. The sequencing analysis revealed a deletion of three nucleotides in the sequence of the RAR alpha gene in HL-60-R9 cells, resulting in deletion of codon 286. No mutation was found in HL-60-R2 cells. Taken together, these data indicate that the resistance to RA is caused by the mutation in RAR alpha of HL-60-R9, but by other factor(s), which also affect the VD-response pathways, in HL-60-R2. The abnormal response to VD may be associated with the abnormal ploidy of the R2 cells.     

c-Cbl interacts with CD38 and promotes retinoic acid-induced differentiation and G0 arrest of human myeloblastic leukemia cells

Retinoic acid (RA) is known to regulate cell growth and differentiation. In HL-60 human myeloblastic leukemia cells, it causes mitogen-activated protein kinase (MAPK) signaling leading to myeloid differentiation and G(0) cell cycle arrest. This communication reports that expression of the Cbl adaptor caused enhanced extracellular signal-regulated kinase 2 activation and promoted RA-induced differentiation and G(0)-arrest. Stable transfectants ectopically expressing c-Cbl underwent myeloid differentiation faster than wild-type (wt) cells when treated with RA. In contrast, c-Cbl knockdown stable transfectants differentiated slower than wt cells when treated with RA. Cells ectopically expressing c-Cbl had enhanced CD38 expression when treated with RA, and cells ectopically expressing CD38 had enhanced c-Cbl expression, even without with RA, suggesting an interaction between c-Cbl and CD38. Fluorescence resource energy transfer and coimmunoprecipitation showed that c-Cbl and CD38 bind each other. RA causes the gradual down-regulation and eventual loss of c-Cbl expression, resulting in loss of the Cbl-CD38 interaction, suggesting that c-Cbl plays a relatively early role in promoting RA-induced differentiation. RA-induced differentiation can thus be propelled by c-Cbl and by CD38, both of which bind together, enhance the expression of each other, and cause MAPK signaling. There thus seems to be a cooperative role for c-Cbl and CD38, reflected in their direct binding, in propulsion of RA-induced differentiation.     

Suppression of mammary carcinoma cell growth by retinoic acid: the cell cycle control gene Btg2 is a direct target for retinoic acid receptor signaling

The anticarcinogenic activities of retinoic acid (RA) are believed to be mediated by the nuclear RA receptor (RAR) and by the RA-binding protein cellular RA-binding protein-II (CRABP-II). In MCF-7 mammary carcinoma cells, growth inhibition by RA entails an early cell cycle arrest followed by induction of apoptosis. Here, we aimed to obtain insights into the initial cell cycle response. We show that a 3- to 5-h RA pulse is sufficient for inducing a robust growth arrest 2 to 4 days later, demonstrating inhibition of the G1-S transition by RA is triggered by immediate-early RAR targets and does not require the continuous presence of the hormone throughout the arrest program. Expression array analyses revealed that RA induces the expression of several genes involved in cell cycle regulation, including the p53-controlled antiproliferative gene B-cell translocation gene, member 2 (Btg2) and the BTG family member Tob1. We show that induction of Btg2 by RA does not require de novo protein synthesis and is augmented by overexpression of CRABP-II. Additionally, we identify a RA response element in the Btg2 promoter and show that the element binds retinoid X receptor/RAR heterodimers in vitro, is occupied by the heterodimers in cells, and can drive RA-induced activation of a reporter gene. Hence, Btg2 is a novel direct target for RA signaling. In concert with the reports that Btg2 inhibits cell cycle progression by down-regulating cyclin D1, induction of Btg2 by RA was accompanied by a marked decrease in cyclin D1 expression. The observations thus show that the antiproliferative activity of RA in MCF-7 cells is mediated, at least in part, by Btg2.     

Formation of PML/RAR alpha high molecular weight nuclear complexes through the PML coiled-coil region is essential for the PML/RAR alpha-mediated retinoic acid response

Retinoic Acid (RA) treatment induces disease remission of Acute Promyelocytic Leukaemia (APL) patients by triggering terminal differentiation of neoplastic cells. RA-sensitivity in APL is mediated by its oncogenic protein, which results from the recombination of the PML and the RA receptor alpha (RAR alpha) genes (PML/RAR alpha fusion protein). Ectopic expression of PML/RAR alpha into haemopoietic cell lines results in increased response to RA-induced differentiation. By structure-function analysis of PML/RAR alpha-mediated RA-differentiation, we demonstrated that fusion of PML and RAR alpha sequences and integrity of the PML dimerization domain and of the RAR alpha DNA binding region are required for the effect of PML/RAR alpha on RA-differentiation. Indeed, direct fusion of the PML dimerization domain to the N- or C-terminal extremities of RAR alpha retained full biological activity. All the biologically active PML/RAR alpha mutants formed high molecular weight complexes in vivo. Functional analysis of mutations within the PML dimerization domain revealed that the capacity to form PML/RAR alpha homodimers, but not PML/RAR alpha-PML heterodimers, correlated with the RA-response. These results suggest that targeting of RAR alpha sequences by the PML dimerization domain and formation of nuclear PML/RAR alpha homodimeric complexes are crucial for the ability of PML/RAR alpha to mediate RA-response.