The autophagy scaffold protein ALFY is critical for the granulocytic differentiation of AML cells.

Acute myeloid leukemia (AML) is a malignancy of myeloid progenitor cells that are blocked in differentiation. Acute promyelocytic leukemia (APL) is a rare form of AML, which generally presents with a t(15;17) translocation causing expression of the fusion protein PML-RARA. Pharmacological doses of all-trans retinoic acid (ATRA) induce granulocytic differentiation of APL cells leading to cure rates of >80% if combined with conventional chemotherapy. Autophagy is a lysosomal degradation pathway for the removal of cytoplasmic content and recycling of macromolecules. ATRA induces autophagy in ATRA-sensitive AML and APL cells and autophagy inhibition attenuates ATRA-triggered differentiation. In this study, we aimed at identifying if the autophagy-linked FYVE-domain containing protein (ALFY/WDFY3) is involved in autophagic degradation of protein aggregates contributes to ATRA therapy-induced autophagy. We found that ALFY mRNA levels increase significantly during the course of ATRA-induced differentiation of APL and AML cell lines. Importantly ALFY depletion impairs ATRA-triggered granulocytic differentiation of these cells. In agreement with its function in aggrephagy, knockdown of ALFY results in reduced ATRA-induced proteolysis. Our data further suggest that PML-RARα is an autophagy substrate degraded with the help of ALFY. In summary, we present a crucial role for ALFY in retinoid triggered maturation of AML cells.

Network-guided modeling allows tumor-type independent prediction of sensitivity to all-trans-retinoic acid.

Background: All-trans-retinoic acid (ATRA) is a differentiating agent used in the treatment of acute-promyelocytic-leukemia (APL) and it is under-exploited in other malignancies despite its low systemic toxicity. A rational/personalized use of ATRA requires the development of predictive tools allowing identification of sensitive cancer types and responsive individuals. Materials and methods: RNA-sequencing data for 10 080 patients and 33 different tumor types were derived from the TCGA and Leucegene datasets and completely re-processed. The study was carried out using machine learning methods and network analysis. Results: We profiled a large panel of breast-cancer cell-lines for in vitro sensitivity to ATRA and exploited the associated basal gene-expression data to initially generate a model predicting ATRA-sensitivity in this disease. Starting from these results and using a network-guided approach, we developed a generalized model (ATRA-21) whose validity extends to tumor types other than breast cancer. ATRA-21 predictions correlate with experimentally determined sensitivity in a large panel of cell-lines representative of numerous tumor types. In patients, ATRA-21 correctly identifies APL as the most sensitive acute-myelogenous-leukemia subtype and indicates that uveal-melanoma and low-grade glioma are top-ranking diseases as for average predicted responsiveness to ATRA. There is a consistent number of tumor types for which higher ATRA-21 predictions are associated with better outcomes. Conclusions: In summary, we generated a tumor-type independent ATRA-sensitivity predictor which consists of a restricted number of genes and has the potential to be applied in the clinics. Identification of the tumor types that are likely to be generally sensitive to the action of ATRA paves the way to the design of clinical studies in the context of these diseases. In addition, ATRA-21 may represent an important diagnostic tool for the selection of individual patients who may benefit from ATRA-based therapeutic strategies also in tumors characterized by lower average sensitivity.

Activation of RARα induces autophagy in SKBR3 breast cancer cells and depletion of key autophagy genes enhances ATRA toxicity.

All-trans retinoic acid (ATRA), a pan-retinoic acid receptor (RAR) agonist, is, along with other retinoids, a promising therapeutic agent for the treatment of a variety of solid tumors. On the one hand, preclinical studies have shown promising anticancer effects of ATRA in breast cancer; on the other hand, resistances occurred. Autophagy is a cellular recycling process that allows the degradation of bulk cellular contents. Tumor cells may take advantage of autophagy to cope with stress caused by anticancer drugs. We therefore wondered if autophagy is activated by ATRA in mammary tumor cells and if modulation of autophagy might be a potential novel treatment strategy. Indeed, ATRA induces autophagic flux in ATRA-sensitive but not in ATRA-resistant human breast cancer cells. Moreover, using different RAR agonists as well as RARα-knockdown breast cancer cells, we demonstrate that autophagy is dependent on RARα activation. Interestingly, inhibition of autophagy in breast cancer cells by either genetic or pharmacological approaches resulted in significantly increased apoptosis under ATRA treatment and attenuated epithelial differentiation. In summary, our findings demonstrate that ATRA-induced autophagy is mediated by RARα in breast cancer cells. Furthermore, inhibition of autophagy results in enhanced apoptosis. This points to a potential novel treatment strategy for a selected group of breast cancer patients where ATRA and autophagy inhibitors are applied simultaneously.

p38αMAPK interacts with and inhibits RARα: suppression of the kinase enhances the therapeutic activity of retinoids in acute myeloid leukemia cells.

All-trans retinoic acid (ATRA) is the only clinically useful differentiating agent, being used in the treatment of acute promyelocytic leukemia (APL). The use of ATRA in other types of acute myelogenous leukemia (AML) calls for the identification of novel strategies aimed at increasing its therapeutic activity. Here, we provide evidence that pharmacological inhibition of the mitogen-activated protein kinase, p38α, or silencing of the corresponding gene sensitizes APL and AML cell lines, as well as primary cultures of AML blasts to the anti-proliferative and cyto-differentiating activity of ATRA and synthetic retinoids. P38α inhibits ligand-dependent transactivation of the nuclear retinoic acid receptor, RARα, and the derived chimeric protein expressed in the majority of APL cases, PML-RARα. Inhibition is the consequence of ligand-independent binding of p38α, which results in stabilization of RARα and PML-RARα via blockade of their constitutive degradation by the proteasome. The inhibitory effect requires a catalytically active p38α and direct physical interaction with RARα and PML-RARα. Ser-369 in the E-region of RARα is essential for the binding of p38α and the ensuing functional effects on the activity of the receptor.

Synergistic antitumor activity of lapatinib and retinoids on a novel subtype of breast cancer with coamplification of ERBB2 and RARA.

All-trans retinoic acid (ATRA), the only clinically available cyto-differentiating agent, has potential for the therapy/chemoprevention of breast carcinoma. Given the heterogeneous nature of this tumor, a rational use of ATRA and derivatives (retinoids) in the clinic requires the identification of patients that would benefit from retinoid-based protocols. Here, we demonstrate that 23-32% of the human ERBB2(+) breast cancers show coamplification of retinoic acid receptor alpha (RARA), encoding the retinoic acid receptor, RARα. This represents a novel subtype of breast cancer characterized by remarkable sensitivity to ATRA and RARα agonists, regardless of positivity to the estrogen receptor, a known modulator of retinoid sensitivity. In estrogen-receptor-negative cellular models showing coamplification of ERBB2 and RARA, simultaneous targeting of the corresponding gene products with combinations of lapatinib and ATRA causes synergistic growth inhibition, cyto-differentiation and apoptosis. This provides proof-of-principle that coamplification of ERBB2 and RARA can be exploited for the stratified and targeted therapy of a novel subtype of breast cancer patients, with an approach characterized by tumor cell selectivity and low predicted toxicity. The available cellular models were exploited to define the molecular mechanisms underlying the antitumor activity of combinations between lapatinib and ATRA. Global gene expression and functional approaches provide evidence for three components of the antiproliferative/apoptotic responses triggered by lapatinib+ATRA. Induction of the retinoid-dependent RARRES3 protein by ATRA stabilizes the effect of lapatinib inhibiting ERBB2 phosphorylation. Upregulation and activation of the transcription factor FOXO3A integrates ATRA-dependent transcriptional and lapatinib-dependent posttranscriptional signals, controlling the levels of effector proteins like the antiapoptotic factor, BIRC5. Stimulation of the TGFß pathway by ATRA mediates other components of the apoptotic process set in motion by simultaneous targeting of ERBB2 and RARα.

Adult cystic fibrosis care in the 21st century.

Cystic fibrosis (CF) is the most common autosomal recessive inherited disease of Caucasian populations. As a result of a variety of diagnostic and therapeutic strategies there has been a dramatic increase in the life expectancy of patients with CF in the last decades and 50% of patients are now adults. This review will focus on the disease in adults and the provision of appropriate care. The complex care required to improve the survival and quality of life in the adult patients can best be provided in a dedicated adult cystic fibrosis unit. These units currently exist in many European countries, but more are needed in Italy.

Induction of apoptosis and stress response in ovarian carcinoma cell lines treated with ST1926, an atypical retinoid.

To understand the molecular mechanisms mediating apoptosis induction by a novel atypical retinoid, ST1926, the cellular response to drug treatment was investigated in IGROV-1 ovarian carcinoma cells carrying wild-type p53 and a cisplatin-resistant p53 mutant subline (IGROV-1/Pt1). Despite a similar extent of drug-induced DNA strand breaks, the level of apoptosis was substantially higher in p53 wild-type cells. p53 activation and early upregulation of p53-target genes were consistent with p53-dependent apoptosis in IGROV-1 cells. Stress-activated protein kinases were activated in both cell lines in response to ST1926. This event and activation of AP-1 were more pronounced in IGROV-1/Pt1 cells, in which the modulation of DNA repair-associated genes suggests an increased ability to repair DNA damage. Inhibition of JNK or p38 stimulated ST1926-induced apoptosis only in IGROV-1 cells, whereas inhibition of ERKs enhanced apoptosis in both the cell lines. Such a pattern of cellular response and modulation of genes implicated in DNA damage response supports that the genotoxic stress is a critical event mediating drug-induced apoptosis. The results are consistent with apoptosis induction through p53-dependent and -independent pathways, regulated by MAP kinases, which likely play a protective role.

Atypical retinoids: an expanding series of anti-leukemia and anti-cancer agents endowed with selective apoptotic activity.

Retinoid-related molecules (RRMs) are an interesting class of novel molecules endowed with remarkable and selective apoptotic activity against various leukemia and cancer cell types. ST1926 is the most promising molecule and is active in vivo on numerous experimental models of leukemia and cancer. This has led to the development of a compound that will soon undergo phase I clinical trials. Although the primary molecular targets of RRMs' apoptotic activity are still unknown, ST1926 and congeners are characterized by a peculiar mechanism of action that centers on the mitochondrion and is associated with alterations in the homeostasis of intracellular calcium.

Cytodifferentiation: a novel approach to cancer treatment and prevention.

Cytodifferentiation therapy promises to control cancer growth and progression with less serious side effects than cytotoxic chemotherapy. Despite recent progress, the molecular mechanisms regulating the differentiation of many cell types are still obscure and the number of active cytodifferentiating agents is limited. Rational ways to develop these types of agents are necessary.

Purification of the aldehyde oxidase homolog 1 (AOH1) protein and cloning of the AOH1 and aldehyde oxidase homolog 2 (AOH2) genes. Identification of a novel molybdo-flavoprotein gene cluster on mouse chromosome 1.

We report the cloning of the AOH1 and AOH2 genes, which encode two novel mammalian molybdo-flavoproteins. We have purified the AOH1 protein to homogeneity in its catalytically active form from mouse liver. Twenty tryptic peptides, identified or directly sequenced by mass spectrometry, confirm the primary structure of the polypeptide deduced from the AOH1 gene. The enzyme contains one molecule of FAD, one atom of molybdenum, and four atoms of iron per subunit and shows spectroscopic features similar to those of the prototypic molybdo-flavoprotein xanthine oxidoreductase. The AOH1 and AOH2 genes are 98 and 60 kilobases long, respectively, and consist of 35 coding exons. The AOH1 gene has the potential to transcribe an extra leader non-coding exon, which is located downstream of exon 26, and is transcribed in the opposite orientation relative to all the other exons. AOH1 and AOH2 map to chromosome 1 in close proximity to each other and to the aldehyde oxidase gene, forming a molybdo-flavoenzyme gene cluster. Conservation in the position of exon/intron junctions among the mouse AOH1, AOH2, aldehyde oxidase, and xanthine oxidoreductase loci indicates that these genes are derived from the duplication of an ancestral precursor.

Tyrosine kinase inhibitor STI571 potentiates the pharmacologic activity of retinoic acid in acute promyelocytic leukemia cells: effects on the degradation of RARalpha and PML-RARalpha.

The 2-phenylaminopyrimidine derivative STI571 is a selective inhibitor of c-Abl, c-kit, and platelet-derived growth factor-receptor tyrosine kinases and is presently in phase II-III clinical studies. Here, this study reports on a novel pharmacologic activity of the compound, ie, enhancement of the cyto-differentiating, growth-inhibitory, and apoptogenic actions of all-trans-retinoic acid (ATRA). Whereas STI571 is not a cytodifferentiating agent by itself, the compound interacts with ATRA and enhances the myeloid maturation program set in motion by the retinoid in the PML-RARalpha(+) acute promyelocytic leukemia NB4 and the PML-RARalpha(-) myeloblastic HL60 and U937 cell lines. In addition, STI571 relieves the cyto-differentiation block observed in the ATRA-resistant cell lines, NB4.R1, NB4.306, and NB4.007. In NB4 promyelocytes, a RARalpha agonist, but not an RXR agonist, can substitute for ATRA and interact with STI571. By contrast, STI571 is unique among c-Abl-specific tyrosine kinase inhibitors in modulating the pharmacologic activity of ATRA. In NB4 cells, enhanced cyto-differentiation results in increased up-regulation of the expression of a number of genes coding for myeloid differentiation markers, including CD11b, CD11c, and some of the components of the nicotinamide adenine dinucleotide phosphate-oxidase enzymatic complex. All this is accompanied by inhibition of c-Abl tyrosine phosphorylation and retardation of the retinoid-dependent degradation of PML-RARalpha and RARalpha. Stabilization of the 2 retinoic acid receptors is likely to be the result of augmented and accelerated inhibition of the proteasome-dependent proteolytic activity observed on ATRA treatment.

Cloning of the cDNAs coding for two novel molybdo-flavoproteins showing high similarity with aldehyde oxidase and xanthine oxidoreductase.

The cDNAs coding for two novel mouse molybdo-flavoproteins, AOH1 and AOH2 (aldehyde oxidase homolog 1 and 2), were isolated. The AOH1 and AOH2 cDNAs code for polypeptides of 1336 amino acids. The two proteins have similar primary structure and show striking amino acid identity with aldehyde oxidase and xanthine oxidoreductase, two other molybdo-flavoenzymes. AOH1 and AOH2 contain consensus sequences for a molybdopterin-binding site and two distinct 2Fe-2S redox centers. In its native conformation, AOH1 has a molecular weight consistent with a homotetrameric structure. Transfection of the AOH1 and AOH2 cDNAs results in the production of proteins with phenanthridine but not hypoxanthine oxidizing activity. Furthermore, the AOH1 protein has benzaldehyde oxidizing activity with electrophoretic characteristics identical to those of a previously identified aldehyde oxidase isoenzyme (Holmes, R. S. (1979) Biochem. Genet. 17, 517-528). The AOH1 transcript is expressed in the hepatocytes of the adult and fetal liver and in spermatogonia. In liver, the AOH1 protein is synthesized in a gender-specific fashion. The expression of AOH2 is limited to keratinized epithelia and the basal layer of the epidermis and hair folliculi. The selective cell and tissue distribution of AOH1 and AOH2 mRNAs is consistent with the localization of the respective protein products.

Retinoid-dependent growth inhibition, differentiation and apoptosis in acute promyelocytic leukemia cells. Expression and activation of caspases.

In the NB4 model of acute promyelocytic leukemia (APL), ATRA, 9-cis retinoic acid (9-cis RA), the pan-RAR and RARalpha-selective agonists, TTNPB and AM580, induce growth inhibition, granulocytic differentiation and apoptosis. By contrast, two RXR agonists, a RARbeta agonist and an anti-AP1 retinoid have very limited activity, ATRA- and AM580-dependent effects are completely inhibited by RAR antagonistic blockade, while 9-cis RA-induced cell-growth-inhibition and apoptosis are equally inhibited by RAR and RXR antagonists. ATRA, 9-cis RA and AM580 cause upregulation of the mRNAs coding for pro-caspase-1, -7, -8, and -9, which, however, results in increased synthesis of only pro-caspase-1 and -7 proteins. These phenomena are associated with activation of pro-caspase-6, -7 and -8, cytochrome c release from the mitochondria, inversion of Bcl-2/Bax ratio and degradation of PML-RARalpha. Caspase activation is fundamental for retinoid-induced apoptosis, which is suppressed by the caspase-inhibitor z-VAD.

Isolation and characterization of an acute promyelocytic leukemia cell line selectively resistant to the novel antileukemic and apoptogenic retinoid 6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid.

6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) is a novel compound that represents the prototype of a new class of synthetic retinoids with apoptogenic properties in acute promyelocytic leukemia (APL) and other types of leukemia. In this article, using SCID mice xenografted with APL-derived NB4 cells, we demonstrate that CD437 has significant antileukemic activity in vivo. In addition, we report on the isolation and characterization of an APL cell line (NB4.437r) resistant to CD437. The cell line retains expression of PML-RARalpha and is approximately 33-fold more resistant than the parental counterpart to the apoptogenic effects of the retinoid. Resistance is relatively specific to CD437 and structural congeners because the NB4.437r cell line is still sensitive to various types of apoptogenic compounds. The CD437-resistant cell line maintains sensitivity to the antiproliferative and apoptotic action of all-trans-retinoic acid, AM580, and fenretinide, though it shows partial resistance to the cytodifferentiating effects of the first 2 compounds. Resistance to CD437 lays upstream of the CD437-induced release of cytochrome c from the mitochondria and the activation of caspase-3, -7, -8, and -9. Furthermore, NB4.437r cells are deficient in the CD437-dependent activation of nuclear NFkb and AP1-binding activities and in the phosphorylation of the protein kinase Akt. In the case of AP1, deficient assembly of the complex is not caused by the lack of activation of the Jun N-terminal kinase (JNK) family of kinases. The novel cell line will be useful in the elucidation of the molecular mechanisms underlying the apoptogenic action of CD437 and structurally related retinoids. (Blood. 2000;95:2672-2682)

Molecular cloning of the cDNA coding for mouse aldehyde oxidase: tissue distribution and regulation in vivo by testosterone.

The cDNA coding for mouse aldehyde oxidase (AO), a molybdoflavoprotein, has been isolated and characterized. The cDNA is 4347 nt long and consists of an open reading frame predicting a polypeptide of 1333 amino acid residues, with 5' and 3' untranslated regions of 13 and 335 nt respectively. The apparent molecular mass of the translation product in vitro derived from the corresponding cRNA is consistent with that of the monomeric subunit of the AO holoenzyme. The cDNA codes for a catalytically active form of AO, as demonstrated by transient transfection experiments conducted in the HC11 mouse mammary epithelial cell line. The deduced primary structure of the AO protein contains consensus sequences for two distinct 2Fe-2S redox centres and a molybdopterin-binding site. The amino acid sequence of the mouse AO has a high degree of similarity with the human and bovine counterparts, and a significant degree of relatedness to AO proteins of plant origin. Northern blot and in situ hybridization analyses demonstrate that hepatocytes, cardiocytes, lung endothelial or epithelial cells and oesophagus epithelial cells express high levels of AO mRNA. In the various tissues and organs considered, the level of AO mRNA expression is not strictly correlated with the amount of the corresponding protein, suggesting that the synthesis of the AO enzyme is under translational or post-translational control. In addition, we observed sex-related regulation of AO protein synthesis. In the liver of male animals, despite similar amounts of AO mRNA, the levels of the AO enzyme and corresponding polypeptide are significantly higher than those in female animals. Treatment of female mice with testosterone increases the amounts of AO mRNA and of the relative translation product to levels similar to those in male animals.

Leucocyte alkaline phosphatase identifies terminally differentiated normal neutrophils and its lack in chronic myelogenous leukaemia is not dependent on p210 tyrosine kinase activity.

Leucocyte alkaline phosphatase (LAP) is a marker of post-mitotic granulocytes and its activity is reduced or absent in chronic myelogenous leukaemia (CML) granulocytes as a consequence of LAP messenger RNA (mRNA) deficiency. We provide evidence that along the granulocytic maturation in normal marrow, the acquisition of LAP surface expression, identified by the monoclonal antibody 1B12.1, was restricted to CD11bbright/CD16bright positive cells. Moreover, in normal granulocytes, exposure to granulocyte colony-stimulating factor (G-CSF) in vitro and in vivo increased the cell surface expression of LAP. Although G-CSF was able to induce the LAP surface expression in CML granulocytes, the inhibition of p210 tyrosine kinase activity by genistein or CGP75148B failed to restore LAP mRNA expression and LAP protein synthesis. In conclusion, the acquisition of LAP protein on the cell surface of granulocytes follows CD16 antigen expression and can be considered as the last marker of terminally differentiated neutrophils. G-CSF is a potent regulator of the LAP mRNA expression and protein synthesis in normal and CML-derived neutrophils. The lack of direct activity of p210 tyrosine kinase on LAP mRNA expression in CML neutrophils supports the notion that the LAP defect in this disease could be related to a precocious and uncontrolled release of white blood cells from the bone marrow into the blood stream.

The novel synthetic retinoid 6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) causes apoptosis in acute promyelocytic leukemia cells through rapid activation of caspases.

The synthetic retinoid 6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437), which was originally developed as an retinoic acid receptor (RAR)-gamma agonist, induces rapid apoptosis in all-trans retinoic acid (ATRA)-sensitive and ATRA-resistant clones of the NB4 cell line, a widely used experimental model of acute promyelocytic leukemia (APL). In addition, the compound is apoptogenic in primary cultures of freshly isolated APL blasts obtained from a newly diagnosed case and an ATRA-resistant relapsed patient. NB4 cells in the S-phase of the cycle are most sensitive to CD437-triggered apoptosis. CD437-dependent apoptosis does not require de novo protein synthesis and activation of RAR-gamma or any of the other nuclear retinoic acid receptors. The process is preceded by rapid activation of a caspase-like enzymatic activity capable of cleaving the fluorogenic DEVD but not the fluorogenic YVAD tetrapeptide. Increased caspase activity correlates with caspase-3 and caspase-7 activation. Inhibition of caspases by z-VAD suppresses the nuclear DNA degradation observed in NB4 cells treated with CD437, as well as the degradation of pro-caspase-3 and pro-caspase-7. CD437-dependent activation of caspases is preceded by release of cytochrome c from the mitochondria into the cytosol of treated cells. Leakage of cytochrome c lays upstream of caspase activation, because the phenomenon is left unaffected by pretreatment of NB4 cells with z-VAD. Treatment of APL cells with CD437 is associated with a caspase-dependent degradation of promyelocytic leukemia-RAR-alpha, which can be completely inhibited by z-VAD.

The mouse aldehyde oxidase gene: molecular cloning, chromosomal mapping and functional characterization of the 5'-flanking region.

In this article, we report on the chromosome mapping and molecular cloning of the genetic locus encoding the mouse molybdo-iron/sulfur-flavoprotein aldehyde oxidase. The aldehyde oxidase locus maps to mouse chromosome 1 band C1-C2, as determined by fluorescence in situ hybridization experiments conducted on metaphase chromosomes. The gene is approximately 83 kb long and consists of 35 exons. The exon/intron boundaries are perfectly conserved relative to the corresponding human homolog and almost completely conserved relative to the mouse xanthine oxidoreductase gene. This further supports the concept that the aldehyde oxidase and xanthine oxidoreductase loci evolved from the same ancestral precursor by a gene duplication event. The position of a major transcription start site was defined by primer extension and RNase mapping analysis. The 5'-flanking region of the mouse aldehyde oxidase gene contains a functional and orientation-dependent promoter as well as several putative binding sites for known cell-specific and general transcription factors. Deletion analysis of the 5'-flanking region defines an approximately 470 bp DNA stretch which is necessary and sufficient for the transcription of the mouse aldehyde oxidase gene.

Cross-talk between retinoic acid and interferons: molecular mechanisms of interaction in acute promyelocytic leukemia cells.

All-trans retinoic acid (ATRA) and interferons (IFNs) are active anticancer agents. ATRA is capable of inducing complete remission in acute promyelocytic leukemia (APL) patients, whereas IFNalpha is successfully used in the treatment of the stable phase of chronic myeloid leukemia. ATRA and IFNs have shown synergistic interactions in various experimental conditions and represent a potentially useful therapeutic combination in the treatment of various types of leukemias and solid tumors. The molecular basis of these interactions are poorly understood and need to be elucidated. In this review, we summarize a series of recent observations concerning the molecular mechanisms underlying the cross-talk between the intracellular pathways activated by ATRA and IFNs in APL cells. In APL blasts, IFNs regulate the expression of retinoic acid receptors, and ATRA, in turn, modulates the levels and the state of activation of members of the Jak-STAT second messenger pathway. This demonstrates a two-way interaction between ATRA and IFNs, which leads to cross-modulation of genes normally under the control of the retinoid and the cytokine. These data may be relevant in the context of a rational use of the combination between ATRA and IFNs in the clinical management of myeloid leukemias.

Cancer procoagulant and tissue factor are differently modulated by all-trans-retinoic acid in acute promyelocytic leukemia cells.

All-trans-retinoic acid (ATRA) downregulates the expression of two cellular procoagulants, tissue factor (TF) and cancer procoagulant (CP), in human promyelocytic leukemia cells. To evaluate whether or not changes of the procoagulant activities (PCAs) may share mechanisms with the ATRA-induced cyto-differentiation process, we have characterized the effect of ATRA on the TF and CP expression by NB4 cells, an ATRA maturation-inducible cell line, and two NB4-derived cell lines resistant to ATRA-induced maturation, the NB4. 306 and NB4.007/6 cells. Next, we evaluated the effect on the PCAs of the NB4 parental cells of three synthetic retinoid analogues, ie: AM580 (selective for the retinoic acid receptor [RAR] alpha), capable to induce the granulocytic differentiation of NB4 cells; and CD2019 (selective for RARbeta) and CD437 (selective for RARgamma), both lacking this capability. Cells were treated with either ATRA or the analogues (10(-6) to 10(-8) mol/L) for 96 hours. The effect on cell differentiation was evaluated by morphologic changes, cell proliferation, nitro blue tetrazolium reduction assay, and flow cytometry analysis of the CD33 and CD11b surface-antigen expression. PCA was first measured in 20 mmol/L Veronal Buffer cell extracts by the one-stage clotting assay of normal and FVII-deficient plasmas. Further TF and CP have been characterized and quantified in cell-sample preparations by chromogenic and immunological assays. In the first series of experiments, ATRA downregulates both TF and CP in NB4 parental cells, as expected. However, in the differentiation-resistant cell lines, it induced a significant loss of TF but had little or no effect on CP. In a second series of experiments, in the NB4 parental cells, the RARalpha agonist (AM580) induced cell maturation and reduced 91% CP expression, whereas CD437 and CD2019 had no cyto-differentiating effects and did not affect CP levels. On the other hand, in the same cells the TF expression was reduced by ATRA and AM580, but also by the RARbeta agonist CD2019, which did not induce cell maturation. These data indicate that in NB4 cells, ATRA modulation of CP occurs in parallel with signs of cell differentiation, while the regulation of TF appears to be at least in part independent from these processes, and involves both alpha and beta nuclear retinoid receptors.