Extramedullary relapse in acute promyelocytic leukemia treated with all-trans retinoic acid and chemotherapy

We analyzed the incidence, presenting features, risk factors of extramedullary (EM) relapse occurring in acute promyelocytic leukemia (APL) treated with all-trans retinoic acid (ATRA) and chemotherapy by using a competing-risk method. In total, 740/ 806 (92%) patients included in three multicenter trials (APL91, APL93 trials and PETHEMA 96) achieved CR, of whom 169 (23%) relapsed, including 10 EM relapses. Nine relapses involved the central nervous system (CNS) and one the skin, of which two were isolated EM relapse. In patients with EM disease, median WBC count was 26950/mm3 (7700-162000). The 3-year cumulative incidence of EM disease at first relapse was 5.0%. Univariate analysis identified age <45 years (P=0.05), bcr3 PML-RARalpha isoform (P= 0.0003) and high WBC counts (> or = 10,000/ mm3) (P<0.0001) as risk factors for EM relapse. In multivariate analysis, only high WBC count remained significant (P= 0.001). Patients with EM relapse had a poorer outcome since median survival from EM relapse was 6.7 months as compared to 26.3 months for isolated BM relapse (P=0.04). In conclusion, EM relapse in APL occurs more frequently in patients with increased WBC counts (> or = 10,000/mm3) and carries a poor prognosis. Whether CNS prophylaxis should be systematically performed in patients with WBC > or = 10,000/mm3 at diagnosis remains to be established.

Human basophils activated by mast cell-derived IL-3 express retinaldehyde dehydrogenase-II and produce the immunoregulatory mediator retinoic acid

The vitamin A metabolite retinoic acid (RA) plays a fundamental role in cellular functions by activating nuclear receptors. Retinaldehyde dehydrogenase-II (RALDH2) creates localized RA gradients needed for proper embryonic development, but very little is known regarding its regulated expression in adults. Using a human ex vivo model of allergic inflammation by coincubating IgE receptor-activated mast cells (MCs) with blood basophils, we observed prominent induction of a protein that was identified as RALDH2 by mass spectroscopy. RALDH2 was selectively induced in basophils by MC-derived interleukin-3 (IL-3) involving PI3-kinase and NF-kappaB pathways. Importantly, neither constitutive nor inducible RALDH2 expression was detectable in any other human myeloid or lymphoid leukocyte, including dendritic cells. RA generated by RALDH2 in basophils modulates IL-3-induced gene expression in an autocrine manner, providing positive (CD25) as well as negative (granzyme B) regulation. It also acts in a paracrine fashion on T-helper cells promoting the expression of CD38 and alpha4/beta7 integrins. Furthermore, RA derived from IL-3-activated basophils provides a novel mechanism of Th2 polarization. Thus, RA must be viewed as a tightly controlled basophil-derived mediator with a high potential for regulating diverse functions of immune and resident cells in allergic diseases and other Th2-type immune responses.

Retinoic acid signaling organizes endodermal organ specification along the entire antero-posterior axis

BACKGROUND: Endoderm organ primordia become specified between gastrulation and gut tube folding in Amniotes. Although the requirement for RA signaling for the development of a few individual endoderm organs has been established a systematic assessment of its activity along the entire antero-posterior axis has not been performed in this germ layer. METHODOLOGY/PRINCIPAL FINDINGS: RA is synthesized from gastrulation to somitogenesis in the mesoderm that is close to the developing gut tube. In the branchial arch region specific levels of RA signaling control organ boundaries. The most anterior endoderm forming the thyroid gland is specified in the absence of RA signaling. Increasing RA in anterior branchial arches results in thyroid primordium repression and the induction of more posterior markers such as branchial arch Hox genes. Conversely reducing RA signaling shifts Hox genes posteriorly in endoderm. These results imply that RA acts as a caudalizing factor in a graded manner in pharyngeal endoderm. Posterior foregut and midgut organ primordia also require RA, but exposing endoderm to additional RA is not sufficient to expand these primordia anteriorly. We show that in chick, in contrast to non-Amniotes, RA signaling is not only necessary during gastrulation, but also throughout gut tube folding during somitogenesis. Our results show that the induction of CdxA, a midgut marker, and pancreas induction require direct RA signaling in endoderm. Moreover, communication between CdxA(+) cells is necessary to maintain CdxA expression, therefore synchronizing the cells of the midgut primordium. We further show that the RA pathway acts synergistically with FGF4 in endoderm patterning rather than mediating FGF4 activity. CONCLUSIONS/SIGNIFICANCE: Our work establishes that retinoic acid (RA) signaling coordinates the position of different endoderm organs along the antero-posterior axis in chick embryos and could serve as a basis for the differentiation of specific endodermal organs from ES cells.

Retinoic acid reduces glucocorticoid sensitivity in C2C12 myotubes by decreasing 11beta-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor activities

Vitamin A is a nutrient with remarkable effects on adipose tissue and skeletal muscles, and plays a role in controlling energy balance. Retinoic acid (RA), the carboxylic form of vitamin A, has been associated with improved glucose tolerance and insulin sensitivity. In contrast, elevated glucocorticoids have been implicated in the development of insulin resistance and impaired glucose tolerance. Here, we investigated whether RA might counteract glucocorticoid effects in skeletal muscle cells by lowering 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1)-dependent local glucocorticoid activation and/or activation of glucocorticoid receptor (GR). We found a dose-dependent down-regulation of 11beta-HSD1 mRNA expression and activity upon incubation of fully differentiated mouse C2C12 myotubes with RA. In addition, RA inhibited GR transactivation by an 11beta-HSD1-independent mechanism. The presence of RA during myogenesis did not prevent myotube formation but resulted in relatively glucocorticoid-resistant myotubes, exhibiting very low 11beta-HSD1 expression and GR activity. The use of selective retinoic acid receptor (RAR) and retinoid X receptor ligands provided evidence that these effects were mediated through RARgamma. Importantly, short hairpin RNA against RARgamma abolished the effect of RA on 11beta-HSD1 and GR. In conclusion, we provide evidence for an important role of RA in the control of glucocorticoid activity during myogenesis and in myotubes. Disturbances of the nutrient and hormonal regulation of glucocorticoid action in skeletal muscles might be relevant for metabolic diseases.

Sweet's syndrome involoving the musculoskeletal system during treatment of promyelocytic leukemia with all-trans retinoic acid

Induction therapy of promyelocytic leukemia with all-trans retinoic acid is a standard therapy despite significant side-effects. The most important, the "retinoic acid syndrome", consists of a hyperinflammatory reaction with capillary leakage (edema, pleural, and pericardial effusion), infiltration of myeloid cells into internal organs and systemic signs of inflammation. We describe here two cases of another hyperinflammatory reaction during all-trans retinoic acid therapy, the Sweet's syndrome, consisting of infiltrates of the skin and internal organs by neutrophilic granulocytes. Fever, painful erythematous cutaneous plaques, prominent musculoskeletal involvement (myositis, fasciitis), a sterile pulmonary infiltration and intercurrent proteinuria characterized the clinical course of all-trans retinoic acid-associated Sweet's syndrome. Treatment with glucocorticoids led to resolution of the syndrome within 48 h. Three other cases of all-trans retinoic acid-associated Sweet's syndrome without involvement of internal organs, prominent on our cases, were published previously. Recognition of ATRA-associated Sweet's syndrome is of practical importance.

ROLE OF VITAMIN-D3 AND RETINOIC ACID IN A HUMAN THP-1 MACROPHAGE MODEL OF MYCOBACTERIUM TUBERCULOSIS INFECTION

Mycobacterium tuberculosis (Mtb) replicates within the human macrophages and we investigated the activating effects of retinoic acid (RA) and vitamin D3 (VD) on macrophages in relation to the viability of Mtb. A combination of these vitamins (RAVD) enhanced the receptors on THP-1 macrophage (Mannose receptor and DC-SIGN) that increased mycobacterial uptake but inhibited thesubsequent intracellular growth of Mtb by inducing reactive oxygen species (ROS) and autophagy. RAVD also enhanced antigen presenting and homing receptors in THPs that suggested an activated phenotype for THPs following RAVD treatment. RAVD mediated activation was also associated with a marked phenotypic change in Mtb infected THPs that fused with adjacent cells to formmultinucleate giant cells (MNGCs). Typically MNGCs occurred over 30 days of in vitro culture and contained non-replicating persisting Mtb for as long as 60 days in culture. We propose that the RAVD mediated inhibition of replicating Mtb leading to persistence of non-replicating Mtb within THPs may provide a novel human macrophage model simulating formation of MNGCs in humanlungs.

Regulation of mucin gene expression by CREB via a nonclassical retinoic acid signaling pathway.

Vitamin A and its metabolite retinoic acid (RA) are essential elements for normal lung development and the differentiation of lung epithelial cells. We previously showed that RA rapidly activated cyclic AMP response element-binding protein (CREB) in a nonclassical manner in normal human tracheobronchial epithelial (NHTBE) cells. In the present study, we further demonstrated that this nonclassical signaling of RA on the activation of CREB plays a critical role in regulating the expression of airway epithelial cell differentiation markers, the MUC2, MUC5AC, and MUC5B genes. We found that RA rapidly activates the protein kinase Calpha isozyme and transmits the activation signal to CREB via the Raf/MEK/extracellular signal-regulated kinase/p90 ribosomal S6 kinase (RSK) pathway. Activated RSK translocated from the cytoplasm to the nucleus, where it phosphorylates CREB. Activated CREB then binds to a cis-acting replication element motif on the promoter (at nucleotides [nt] -878 to -871) of the MUC5AC gene. The depletion of CREB using small interfering RNA abolished not only the RA-induced MUC5AC but also RA-induced MUC2 and MUC5B. Taken together, our findings demonstrate that CREB activation via this nonclassical RA signaling pathway may play an important role in regulating the expression of mucin genes and mediating the early biological effects of RA during normal mucous differentiation in NHTBE cells.

Sp1 up-regulates cAMP-response-element-binding protein expression during retinoic acid-induced mucous differentiation of normal human bronchial epithelial cells.

CREB [CRE (cAMP-response element)-binding protein] is an important transcription factor that is differentially regulated in cells of various types. We recently reported that RA (retinoic acid) rapidly activates CREB without using RARs (RA receptors) or RXRs (retinoid X receptors) in NHTBE cells (normal human tracheobronchial epithelial cells). However, little is known about the role of RA in the physiological regulation of CREB expression in the early mucous differentiation of NHTBE cells. In the present study, we report that RA up-regulates CREB gene expression and that, using 5'-serial deletion promoter analysis and mutagenesis analyses, two Sp1 (specificity protein 1)-binding sites located at nt -217 and -150, which flank the transcription initiation site, are essential for RA induction of CREB gene transcription. Furthermore, we found that CREs located at nt -119 and -98 contributed to basal promoter activity. Interestingly, RA also up-regulated Sp1 in a time- and dose-dependent manner. Knockdown of endogenous Sp1 using siRNA (small interfering RNA) decreased RA-induced CREB gene expression. However, the converse was not true: knockdown of CREB using CREB siRNA did not affect RA-induced Sp1 gene expression. We conclude that RA up-regulates CREB gene expression during the early stage of NHTBE cell differentiation and that RA-inducible Sp1 plays a major role in up-regulating human CREB gene expression. This result implies that co-operation of these two transcription factors plays a crucial role in mediating early events of normal mucous cell differentiation of bronchial epithelial cells.

Association of retinoic acid receptor genes with meningomyelocele.

BACKGROUND: Neural tube defects (NTDs) occur in as many as 0.5-2 per 1000 live births in the United States. One of the most common and severe neural tube defects is meningomyelocele (MM) resulting from failed closure of the caudal end of the neural tube. MM has been induced by retinoic acid teratogenicity in rodent models. We hypothesized that genetic variants influencing retinoic acid (RA) induction via retinoic acid receptors (RARs) may be associated with risk for MM. METHODS: We analyzed 47 single nucleotide polymorphisms (SNPs) that span across the three retinoic acid receptor genes using the SNPlex genotyping platform. Our cohort consisted of 610 MM families. RESULTS: One variant in the RARA gene (rs12051734), three variants in the RARB gene (rs6799734, rs12630816, rs17016462), and a single variant in the RARG gene (rs3741434) were found to be statistically significant at p < 0.05. CONCLUSION: RAR genes were associated with risk for MM. For all associated SNPs, the rare allele conferred a protective effect for MM susceptibility.

The molecular mechanism of retinoic acid action: Regulation of tissue transglutaminase gene expression

Retinoic acid is a small lipophilic molecule that exerts profound effects on the growth and differentiation of both normal and transformed cells. It is also a natural morphogen that is critical in the development of embryonic structures. The molecular effects of retinoic acid involve alterations in the expression of several proteins and these changes are presumably mediated in part by alterations in gene expression. For instance, retinoic acid causes a rapid induction of tissue transglutaminase, an enzyme involved in protein cross-linking. The molecular mechanisms responsible for the effects of retinoic acid on gene expression have not been characterized. To approach this question, I have isolated and characterized tissue transglutaminase of cDNA clones. The deduced amino acid sequences of tissue transglutaminase and of factor XIIIa showed a relatively high degree of homology in their putative calcium binding domains.^ To explore the mechanism of induction of this enzyme, both primary (macrophages) and cultured cells (Swiss 3T3-C2 and CHO fibroblasts) were used. I found that retinoic acid is a general inducer of tissue transglutaminase mRNA in these cells. In murine peritoneal macrophages retinoic acid causes a rapid accumulation of this mRNA and this effect is independent of concurrent protein synthesis. The retinoic acid effect is not mediated by a post-transcriptional increase in the stability of the tissue transglutaminase mRNA, but appears to involve an increase in the transcription rate of the tissue transglutaminase gene. This provides the first example of regulation by retinoic acid of a specific gene, supporting the hypothesis that these molecules act by directly regulating the transcriptional activity of specific genes. A molecular model for the effects of retinoic acid on the expression of genes linked to cellular proliferation and differentiation is proposed. ^

Molecular basis of retinoid action: The role of retinoic acid receptors in retinoic acid-induced tissue transglutaminase expression

Retinoic acid has profound effects on the cellular growth and differentiation of a variety of cells. However, the molecular basis of retinoic acid action has, until recently, not been well understood. The identification of retinoic acid receptors which bear a high degree of homology to members of the steroid receptor super-family has dramatically altered our understanding of the biology of retinoids. The focus of this dissertation has been toward identification of retinoic acid binding proteins responsible for the effects of this molecule on gene expression.^ We have characterized in detail the retinoic acid-dependent induction of tissue transglutaminase gene expression in a myeloid cell line, human promyelocytic leukemia cells (HL-60 cells). Using cDNA probes specific for tissue transglutaminase, we have determined that the retinoic acid induced increase in enzyme level is due to an increase in the level of tissue transglutaminase mRNA. We have used this model as a probe to investigate the molecular basis of retinoid regulated gene expression.^ This thesis demonstrates that retinoic acid receptors are expressed in cells which induce tissue transglutaminase expression in response to retinoic acid. In Hl-60 cells retinoic acid-induced transglutaminase expression is associated with saturable nuclear retonic acid binding. Transcripts for both the alpha and beta forms of the retinoic acid receptors can be detected in these cells. Pretreatment of HL-60 cells with agents that potentiate retinoic acid-induced transglutaminase expression also modestly induced the alpha form of the retinoic acid receptor. Studies in macrophages and umbilical vein endothelial cells have also associated expression of the beta form of the retinoic acid with retinoic acid induced tissue transglutaminase expression.^ To investigate directly if retinoic acid receptors regulate retinoic acid-induced tissue transglutaminase expression we developed a series of stably transfected Balb-c 3T3 cells expressing different levels of the beta or gamma form of the retinoic acid receptor. These studies indicated that either the beta or gamma receptor can stimulate endogenous tissue transglutaminase expression in response to retinoic acid. These are among the first studies in the steroid field to describe regulation of an endogenous gene by a transfected receptor. ^

Analysis of endogenous retinoic acid receptor expression and function in murine melanoma cells

There have been numerous reports over the past several years on the ability of vitamin A analogs (retinoids) to modulate cell proliferation, malignant transformation, morphogenesis, and differentiation in a wide variety of cell types and organisms. Two families of nuclear retinoid-inducible, trans-acting, transcription-enhancing receptors that bear strong DNA sequence homology to thyroid and steroid hormone receptors have recently been discovered. The retinoic acid receptors (RARs) and retinoid X receptors (RXRs) each have at least three types designated $\alpha,$ $\beta,$ and $\gamma,$ which are encoded by separate genes and expressed in a tissue and cell type-specific manner. We have been interested in the mechanism by which retinoids inhibit tumor cell proliferation and induce differentiation. As a model system we have employed several murine melanoma cell lines (S91-C2, K1735P, and B16-F1), which are sensitive to the growth-inhibitory and differentiation-inducing effects of RA, as well as a RA-resistant subclone of one of the cell lines (S91-C154), in order to study the role of the nuclear RARs in these effects. The initial phase of this project consisted of the characterization of the expression pattern of the three known RAR and RXR types in the murine melanoma cell lines in order to determine whether any differences exist which may elucidate a role for any of the receptors in RA-induced growth inhibition and differentiation. The novel finding was made that the RAR-$\beta$ gene is rapidly induced from undetectable levels by RA treatment at the mRNA and protein level, and that the induction of RAR-$\beta$ by other biologically active retinoids correlated with their ability to inhibit the growth of the highly RA-sensitive S91-C2 cell line. This suggests a role for RAR-$\beta$ in the growth inhibiting effect of retinoids. The second phase of this project involves the stable expression of RAR-$\beta$ in the S91-C2 cells and the RAR-$\beta$ receptor-null cell line, K1735P. These studies have indicated an inverse correlation between RAR-$\beta$ expression and proliferation rate. ^

Mechanisms of liposomal all-{\it trans\/} retinoic acid and vitamin D3 induced inhibition of cell proliferation and stimulation of apoptosis in aggressive B-cell non-Hodgkin's lymphoma

The Non-Hodgkin's Lymphoma (NHLs) are neoplasms of the immune system. Currently, less than 1% of the etiology of the 22,000 newly diagnosed lymphoma cases in the U.S.A. every year is known. This disease has a significant prevalence and high mortality rate. Cell growth in lymphomas has been shown to be an important parameter in aggressive NHL when establishing prognosis, as well as an integral part in the pathophysiology of the disease process. While many aggressive B cell NHLs respond initially to chemotherapeutic regimens such as CHOP-bleo (adriamycin, vincristine and bleomycin) etc., relapse is common, and the patient is then often refractory to further salvage treatment regimens.^ To assess their potential to inhibit aggressive B cell NHLs and induce apoptosis (also referred to as programmed cell death (PCD)), it was proposed to utilize the following biological agents-liposomal all-trans retinoic acid (L-ATRA) which is a derivative of Vitamin A in liposomes and Vitamin D3. Preliminary evidence indicates that L-ATRA may inhibit cell growth in these cells and may induce PCD as well. Detailed studies were performed to understand the above phenomena by L-ATRA and Vitamin D3 in recently established NHL-B cell lines and primary cell cultures. The gene regulation involved in the case of L-ATRA was also delineated. ^

Study of retinoic acid signaling in cancer cells: Cloning and characterization of retinoic acid responsive genes

Retinoids such as all-trans-retinoic acid (ATRA) are promising agents for cancer chemoprevention and therapy. ATRA can cause growth inhibition, induction of differentiation and apoptosis of a variety of cancer cells. These effects are thought to be mediated by nuclear retinoids receptors which are involved in ligand-dependent transcriptional activation of downstream target genes. Using differential display, we identified several retinoic acid responsive genes in the head and neck squamous carcinoma cells and lung cancer cells, including tissue type transglutaminase, cytochrome P450-related retinoic acid hydroxylase, and a novel gene, designated RAIG1. RAIG1 has two transcripts of 2.4 and 6.8 kbp, respectively, that are generated by alternative selection of polyadenylation sites. Both transcripts have the same open reading frame that encodes a protein comprised of 357 amino acid residues. The deduced RAIG1 protein sequence contains seven transmembrane domains, a signature structure of G protein-coupled receptors. RAIG1 mRNA is expressed at high level in fetal and adult lung tissues. Induction of RAIG1 expression by ATRA is rapid and dose-dependent. A fusion protein of RAIG1 and the green fluorescent protein was localized in the cell surface membrane and perinuclear vesicles in transiently transfected cells. The locus for RAIG1 gene was mapped to a region between D12S358 and D12S847 on chromosome 12p12.3-p13. Our study of the novel retinoic acid induced gene RAIG1 provide evidence for a possible interaction between retinoid and G protein signaling pathways.^ We further examined RAIG1 expression pattern in a panel of 84 cancer cell lines of different origin. The expression level varies greatly from very high to non-detectable. We selected a panel of different cancer cells to study the effects of retinoids and other differentiation agents. We observed: (1) In most cases, retinoids (including all-trans retinoic acid, 4HPR, CD437) could induce the expression of RAIG-1 in cells from cancers of the breast, colon, head and neck, lung, ovarian and prostate. (2) Compare to retinoids, butyrate is often a more potent inducer of RAIG-1 expression in many cancer cells. (3) Butyrate, Phenylacetate butyrate, (R)P-Butyrate and (S)P-Butyrate have different impact on RAIG1 expression which varies among different cell lines. Our results indicate that retinoids could restore RAIG1 expression that is down-regulated in many cancer cells.^ A mouse homologous gene, mRAIG1, was cloned by 5$\sp\prime$ RACE reaction. mRAIG1 cDNA has 2105 bp and shares 63% identity with RAIG1 cDNA. mRAIG1 encodes a polypeptide of 356 amino acid which is 76% identity with RAIG1 protein. mRAIG1 protein also has seven transmembrane domains which are structurally identical to those of RAIG1 protein. Only one 2.2 kbp mRAIG1 transcript could be detected. The mRAIG1 mRNA is also highly expressed in lung tissue. The expression of mRAIG1 gene could be induced by ATRA in several mouse embryonal carcinoma cells. The induction of mRAIG1 expression is associated with retinoic acid-induced neuroectoderm differentiation of P19 cells. Similarity in cDNA and protein sequence, secondary structure, tissue distribution and inducible expression by retinoic acid strongly suggest that the mouse gene is the homologue of the human RAIG1 gene. ^