All-trans Retinoic Acid Receptors Research Articles

All-trans retinoic acid improves NSD2-mediated RARα phase separation and efficacy of anti-CD38 CAR T-cell therapy in multiple myeloma

BackgroundImmunotherapies targeting CD38 have demonstrated salient efficacy in relapsed/refractory multiple myeloma (MM). However, loss of CD38 antigen and outgrowth of CD38 negative plasma cells have emerged as a major obstacle...

Abstract MP01: Retinoic Acid Receptor Alpha (Rarα) In Macrophages Protects From Diet-induced Atherosclerosis In Mice

Atherosclerosis is a chronic vascular disease caused by inflammation and accumulation of lipids in the blood vessels. Macrophages in atherosclerotic lesions participate in lipid accumulation giving rise to formation of foam cells and production of mediating inflammatory cytokines. This makes them an attractive target for therapy. Previous studies have shown that retinoid signaling plays a broader role in modulating macrophage lipid metabolism and its inflammatory phenotype. Our preliminary results showed that when macrophages (RAW267.4 cells and mouse peritoneal macrophages) were treated with all-trans retinoic acid or retinoic acid receptor alpha (Rarα)-specific agonist, cholesterol efflux genes like Abca1 And Abcg1 and anti-inflammatory gene like Arg-1 were significantly increased. We hypothesized that retinoic acid receptor alpha (Rarα) may play a role in macrophages to protect against diet-induced atherosclerosis. To test this hypothesis, we conducted in-vivo studies using macrophage-specific Rarα knockout mice. Rarα floxed mice were crossed with lysozyme-cre transgenic mice to generate macrophage specific knockout mice (m-Rarα -/- ). Rarα fl/fl (control) and m-Rarα -/- were given high fat/high cholesterol (HFHC) diet for 16 weeks. Intracellular triglyceride and cholesterol levels were significantly increased in peritoneal macrophages of m-Rarα -/- compared to the control. Analysis of macrophage gene expression showed increase in the expression of pro-inflammatory genes (Il-1β, Tnfα and Tgm-2) with decrease in genes involved in cholesterol efflux (Abca1 and Abcg1) and anti-inflammation (Arg-1 and Mrc-1) in m-Rarα -/- macrophages. This implies that loss of Rarα in macrophages can aggravate atherosclerosis. To test this hypothesis, we generated mice deficient in macrophage Rarα and low-density lipoprotein receptor and their control mice. These mice were then fed an HFHC diet for 16 weeks. The Oil Red O staining of aortas showed that mice lacking macrophage Rar had a significant increase in atherosclerotic plaques compared to the control mice. Our data demonstrate that Rarα in macrophages is protective from atherosclerosis.

Maternal vitamin A deficiency during pregnancy affects vascularized islet development

Vitamin A deficiency is known to affect 20 million pregnant women worldwide. However, the prenatal effects of maternal vitamin A deficiency on pancreas development have not been clearly determined. The present study examined how maternal vitamin A deficiency affects fetal islet development. Vitamin A-deficient mice were generated by feeding female mice with a chemically defined diet lacking vitamin A prior to mating as well as during pregnancy. We found that maternal vitamin A deficiency during pregnancy affected fetal pancreas development. Although the exocrine differentiation appeared normal, development of islet tissue was impaired. In the pancreas of neonatal mice, only a few endocrine cell clusters were formed, and these cell clusters lacked capillary endothelial cells. To further determine how vitamin A metabolites, such as retinoic acid, regulate vascularized islet development, ex vivo culture of embryonic pancreas either in the presence of 4-diethylaminobenzaldehyde (DEAB; an inhibitor of retinaldehyde dehydrogenase), all-trans retinoic acid (atRA) or retinoic acid receptor agonist (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthylenyl)-1-propenyl] benzoic acid (TTNPB) was carried out. We found that the addition of DEAB blocked vascularization and suppressed β-cell differentiation. Conversely, atRA or TTNPB promoted β-cell differentiation accompanied by enhanced expression of vascular basement component, laminin. We further demonstrated that atRA regulated vascularization via upregulating vascular endothelial growth factor-A (VEGF-A) secretion in embryonic pancreas and treatment with VEGF-A was able to partially rescue vascularization and β-cell differentiation in DEAB-treated embryonic pancreas cultures. The findings explain why maternal vitamin A deficiency affects fetal islet development and support an essential role of retinoid signaling in regulating vascularized islet development.

Retinoic Acid Receptors Control Spermatogonia Cell-Fate and Induce Expression of the SALL4A Transcription Factor.

All-trans retinoic acid (ATRA) is instrumental to male germ cell differentiation, but its mechanism of action remains elusive. To address this question, we have analyzed the phenotypes of mice lacking, in spermatogonia, all rexinoid receptors (RXRA, RXRB and RXRG) or all ATRA receptors (RARA, RARB and RARG). We demonstrate that the combined ablation of RXRA and RXRB in spermatogonia recapitulates the set of defects observed both upon ablation of RAR in spermatogonia. We also show that ATRA activates RAR and RXR bound to a conserved regulatory region to increase expression of the SALL4A transcription factor in spermatogonia. Our results reveal that this major pluripotency gene is a target of ATRA signaling and that RAR/RXR heterodimers are the functional units driving its expression in spermatogonia. They add to the mechanisms through which ATRA promote expression of the KIT tyrosine kinase receptor to trigger a critical step in spermatogonia differentiation. Importantly, they indicate also that meiosis eventually occurs in the absence of a RAR/RXR pathway within germ cells and suggest that instructing this process is either ATRA-independent or requires an ATRA signal originating from Sertoli cells.

Transcriptional regulation of Tal2 gene by all-trans retinoic acid (atRA) in P19 cells.

TAL2 is a transcription factor required in the normal development of mouse brain. In a previous study, we demonstrated that the expression of Tal2 gene is induced by the complex of all-trans retinoic acid (atRA) and retinoic acid receptor α (RARα) in mouse embryonal carcinoma P19 cells. atRA is also known to be important in inducing P19 cells to differentiate into the neural lineage. Therefore, we believe that the function of TAL2 in neural differentiation may be clarified by utilizing P19 cells. As the atRA-RARα complex induced the expression of Tal2, we focused on the regulatory region that is involved in its transcription. The atRA-RARα complex occupies a characteristic retinoic acid response element (RARE) located in the promoter of target genes. Therefore, we searched for RARE on the mouse Tal2 and found that a RARE-like element was located in the intron. We also found that a TATA-box-like element was located in the 5'-region of Tal2. Involvement between transcriptional activity and the TATA-box-like element was confirmed in the luciferase assay, and TATA-box binding protein was bound to this element upstream of Tal2 in P19 cells. atRA signaling activated the transcription through the RARE-like element, and RARα was bound to this element on Tal2 in P19 cells. In addition, the interaction between these elements on Tal2 was shown in the chromatin immunoprecipitation assay. These results suggest that the transcription of Tal2 is coordinately mediated by two distal regulatory elements.

Long-distance retinoid signaling in the zebra finch brain.

All-trans retinoic acid (ATRA), the main active metabolite of vitamin A, is a powerful signaling molecule that regulates large-scale morphogenetic processes during vertebrate embryonic development, but is also involved post-natally in regulating neural plasticity and cognition. In songbirds, it plays an important role in the maturation of learned song. The distribution of the ATRA-synthesizing enzyme, zRalDH, and of ATRA receptors (RARs) have been described, but information on the distribution of other components of the retinoid signaling pathway is still lacking. To address this gap, we have determined the expression patterns of two obligatory RAR co-receptors, the retinoid X receptors (RXR) α and γ, and of the three ATRA-degrading cytochromes CYP26A1, CYP26B1, and CYP26C1. We have also studied the distribution of zRalDH protein using immunohistochemistry, and generated a refined map of ATRA localization, using a modified reporter cell assay to examine entire brain sections. Our results show that (1) ATRA is more broadly distributed in the brain than previously predicted by the spatially restricted distribution of zRalDH transcripts. This could be due to long-range transport of zRalDH enzyme between different nuclei of the song system: Experimental lesions of putative zRalDH peptide source regions diminish ATRA-induced transcription in target regions. (2) Four telencephalic song nuclei express different and specific subsets of retinoid-related receptors and could be targets of retinoid regulation; in the case of the lateral magnocellular nucleus of the anterior nidopallium (lMAN), receptor expression is dynamically regulated in a circadian and age-dependent manner. (3) High-order auditory areas exhibit a complex distribution of transcripts representing ATRA synthesizing and degrading enzymes and could also be a target of retinoid signaling. Together, our survey across multiple connected song nuclei and auditory brain regions underscores the prominent role of retinoid signaling in modulating the circuitry that underlies the acquisition and production of learned vocalizations.

Inhibition Of LSD1 As a Therapeutic Strategy For The Treatment Of Acute Myeloid Leukemia

Lysine specific demethylase 1 (LSD1) is a histone H3K4me1/2 demethylase found in various transcriptional co-repressor complexes. These complexes include Histone Deacetylases (HDAC1/2) and Co-Repressor for Element-1-Silencing Transcription factor (CoREST). LSD1 mediated H3K4 demethylation can result in a repressive chromatin environment that silences gene expression. LSD1 has been shown to play a role in development in various contexts. LSD1 can interact with pluripotency factors in human embryonic stem cells and is important for decommissioning enhancers in stem cell differentiation. Beyond embryonic settings, LSD1 is also critical for hematopoietic differentiation. LSD1 is overexpressed in multiple cancer types and recent studies suggest inhibition of LSD1 reactivates the all-trans retinoic acid receptor pathway in acute myeloid leukemia (AML). These studies implicate LSD1 as a key regulator of the epigenome that modulates gene expression through post-translational modification of histones and through its presence in transcriptional complexes.The current study describes the anti-tumor effects of a novel LSD1 inhibitor (GSK2879552) in AML. GSK2879552 is a potent, selective, mechanism-based, irreversible inhibitor of LSD1. Screening of over 150 cancer cell lines revealed that AML cells have a unique requirement for LSD1. While LSD1 inhibition did not affect the global levels of H3K4me1 or H3K4me2, local changes in these histone marks were observed near transcriptional start sites of putative LSD1 target genes. This increase in the transcriptionally activating histone modification correlated with a dose dependent increase in gene expression. Treatment with GSK2879552 promoted the expression of cell surface markers, including CD11b and CD86, associated with a differentiated immunophenotype in 12 of 13 AML cell lines. For example, in SKM-1 cells, increases in cell surface expression of CD86 and CD11b occurred after as early as one day of treatment with EC50 values of 13 and 7 nM respectively. In a separate study using an MV-4-11 engraftment model, increases in CD86 and CD11b were observed as early as 8 hours post dosing.GSK2879552 treatment resulted in a potent anti-proliferative growth effect in 19 of 25 AML cell lines (average EC50 = 38 nM), representing a range of AML subtypes. Potent growth inhibition was also observed on AML blast colony forming ability in 4 out of 5 bone marrow samples derived from primary AML patient samples (average EC50 = 205 nM). The effects of LSD1 inhibition were further characterized in an in vivo mouse model of AML induced by transduction of mouse hematopoietic progenitor cells with a retrovirus encoding MLL-AF9 and GFP. Primary AML cells were transplanted into a cohort of secondary recipient mice and upon engraftment, the mice were treated for 17 days. After 17 days of treatment, control treated mice had 80% GFP+ cells in the bone marrow whereas treated mice possessed 2.8% GFP positive cells (p<0.012). The percentage of GFP+ cells continued to decrease to 1.8% by 1-week post therapy. Remarkably, in a preliminary assessment for survival, control-treated mice succumbed to AML by 28 days post transplant, while treated mice showed prolonged survival.Together, these data demonstrate that pharmacological inhibition of LSD1 may provide a promising treatment for AML by promoting differentiation and subsequent growth inhibition of AML blasts. GSK2879552 is currently in late preclinical development and clinical trials are anticipated to start in 2014.All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. Disclosures:Kruger:GlaxoSmithKline Pharmaceuticals: Employment. Mohammad:GlaxoSmithKline Pharmaceuticals: Employment. Smitheman:GlaxoSmithKline Pharmaceuticals: Employment. Liu:GlaxoSmithKline Pharmaceuticals: Employment. Pappalardi:GlaxoSmithKline Pharmaceuticals: Employment. Federowicz:GlaxoSmithKline Pharmaceuticals: Employment. Van Aller:GlaxoSmithKline Pharmaceuticals: Employment. Kasparec:GlaxoSmithKline Pharmaceuticals: Employment. Tian:GlaxoSmithKline Pharmaceuticals: Employment. Suarez:GlaxoSmithKline Pharmaceuticals: Employment. Rouse:GlaxoSmithKline Pharmaceuticals: Employment. Schneck:GlaxoSmithKline Pharmaceuticals: Employment. Carson:GlaxoSmithKline Pharmaceuticals: Employment. McDevitt:GlaxoSmithKline Pharmaceuticals: Employment. Ho:GlaxoSmithKline Pharmaceuticals: Employment. McHugh:GlaxoSmithKline Pharmaceuticals: Employment. Miller:GlaxoSmithKline Pharmaceuticals: Employment. Johnson:GlaxoSmithKline Pharmaceuticals: Employment. Armstrong:Epizyme Inc.: Has consulted for Epizyme Inc. Other. Tummino:GlaxoSmithKline Pharmaceuticals: Employment.

The relationship between renal cell carcinoma and nuclear retinoid/rexinoid receptors

Renal cell carcinoma (RCC) is a urologic malignancy with a steady rise in incidence and high mortality rate. Between 60 to 70% of patients with renal cell carcinoma can only be cured with surgery but despite advances in early diagnostis, in around 20-30% of cases there is metastasis. For these patients, chemotherapy and radiotherapy are ineffective and hence the prognosis is poor. Retinoids are biologically active compounds of either natural or synthetic origin that are involved in complex physiological and developmental processes in many tissues including cell proliferation and activation of tumour suppression genes. This article reviews the role of retinoids and their cognate nuclear retinoid/rexinoid receptors in relation to renal cell carcinoma. A literature search using ScienceDirect and Medline with a focus on the relationship between renal cell carcinoma and nuclear retinoid/rexinoid receptors. Use of retinoids/rexinoids in the treatment of locally advanced and metastatic RCC significantly prolongs median time of tumour progression and overall survival of patients. Combination therapy with other preparations has greater efficacy than treatment with retinoids alone. Patient survival can be predicted on the basis of the expression of different all-trans retinoic acid receptor (RAR) and 9-cis retinoic acid receptor (RXR) subtypes. Since nuclear retinoid receptors play acrucial role as ligand-activated, DNA binding, trans-acting, transcription-modulating proteins involved in ageneral molecular mechanism responsible for transcriptional responses in target genes, retinoids might be an alternative approach for the treatment of renal cell carcinoma.

The Elongation Complex Components BRD4 and MLLT3/AF9 Are Transcriptional Coactivators of Nuclear Retinoid Receptors

Nuclear all-trans retinoic acid receptors (RARs) initiate early transcriptional events which engage pluripotent cells to differentiate into specific lineages. RAR-controlled transactivation depends mostly on agonist-induced structural transitions in RAR C-terminus (AF-2), thus bridging coactivators or corepressors to chromatin, hence controlling preinitiation complex assembly. However, the contribution of other domains of RAR to its overall transcriptional activity remains poorly defined. A proteomic characterization of nuclear proteins interacting with RAR regions distinct from the AF-2 revealed unsuspected functional properties of the RAR N-terminus. Indeed, mass spectrometry fingerprinting identified the Bromodomain-containing protein 4 (BRD4) and ALL1-fused gene from chromosome 9 (AF9/MLLT3), known to associate with and regulates the activity of Positive Transcription Elongation Factor b (P-TEFb), as novel RAR coactivators. In addition to promoter sequences, RAR binds to genomic, transcribed regions of retinoid-regulated genes, in association with RNA polymerase II and as a function of P-TEFb activity. Knockdown of either AF9 or BRD4 expression affected differentially the neural differentiation of stem cell-like P19 cells. Clusters of retinoid-regulated genes were selectively dependent on BRD4 and/or AF9 expression, which correlated with RAR association to transcribed regions. Thus RAR establishes physical and functional links with components of the elongation complex, enabling the rapid retinoid-induced induction of genes required for neuronal differentiation. Our data thereby extends the previously known RAR interactome from classical transcriptional modulators to components of the elongation machinery, and unravel a functional role of RAR in transcriptional elongation.

Ecdysone- and NO-Mediated Gene Regulation by Competing EcR/Usp and E75A Nuclear Receptors during Drosophila Development

The Drosophila ecdysone receptor (EcR/Usp) is thought to activate or repress gene transcription depending on the presence or absence, respectively, of the hormone ecdysone. Unexpectedly, we found an alternative mechanism at work in salivary glands during the ecdysone-dependent transition from larvae to pupae. In the absense of ecdysone, both ecdysone receptor subunits localize to the cytoplasm, and the heme-binding nuclear receptor E75A replaces EcR/Usp at common target sequences in several genes. During the larval-pupal transition, a switch from gene activation by EcR/Usp to gene repression by E75A is triggered by a decrease in ecdysone concentration and by direct repression of the EcR gene by E75A. Additional control is provided by developmentally timed modulation of E75A activity by NO, which inhibits recruitment of the corepressor SMRTER. These results suggest a mechanism for sequential modulation of gene expression during development by competing nuclear receptors and their effector molecules, ecdysone and NO.

Thyroid non-Hodgkin’s lymphoma expression pattern of nuclear retinoid and rexinoid receptor subtypes

Correspondence to: Julius Brtko, Laboratory of Molecular Endocrinology, Institute of Experimental Endocrinology, Centre of Excellence Acknowledged by the European Commission, Slovak Academy of Sciences, Vlarska 3, 833 06 Bratislava, Slovakia E-mail: julius.brtko@savba.sk Thyroid non-Hodgkin’s lymphoma (TNHL) is an uncommon tumour, representing 2–8% of thyroid malignancies and approximately 1–2% of extranodal lymphomas. TNHL is a relative rare, and it occurs most frequently in elderly women and has been linked to Hashimoto’s thyroiditis. The female preponderance is due to its origin from chronic lymphocytic thyroiditis, which tends to occur in elderly women (Evans et al. 1995; Harrington et al. 2005). A number of controversies exist regarding the roles of surgery, radiotherapy and chemotherapy in the management of this disease (Ha et al. 2001). Surgery occupied a pre-eminent place in management of TNHL, in recent years, the appreciation that TNHL is sensitive to radiotherapy and chemotherapy has resulted in a move towards limited number of surgical interventions (Harrington et al. 2005). Retinoids, rexinoids and their biologically active derivatives are involved in a complex arrangement of physiological and developmental responses in many tissues of higher vertebrates. Both retinoids and rexinoids are either natural or synthetic compounds related to retinoic acids that act through interaction with two basic types of nuclear receptors belonging to the nuclear receptor superfamily: all-trans retinoic acid receptors, known as nuclear retinoid receptors (RARα, RARβ, and RARγ) and retinoid X receptors (RXRα, RXRβ and RXRγ) play a crucial role in both physiological and pathophysiological conditions as retinoid-inducible transcription factors (Brtko et al. 2009). Retinoids inhibit carcinogenesis, suppress premalignant epithelial lesions and tumour growth and invasion in a variety of tissues. Natural and synthetic retinoids exert important biological effects due to their antiproliferative and apoptosis-inducing effects. They are also known to cause redifferentiation or to prevent further dedifferentiation of various tumour tissues (Brtko and Thalhamer 2003; Brtko 2007). Acute promyelocytic leukemia (APL), characterized by its distinctive FAB-M3 morphology and its association with a bleeding coagulopathy, is known to be associated with at least four types of non-random reciprocal chromosomal translocation, but always involving the retinoic acid receptor α gene (RARα) on chromosome 17. The majority of APL patients with the promyelocytic gene-RARα fusion 21 and the rare cases of the nucleophosmin gene-RARα fusion 22 respond to retinoic acid-induced differentiation therapy; however, APL patients with the promyelocytic leukemia zinc finger gene-RARα fusion are unresponsive (So et al. 2000). Here, we report the case of a 67-year-old woman in Slovakia who was admited to the Saint Elizabeth Institute of Oncology in Bratislava with a neck stiffness. Magnetic resonance demonstrated thyroid neoplasia with the size of 52 × 63 × 90 mm. The pathological examination after surgery confirmed TNHL of unspecified origin. The patient after tyreoidectomy underwent radiation therapy with the total dose of 60.0 Gy. TNHL tissue from this patient has been analyzed in order to get information about expression of all known subtypes of nuclear retinoid and nuclear retinoid X receptors mediating the effects of retinoic acids at the nuclear/cellular level. Determination of mRNA levels encoding all isoforms of nuclear retinoid receptors (RARα, RARβ, RARγ) and nuclear retinoid X receptors (RXRα, RXRβ, RXRγ) has been performed by the reverse transcription and subsequent PCR analyses by previously established protocols in this laboratory (Szabova et al. 2003). The band intensities were measured using the STS 6220I Documentation System (Ultralum, USA) and normalized to the band intensity of PCR product corresponding to the house keeper gene GAPDH (Fig. 1). As shown in Fig. 1, the TNHL tissue excised from the patient thyroid expressed all isoforms of RARs or RXRs. Gen. Physiol. Biophys. (2010), 29, 411–413

Impact of DNA demethylation of the G0S2 gene on the transcription of G0S2 in squamous lung cancer cell lines with or without nuclear receptor agonists

We recently identified that DNA methylation of the G0S2 gene was significantly more frequent in squamous lung cancer than in non-squamous lung cancer. However, the significance of G0S2 methylation levels on cancer cells is not yet known. We investigated the effect of G0S2 methylation levels on cell growth, mRNA expression, and chromatin structure using squamous lung cancer cell lines and normal human bronchial epithelial cells. DNA methylation and mRNA expression of G0S2 were inversely correlated, and in one of the squamous lung cancer cell lines, LC-1 sq, G0S2 was completely methylated and suppressed. Overexpression of G0S2 in LC-1 sq did not show growth arrest or apoptosis. The G0S2 gene has been reported to be a target gene of all-trans retinoic acid and peroxisome proliferator-activated receptor agonists. We treated LC-1 sq with 5-Aza-2′-deoxycytidine, Trichostatin A, all-trans retinoic acid, Wy 14643, or Pioglitazone either alone or in combination. Only 5-Aza-2′-deoxycytidine restored mRNA expression of G0S2. Chromatin immunoprecipitation revealed that histone H3 lysine 9 was methylated regardless of DNA methylation or mRNA expression. In summary, mRNA expression of G0S2 was regulated mainly by DNA methylation in squamous lung cancer cell lines. When the G0S2 gene was methylated, nuclear receptor agonists could not restore mRNA expression of G0S2 and did not show any additive effect on mRNA expression of G0S2 even after the treatment with 5-Aza-2′-deoxycytidine.

RETINOIDS, REXINOIDS AND THEIR COGNATE NUCLEAR RECEPTORS: CHARACTER AND THEIR ROLE IN CHEMOPREVENTION OF SELECTED MALIGNANT DISEASES

Retinoids, rexinoids and their biologically active derivatives are involved in a complex arrangement of physiological and developmental responses in many tissues of higher vertebrates. Both retinoids and rexinoids are either natural or synthetic compounds related to retinoic acids that act through interaction with two basic types of nuclear receptors belonging to the nuclear receptor superfamily: All-trans retinoic acid receptors (RARalpha, RARbeta, and RARgamma) and retinoid X receptors (RXRalpha, RXRbeta and RXRgamma) as retinoid-inducible transcription factors. Summarization of selected effects of biologically active natural or synthetic retinoids and rexinoids and their exploitation in chemoprevention of various types of cancer. Retinoid receptors play a role as ligand-activated, DNA-binding, trans-acting, transcription-modulating proteins involved in a general molecular mechanism responsible for transcriptional responses in target genes. They exert both beneficial and detrimental activity; they have tumour-suppressive activity but on the other hand they are teratogenic. A number of nuclear receptor selective retinoids and rexinoids, have been successfully tested using a variety of cell lines or animal models. Retinoids inhibit carcinogenesis, suppress premalignant epithelial lesions and tumour growth and invasion in a variety of tissues. Natural and synthetic retinoids exert important biological effects due to their antiproliferative and apoptosis-inducing effects. They are also known to cause redifferentiation or to prevent further dedifferentiation of various tumour tissues.

13-cis Retinoic Acid Induces Apoptosis and Cell Cycle Arrest in Human SEB-1 Sebocytes

Isotretinoin (13-cis retinoic acid (13-cis RA)) is the most potent inhibitor of sebum production, a key component in the pathophysiology of acne, yet its mechanism of action remains largely unknown. The effects of 13-cis RA, 9-cis retinoic acid (9-cis RA), and all-trans retinoic acid (ATRA) on cell proliferation, apoptosis, and cell cycle proteins were examined in SEB-1 sebocytes and keratinocytes. 13-cis RA causes significant dose-dependent and time-dependent decreases in viable SEB-1 sebocytes. A portion of this decrease can be attributed to cell cycle arrest as evidenced by decreased DNA synthesis, increased p21 protein expression, and decreased cyclin D1. Although not previously demonstrated in sebocytes, we report that 13-cis RA induces apoptosis in SEB-1 sebocytes as shown by increased Annexin V-FITC staining, increased TUNEL staining, and increased cleaved caspase 3 protein. Furthermore, the ability of 13-cis RA to induce apoptosis cannot be recapitulated by 9-cis RA or ATRA, and it is not inhibited by the presence of a retinoid acid receptor (RAR) pan-antagonist AGN 193109. Taken together these data indicate that 13-cis RA causes cell cycle arrest and induces apoptosis in SEB-1 sebocytes by a RAR-independent mechanism, which contributes to its sebosuppressive effect and the resolution of acne.

Designed ATRA analogue active against ATRA-resistant acute promyelocytic leukemia cells having a single nucleotide substitution in their retinoic acid receptor

All- trans retinoic acid (ATRA) induces the differentiation of acute promyelocytic leukemia (APL) cells into neutrophils. UF-1 cells were established from an ATRA-resistant APL patient, and were previously shown to possess a single amino acid (or nucleotide) substitution, Arg276Trp, in their ATRA receptor. In the present research, we designed several ATRA derivatives having a hydrophobic alkyl ketone moiety instead of the negatively charged carboxylic acid moiety. Among them the ethyl ketone derivative, Et-ketone ATRA, was shown to induce the differentiation of UF-1 cells when assessed in terms of intracellular ROS production. It also induced the formation of PML NBs and expression of CD11b antigen marker and p21, transcriptional targets of RARα. Et-ketone ATRA did not induce these phenotypic changes in wild-type APL NB4 cells. Furthermore, we found that Et-ketone ATRA induced apoptosis selectively in UF-1 cells, i.e., not in other leukemic cells. The induction of apoptosis was shown to be partly due to the up-regulation of Bax protein. Thus, Et-ketone ATRA selectively induced differentiation and apoptosis in ATRA-resistant APL UF-1 cells, and is likely to be useful for the clinical treatment of the Arg276Trp-type of ATRA-resistant APL.

Increased expression of cyclin A1 protein is associated with all-trans retinoic acid-induced apoptosis

Deregulated cell growth and inhibition of apoptosis are hallmarks of cancer. All-trans retinoic acid induces clinical remission in patients with acute promyelocytic leukemia by inhibiting cell growth and inducing differentiation and apoptosis of the leukemic blasts. An important role of the cell cycle regulatory protein, cyclin A1, in the development of acute myeloid leukemia has previously been demonstrated in a transgenic mouse model. We have recently shown that there was a direct interaction between cyclin A1 and a major all-trans retinoic acid receptor, RARα, following all-trans retinoic acid treatment of leukemic cells. In the present study, we investigated whether cyclin A1 might be involved in all-trans retinoic acid-induced apoptosis in U-937 leukemic cells. We found that all-trans retinoic acid-induced apoptosis was associated with concomitant increase in cyclin A1 expression. However, there was no induction of cyclin A1 mRNA expression following the all-trans retinoic acid-induced apoptosis. Treatment of cells with a caspase inhibitor was not able to prevent all-trans retinoic acid-induced up-regulation of cyclin A1 expression. Interestingly, induced cyclin A1 expression in U-937 cells led to a significant increase in the proportion of apoptotic cells. Further, U-937 cells overexpressing cyclin A1 appeared to be more sensitive to all-trans retinoic acid-induced apoptosis indicating the ability of cyclin A1 to mediate all-trans retinoic acid-induced apoptosis. Induced cyclin E expression was not able to initiate cell death in U-937 cells. Our results indicate that cyclin A1 might have a role in apoptosis by mediating all-trans retinoic acid-induced apoptosis.

Coactivation of nuclear receptors and myogenic factors induces the major BTG1 influence on muscle differentiation

The btg1 (B-cell translocation gene 1) gene coding sequence was isolated from a translocation break point in a case of B-cell chronic lymphocytic leukaemia. We have already shown that BTG1, considered as an antiproliferative protein, strongly stimulates myoblast differentiation. However, the mechanisms involved in this influence remained unknown. In cultured myoblasts, we found that BTG1 stimulates the transcriptional activity of nuclear receptors (T3 and all-trans retinoic acid receptors but not RXRalpha and PPARgamma), c-Jun and myogenic factors (CMD1, Myf5, myogenin). Immunoprecipitation experiments performed in cells or using in vitro-synthesized proteins and GST pull-down assays established that BTG1 directly interacts with T3 and all-trans retinoic acid receptors and with avian MyoD (CMD1). These interactions are mediated by the transactivation domain of each transcription factor and the A box and C-terminal part of BTG1. NCoR presence induces the ligand dependency of the interaction with nuclear receptors. Lastly, deletion of BTG1 interacting domains abrogates its ability to stimulate nuclear receptors and CMD1 activity, and its myogenic influence. In conclusion, BTG1 is a novel important coactivator involved in the regulation of myoblast differentiation. It not only stimulates the activity of myogenic factors, but also of nuclear receptors already known as positive myogenic regulators.

Retinoids: Inducers of Tumor/Growth Suppressors

The process of carcinogenesis involves genomic disasters that result in inactivation or underexpression of tumor/growth suppressor genes and/or overexpression of oncogenes. Decreased levels of tumor suppressors may result from DNA hypermethylation, mutations, translocation, or transcriptional repression. Therefore, logically, one of the most promising chemopreventive and chemotherapeutic targets appears to be the activation of tumor suppressors. The expression of tumor suppressor genes could be activated by reversal of epigenetic gene silencing by using DNA methyltransferase inhibitors and/or histone deacetylase inhibitors (Zelent et al, 2004). Alternatively, the expression of multiple tumor suppressor genes in cancer cells could be induced by ligand-dependent transcription factors, such as nuclear receptors for the active form of vitamin A, all-trans retinoic acid (ATRA) that belong to the superfamily of steroid/thyroid hormone nuclear receptors. Retinoids (ATRA and its synthetic analogs) exert their anti-proliferative and differentiation effects through retinoic acid receptors (RAR) that function as heterodimers with retinoid X receptors (RXR). RXR, a nuclear receptor for 9-cis retinoic acid, is an obligate partner of RAR in mediating retinoid action. Three distinct isotypes of RAR (, and ) and RXR (, and ) are encoded by separate genes. The RXR–RAR heterodimer binds to retinoic acid response elements (RARE) present in the promoter regions of responsive genes. RARE are direct repeat (DR) of 5'-RGKTCA-3' motifs (where R is A or G and K is G or T) separated by either two (DR-2) or five (DR-5) nucleotides (Nagpal and Chandraratna, 1998).

Regulation of the cyclin A1 protein is associated with its differential subcellular localization in hematopoietic and leukemic cells.

An important role of the cell cycle regulatory protein cyclin A1 in the development of acute myeloid leukemia (AML) was previously demonstrated in a transgenic mouse model. We have now turned our attention to study specific aspects of the activity and subcellular distribution of cyclin A1 using bone marrow samples from normal donors and patients with AML, as well as leukemic cell lines. We show that the localization of cyclin A1 in normal hematopoietic cells is nuclear, whereas in leukemic cells from AML patients and cell lines, it is predominantly cytoplasmic. In leukemic cell lines treated with all-trans retinoic acid (ATRA), cyclin A1 localized to the nucleus. Further, there was a direct interaction between cyclin A1 and cyclin-dependent kinase 1, as well as a major ATRA receptor, RARalpha, in ATRA-treated cells but not in untreated leukemic cells. Our results indicate that the altered intracellular distribution of cyclin A1 in leukemic cells correlates with the status of the leukemic phenotype.

Expression of nuclear retinoic acid receptor in peripheral blood mononuclear cells (PBMC) of healthy subjects

In vertebrates, both nuclear all-trans and 9-cis retinoic acid receptors (RAR and RXR) belonging to the steroid/thyroid/retinoid nuclear receptor superfamily play a crucial role in the vitamin A action. Qualitative analysis of all known RAR or RXR subtypes in both pooled and non-pooled peripheral blood mononuclear cells (PBMC) from healthy human subjects has been performed by reverse transcription and polymerase chain reaction (RT–PCR). Our data, based on qualitative RT–PCR analysis has shown that human PBMC are capable to express RAR α, RAR γ, RXR α, and RXR β.