RESUMEN

Evaluation of retinoic acid receptor (RAR) subtype-selective alpha and gamma agonists and antagonists and a retinoid X receptor (RXR) class-selective agonist for efficacy at inhibiting both induction of ornithine decarboxylase (ODC) by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse epidermis and rat tracheal epithelial cells and the appearance of papillomas in mouse epidermis treated in the 2-stage tumor initiation-promotion model indicated that (i) RXR class-selective transcriptional agonists, such as MM11246, were not involved in ODC inhibition; (ii) RAR-selective agonists that induce gene transcription from RA-responsive elements (RAREs) were active at low concentrations; (iii) RAR-selective antagonists that bind RARs and inhibit AP-1 activation on the collagenase promoter but do not activate RAREs to induce gene transcription were less effective inhibitors; and (iv) RARgamma-selective retinoid agonists were more effective inhibitors of TPA-induced ODC activity than RARalpha-selective agonists. These results suggest that RARE activation has a more important role in inhibition of ODC activity than RXR activation or AP-1 inhibition and that RARgamma-selective agonists would be the most useful inhibitors of epithelial cell proliferation induced by tumor promoters. The natural retinoid all-trans-RA induced expression of transcription factor ZBP-89, which represses activation of the GC box in the ODC promoter by the transcription factor Sp1.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Inhibidores de la Ornitina Descarboxilasa , Receptores de Ácido Retinoico/metabolismo , Retinoides/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Factores de Transcripción/fisiología , 9,10-Dimetil-1,2-benzantraceno , Animales , Antineoplásicos/farmacología , Northern Blotting , Western Blotting , Carcinógenos , Supervivencia Celular/efectos de los fármacos , Colagenasas/genética , Relación Dosis-Respuesta a Droga , Epidermis/metabolismo , Células Epiteliales/metabolismo , Femenino , Células HeLa , Humanos , Ratones , Ratones Pelados , Neoplasias Experimentales/metabolismo , Papiloma/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Quinasas/metabolismo , ARN Mensajero/metabolismo , Ratas , Receptores de Ácido Retinoico/química , Elementos de Respuesta , Receptor alfa de Ácido Retinoico , Retinoides/farmacología , Factores de Tiempo , Tráquea/metabolismo , Factor de Transcripción AP-1/antagonistas & inhibidores , Transcripción Genética , Activación Transcripcional , Transfección , Rayos Ultravioleta , Receptor de Ácido Retinoico gamma

RESUMEN

The role of the active metabolite of vitamin D, 1,25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), in cell differentiation is well established. However, its use as a differentiating agent in a clinical setting is precluded due to its hypercalcaemic activity. Recently, we synthesised a relatively non-calcaemic analogue of vitamin D(5), 1alpha-hydroxyvitamin D(5) (1alpha(OH)D(5)), which inhibited the development of carcinogen-induced mammary lesions in culture and suppressed the incidence of chemically induced mammary carcinogmas in rats. In the present study, we determined the differentiating effects of 1alpha-(OH)D(5) in T47D human breast cancer cells and compared its effects with 1,25(OH)(2)D(3). Cells incubated with either 10 or 100 nM of the analogues inhibited cell proliferation in a dose-dependent manner, as measured by the dimethylthiazolyl-2,5-diphenyltetrazolium bromide (MTT) assay. Similar growth-inhibitory effects were also observed for MCF10(neo) cells. Both vitamin D analogues induced cell differentiation, as determined by induction of casein expression and lipid production. However, MCF10(neo) cells failed to respond to either vitamin D analogue and did not undergo cell differentiation. Since the cell differentiating effect of vitamin D is considered to be mediated via the vitamin D receptor (VDR), we examined the induction of VDR using reverse transcriptase-polymerase chain reaction (RT-PCR) in both cells. The results showed that, in T47D cells, both 1,25(OH)(2)D(3) and 1alpha(OH)D(5) induced VDR in a dose-dependent manner. Moreover, both analogues of vitamin D upregulated the expression of vitamin D response element-chloramphenicol acetyl transferase (VDRE-CAT). These results collectively indicate that 1alpha-(OH)D(5) may mediate its cell-differentiating action via VDR in a manner similar to that of 1,25(OH)(2)D(3).

Asunto(s)

Neoplasias de la Mama/patología , Hidroxicolecalciferoles/farmacología , Receptores de Calcitriol/metabolismo , Calcitriol/farmacología , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Femenino , Expresión Génica , Humanos , Hidroxicolecalciferoles/metabolismo , ARN Mensajero/genética , Receptores de Calcitriol/genética , Elementos de Respuesta , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Activación Transcripcional/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacos

RESUMEN

Phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and all-trans-retinoic acid (trans-RA) are potent regulators of growth of cancer cells. In this study, we investigated the effect of TPA and trans-RA alone or their combination on proliferation of human breast cancer ZR75-1 and T47D and lung cancer H460 and H292 cell lines. trans-RA caused various degrees of growth inhibition of these cell lines. However, TPA showed inhibition of proliferation of H460 and H292 cells and induction of ZR75-1 cell growth. Although trans-RA did not significantly regulate the growth inhibitory effect of TPA, it completely prevented its growth stimulating function. The divergent effects of TPA were associated with specific disruption of cell cycle events, an induction of G(0)/G(1) arrest in H460 and H292 cells and inhibition of G(0)/G(1) arrest with increase of S phase in ZR75-1 cells. Induction of G(0)/G(1) arrest was accompanied by induction of p21(WAF1) and ERK activity, whereas inhibition of G(0)/G(1) arrest was associated with enhanced activity of JNK and AP-1 but not ERK. trans-RA did not affect TPA-induced p21(WAF1) expression. However, it inhibited TPA-induced AP-1 activity in ZR75-1 cells and the constitutive AP-1 activity in H460 and H292 cells. Thus, trans-RA modulates TPA activity through its interaction through TPA-induced JNK/AP-1 pathway but not TPA-induced ERK/p21(WAF1) pathway.

Asunto(s)

División Celular/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Tretinoina/farmacología , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor de Transcripción AP-1/antagonistas & inhibidores , Factor de Transcripción AP-1/metabolismo , Células Tumorales Cultivadas

RESUMEN

Four candidate retinoid antagonists (LE135, LE511, LE540, and LE550) were designed on the basis of the ligand superfamily concept and synthesized. Analysis of these related retinoids by transient transfection assay demonstrated that LE135, LE540, and LE550 are effective retinoic acid receptor (RAR) antagonists, whereas LE511 selectively induced RARbeta transcriptional activity. Both LE135 and LE540 inhibited retinoic acid (RA)-induced transcriptional activation of RARbeta, but not RARalpha, RARgamma or retinoid X receptor alpha (RXRalpha), on a variety of RA response elements. The retinoid antagonists also inhibited all-trans-RA-induced transcriptional activation of RARbeta/RXRalpha heterodimers, although they did not show any effect on transactivation activity of RXR/RXR homodimers. In ZR-75-1 human breast cancer cells, cotreatment of LE135 and LE540 with all-trans-RA inhibited all-trans-RA-induced apoptosis of the cells, further demonstrating that RARbeta plays a role in RA-induced apoptosis of breast cancer cells. We also evaluated the effect of these retinoids on AP-1 activity. Our data showed that LE135 and LE540 strongly repressed 12-O-tetradecanoylphorbol-13-acetate-induced AP-1 activity in the presence of RARbeta and RXRalpha. Interestingly, LE550 induced AP-1 activity when RARbeta and RXRalpha were expressed in HeLa cells but not in breast cancer cells. These results demonstrate that LE135 and LE540 were a novel class of RARbeta-selective antagonists and anti-AP-1 retinoids and should be useful tools for studying the role of retinoids and their receptors.

Asunto(s)

Receptores de Ácido Retinoico/antagonistas & inhibidores , Retinoides/farmacología , Factor de Transcripción AP-1/antagonistas & inhibidores , Tretinoina/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Benzoatos/farmacología , Benzodiazepinas/farmacología , Neoplasias de la Mama , Colagenasas/genética , Dimerización , Diseño de Fármacos , Genes Reporteros/genética , Células HeLa , Humanos , Regiones Promotoras Genéticas/efectos de los fármacos , Receptores de Ácido Retinoico/genética , Receptores X Retinoide , Retinoides/química , Acetato de Tetradecanoilforbol/farmacología , Factores de Transcripción/antagonistas & inhibidores , Activación Transcripcional , Transfección , Células Tumorales Cultivadas

RESUMEN

Vitamin D analogs are effective inhibitors of breast cancer cell growth, but many breast cancer cell lines show various degrees of resistance to the growth inhibitory effect of vitamin D. In this study, we investigated the mechanism of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] resistance of the human breast epithelial cell line HBL100, which had been immortalized by Simian virus 40 (SV40) large T antigen. We determined the expression, DNA binding and transactivation activity of vitamin D3 receptor (VDR) in HBL100 and a vitamin D-sensitive ZR75-1 breast cancer cell line. Western blot analysis revealed a comparable expression of VDR gene in both cell lines. However, gel retardation assays demonstrated nuclear proteins from ZR75-1 cells but not from HBL100; cells expressed a 9-fold increase in the binding activity with a vitamin D response element (VDRE). Using a transient transfection assay, we showed that the VDRE was activated by 8-fold in ZR75-1. However, in HBL100 cells there was no activation observed in response to 1,25(OH)2D3. On the other hand, co-transfection of a VDR expression vector could restore 1,25(OH)2D3-induced VDRE transcription in HBL100 cells. Moreover, stable expression of VDR in HBL100 cells resulted in enhanced sensitivity of the cells to the growth inhibitory effect of 1,25(OH)2D3. Since CV-1 cells express very little endogenous VDR, the interactions of VDR and large T antigen were carried out in these cells. By transient co-transfection, we observed that expression of the large T antigen strongly inhibited 1,25(OH)2D3-induced VDRE transcriptional activity in a dose-dependent fashion in CV-1 cells. At 120 ng VDR concentration, the inhibition was completely reversed. Thus the loss of the growth inhibitory effect of vitamin D3 in HBL100 cells may be caused by the expression of the large T antigen in the cells, and provide further evidence that VDR is required for efficient growth inhibition by vitamin D3.

Asunto(s)

Neoplasias de la Mama/patología , Mama/efectos de los fármacos , Calcitriol/farmacología , Receptores de Calcitriol/metabolismo , Antígenos Transformadores de Poliomavirus/genética , Antígenos Transformadores de Poliomavirus/fisiología , Mama/citología , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Resistencia a Medicamentos , Células Epiteliales/efectos de los fármacos , Femenino , Humanos , Proteínas Nucleares/metabolismo , Receptores de Calcitriol/efectos de los fármacos , Receptores de Calcitriol/genética , Receptores de Ácido Retinoico/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Secuencias Reguladoras de Ácidos Nucleicos , Receptores X Retinoide , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Virus 40 de los Simios/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Transfección , Células Tumorales Cultivadas/efectos de los fármacos

RESUMEN

6-[3-(1-Adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN or CD437), originally identified as a retinoic acid receptor gamma-selective retinoid, was previously shown to induce growth inhibition and apoptosis in human breast cancer cells. In this study, we investigated the role of AHPN/CD437 and its mechanism of action in human lung cancer cell lines. Our results demonstrated that AHPN/CD437 effectively inhibited lung cancer cell growth by inducing G0/G1 arrest and apoptosis, a process that is accompanied by rapid induction of c-Jun, nur77, and p21(WAF1/CIP1). In addition, we found that expression of p53 and Bcl-2 was differentially regulated by AHPN/CD437 in different lung cancer cell lines and may play a role in regulating AHPN/CD437-induced apoptotic process. On constitutive expression of the c-JunAla(63,73) protein, a dominant-negative inhibitor of c-Jun, in A549 cells, nur77 expression and apoptosis induction by AHPN/CD437 were impaired, whereas p21(WAF1/CIP1) induction and G0/G1 arrest were not affected. Furthermore, overexpression of antisense nur77 RNA in A549 and H460 lung cancer cell lines largely inhibited AHPN/CD437-induced apoptosis. Thus, expression of c-Jun and nur77 plays a critical role in AHPN/CD437-induced apoptosis. Together, our results reveal a novel pathway for retinoid-induced apoptosis and suggest that AHPN/CD437 or analogs may have a better therapeutic efficacy against lung cancer.

Asunto(s)

Apoptosis , Inhibidores de Crecimiento/farmacología , Retinoides/farmacología , Carcinoma de Pulmón de Células no Pequeñas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Fase G1 , Humanos , Neoplasias Pulmonares , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Receptores Citoplasmáticos y Nucleares , Receptores de Esteroides , Fase de Descanso del Ciclo Celular , Factores de Transcripción/biosíntesis , Tretinoina/farmacología , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/biosíntesis

RESUMEN

Retinoids regulate the growth and differentiation of human tracheobronchial epithelial cells. In this study, we investigated the effects of all-trans-retinoic acid (trans-RA) and receptor class-selective retinoids on the growth and apoptosis of human lung cancer cell lines. Trans-RA significantly inhibited the growth of Calu-6 and H460 cells, accompanied by induction of RA receptor (RAR) beta expression. In contrast, it had little effect on the growth of H292, SK-MES-1 and H661 lung cancer cell lines, in which RAR beta expression was not induced. Stable expression of RAR beta in RAR beta-negative, trans-RA-resistant SK-MES-1 and H661 lung cancer cells led to recovery of trans-RA-induced growth inhibition, which occurred, however, only at low serum concentration. Using fluorescent microscopy and the terminal deoxyribonucleotidyl transferase (TdT) assay, we demonstrated that induction of apoptosis by trans-RA contributed to its growth-inhibitory effect in trans-RA-sensitive lung cancer cell lines. Analysis of RAR-selective and retinoid X receptor (RXR)-selective retionoids showed that activation of both RARs and RXRs could induce growth inhibition in trans-RA-sensitive lung cancer cells. Also, an additive synergistic effect on growth inhibition and RAR beta induction was observed when cells were treated with combinations of RAR-selective and RXR-selective retinoids. Together, our results show that expression of RAR beta plays a role in mediating retinoid response in lung cancer cells and that activation of RARs or RXRs contributes to induction of RAR beta, growth inhibition and apoptosis by retinoids.

Asunto(s)

Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Receptores de Ácido Retinoico/efectos de los fármacos , Tretinoina/farmacología , División Celular/efectos de los fármacos , Humanos , Receptores de Ácido Retinoico/metabolismo , Transducción de Señal , Células Tumorales Cultivadas/efectos de los fármacos , Regulación hacia Arriba

RESUMEN

All-trans-retinoic acid (trans-RA) and other retinoids exert anticancer effects through two types of retinoid receptors, the RA receptors (RARs) and retinoid X receptors (RXRs). Previous studies demonstrated that the growth-inhibitory effects of trans-RA and related retinoids are impaired in certain estrogen-independent breast cancer cell lines due to their lower levels of RAR alpha and RARbeta. In this study, we evaluated several synthetic retinoids for their ability to induce growth inhibition and apoptosis in both trans-RA-sensitive and trans-RA-resistant breast cancer cell lines. Our results demonstrate that RXR-selective retinoids, particularly in combination with RAR-selective retinoids, could significantly induce RARbeta and inhibit the growth and induce the apoptosis of trans-RA-resistant, RAR alpha-deficient MDA-MB-231 cells but had low activity against trans-RA-sensitive ZR-75-1 cells that express high levels of RAR alpha. Using gel retardation and transient transfection assays, we found that the effects of RXR-selective retinoids on MDA-MB-231 cells were most likely mediated by RXR-nur77 heterodimers that bound to the RA response element in the RARbeta promoter and activated the RARbeta promoter in response to RXR-selective retinoids. In contrast, growth inhibition by RAR-selective retinoids in trans-RA-sensitive, RAR alpha-expressing cells most probably occurred through RXR-RAR alpha heterodimers that also bound to and activated the RARbeta promoter. In MDA-MB-231 clones stably expressing RAR alpha, both RARbeta induction and growth inhibition by RXR-selective retinoids were suppressed, while the effects of RAR-selective retinoids were enhanced. Together, our results demonstrate that activation of RXR can inhibit the growth of trans-RA-resistant MDA-MB-231 breast cancer cells and suggest that low cellular RAR alpha may regulate the signaling switch from RAR-mediated to RXR-mediated growth inhibition in breast cancer cells.

Asunto(s)

Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Receptores de Ácido Retinoico/metabolismo , Retinoides/farmacología , Factores de Transcripción/metabolismo , Tretinoina/farmacología , Apoptosis/efectos de los fármacos , Unión Competitiva , División Celular/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Resistencia a Medicamentos , Estudios de Evaluación como Asunto , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Isomerismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Receptores Citoplasmáticos y Nucleares , Receptores de Esteroides , Receptores X Retinoide , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas

RESUMEN

Retinyl methyl ether (RME) is known to prevent the development of mammary cancer. However, the mechanism by which RME exerts its anticancer effect is presently unclear. The diverse biological functions of retinoids, the vitamin A derivatives, are mainly mediated by their nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RARs and RXRs are ligand-dependent transcriptional factors that either activate gene transcription through their binding to retinoic acid response elements or repress transactivation of genes containing the activator protein 1 (AP-1) binding site. Previous studies demonstrated that RME can modulate transcriptional activity of retinoid receptors on retinoic acid response elements, suggesting that regulation of retinoid receptor activity may mediate the anticancer effect of RME. In this study, we present evidence that RME can down-regulate AP-1 activity induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, insulin, growth factors, and the nuclear proto-oncogenes c-Jun and c-Fos. Transient transfection assays demonstrate that inhibition of AP-1 activity occurs on the human collagenase promoter containing an AP-1 binding site or the thymidine kinase promoter linked with an AP-1 binding site. In HeLa cells, the inhibition is observed when RAR-alpha and/or RXR-alpha but not RAR-beta or RAR-gamma expression vectors are cotransfected, whereas the endogenous retinoid receptors in breast cancer cells T-47D and ZR-75-1 were sufficient to confer the inhibition by RME. Furthermore, using gel retardation assay, we show that 12-O-tetradecanoylphorbol-13-acetate- and epidermal growth factor-induced AP-1 binding activity in breast cancer cells is inhibited by RME. These results suggest that one of the mechanisms by which RME prevents cancer development may be due to the repression of AP-1-responsive genes.

Asunto(s)

Neoplasias de la Mama/metabolismo , Regulación hacia Abajo , Receptores de Ácido Retinoico/efectos de los fármacos , Factor de Transcripción AP-1/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Vitamina A/análogos & derivados , Neoplasias de la Mama/patología , Carcinógenos/antagonistas & inhibidores , Colagenasas/efectos de los fármacos , Colagenasas/metabolismo , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Activación Enzimática/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Genes Reporteros/efectos de los fármacos , Células HeLa , Humanos , Proteínas Proto-Oncogénicas c-fos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-jun/metabolismo , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Receptores X Retinoide , Acetato de Tetradecanoilforbol/antagonistas & inhibidores , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Células Tumorales Cultivadas , Vitamina A/farmacología

RESUMEN

The diverse function of retinoic acid (RA) is mediated by its nuclear receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). However, the RA response is often lost in cancer cells that express the receptors. Previously, it was demonstrated that the RA response is regulated by the COUP-TF orphan receptors. Here, we present evidence that nur77, another orphan receptor whose expression is highly induced by phorbol esters and growth factors, is involved in modulation of the RA response. Expression of nur77 enhances ligand-independent transactivation of RA response elements (RAREs) and desensitizes their RA responsiveness. Conversely, expression of COUP-TF sensitizes RA responsiveness of RAREs by repressing their basal transactivation activity. Unlike the effect of COUP-TFs, the function of nur77 does not require direct binding of nur77 to the RAREs, but is through interaction between nur77 and COUP-TFs. The interaction occurs in solution and results in inhibition of COUP-TF RARE binding and transcriptional activity. Unlike other nuclear receptors, a large portion of the carboxy-terminal end of nur77 is not required for its interaction with COUP-TF. In human lung cancer cell lines, COUP-TF is highly expressed in RA-sensitive cell lines while nur77 expression is associated with RA resistance. Stable expression of COUP-TF in nur77-positive, RA-resistant lung cancer cells enhances the inducibility of RARbeta gene expression and growth inhibition by RA. These observations demonstrate that a dynamic equilibrium between orphan receptors nur77 and COUP-TF, through their heterodimerization that regulates COUP-TF RARE binding, is critical for RA responsiveness of human lung cancer cells.

Asunto(s)

Antineoplásicos/toxicidad , Proteínas de Unión al ADN/fisiología , Factores de Transcripción/fisiología , Tretinoina/toxicidad , Animales , Sitios de Unión , Factor de Transcripción COUP I , Línea Celular , Chlorocebus aethiops , Clonación Molecular , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/efectos de los fármacos , Dimerización , Expresión Génica/efectos de los fármacos , Glutatión Transferasa/biosíntesis , Humanos , Neoplasias Pulmonares , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Receptores Citoplasmáticos y Nucleares , Receptores de Glucocorticoides/fisiología , Receptores de Esteroides/fisiología , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/biosíntesis , Factores de Transcripción/efectos de los fármacos , Transfección , Células Tumorales Cultivadas

RESUMEN

PURPOSE: This study investigated the in vitro pharmacologic behavior and disposition kinetics of all-trans retinoic acid (ATRA) in acute myeloid leukemic (AML) cells, their sensitivity to its differentiating effect, and the in vivo response of acute promyelocytic leukemia (APL) patients after therapy. PATIENTS AND METHODS: Fresh leukemic cells from 14 AML patients (nine APL and five non-APL), were incubated in suspension culture in the absence or presence of 10(-6) mol/L ATRA. Intracellular ATRA concentration and ATRA metabolism was determined by high-performance liquid chromatography (HPLC). RESULTS: Immediate uptake is observed with maximal intracellular levels (Cmax) achieved after 24 hours of incubation. At this time, ATRA levels were variable, ranging from 20 to 230 pmol/10(6) cells (median, 100 pmol/10(6) cells). Comparison of ATRA intracellular levels with the in vitro response of patients' cell samples as measured by the percentage of nitro blue tetrazolium (NBT)-positive cells after a 3-day incubation period allowed us to discriminate a group of APL patients (n = 6) with high Cmax (group A; median, 200 pmol/10(6) cells) and maximal differentiation at day 3 (median, 80%), and a group of patients (n = 8, three APL and five non-APL) with low Cmax (group B; median, 35 pmol/10(6) cells) and poor in vitro response (median, 40%; APL cases only). Interestingly, all APL patients, except one included in group A (rapid in vitro ATRA uptakers), achieved a complete remission. CONCLUSION: These findings suggest that intracellular ATRA concentrations are determinant for ATRA response and should be taken into account when monitoring the efficacy of ATRA differentiation therapeutic trials in malignant disorders.

Asunto(s)

Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Leucemia Promielocítica Aguda/tratamiento farmacológico , Tretinoina/farmacocinética , Tretinoina/uso terapéutico , Adolescente , Adulto , Anciano , Disponibilidad Biológica , Cromatografía Líquida de Alta Presión , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Estudios de Seguimiento , Humanos , Técnicas In Vitro , Leucemia Promielocítica Aguda/metabolismo , Masculino , Persona de Mediana Edad , Inducción de Remisión , Resultado del Tratamiento , Células Tumorales Cultivadas

RESUMEN

The diverse effects of all-trans retinoic acid (ATRA) on growth, differentiation and homeostasis of vertebrate organisms are mediated by three distinct isoforms of retinoic acid receptors (RARs). Although it is not known to what extent each RAR contributes to the different effects of ATRA, several studies have demonstrated that ATRA induced granulocytic differentiation in human myeloid leukemic cell lines is mediated by RAR alpha. In this study, we investigated ATRA binding affinity of the endogenous nuclear receptors of HL-60 and NB4 leukemic cells. Scatchard plot analysis yielded an apparent dissociation constant of 5 +/- 0.3 nM and 1400 +/- 80 receptor sites per cell in HL-60 cells, whereas the NB4 promyelocytic leukemic cell line showed a lower affinity (8.5 +/- 0.5 nM and 900 +/- 30 receptor sites per cell). Modulation of RAR alpha protein (5 fold excess) was found in NB4 cells after 24 hours ATRA exposure, whereas HL-60 cells required a 72-hour culture period to weakly increase the RAR alpha protein level. These data were closely related to the ATRA intracellular concentration and kinetics of terminal differentiation of the cells.

Asunto(s)

Receptores de Ácido Retinoico/metabolismo , Tretinoina/metabolismo , Tretinoina/farmacología , Animales , Anticuerpos , Anticuerpos Monoclonales , Transporte Biológico , Western Blotting , Diferenciación Celular/efectos de los fármacos , Línea Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Humanos , Cinética , Leucemia Promielocítica Aguda , Peso Molecular , Receptores de Ácido Retinoico/análisis , Receptores de Ácido Retinoico/aislamiento & purificación , Proteínas Recombinantes/análisis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Receptor alfa de Ácido Retinoico , Transfección , Células Tumorales Cultivadas

RESUMEN

All-trans retinoic acid (all-trans RA), the active metabolite of vitamin A, has been demonstrated to be an efficient alternative to chemotherapy in the treatment of acute promyelocytic leukemia (APL), the AML3 subtype of the FAB cytological classification. Complete remission is obtained by inducing terminal granulocytic differentiation of the leukemic cells. To study all-trans RA pharmacokinetics in patients with APL, a rapid, precise and selective high-performance liquid chromatographic (HPLC) assay was developed. This method is easy and shows good repeatability (C.V. = 8.41-12.44%), reproducibility (C.V. = 9.19-14.73%), accuracy (C.V. = 3.5-11%) and sensitivity with a detection limit of 5 pmol/ml. The analysis is performed using normal-phase HPLC in an isocratic mode with UV detection after solid-phase extraction on octadecyl (C18) columns. The mobile phase is hexane-dichloromethane-dioxane (78:18:4, v/v) containing 1% acetic acid.

Asunto(s)

Cromatografía Líquida de Alta Presión/métodos , Isotretinoína/sangre , Tretinoina/sangre , Adsorción , Humanos , Leucemia Promielocítica Aguda/sangre , Reproducibilidad de los Resultados , Espectrofotometría Ultravioleta

RESUMEN

All-trans retinoic acid (ATRA) has been demonstrated to be an efficient alternative to chemotherapy in the treatment of acute promyelocytic leukemia (APL or AML3). Complete remission is obtained by inducing granulocytic differentiation of the leukemic cells. To date, the exact mechanism through which ATRA exerts its differentiating effect is not known. The present investigation was initiated to characterize ATRA intracellular concentrations achieved in human myeloid leukemic cells in relation to their different sensitivity to ATRA differentiating effect. During the first 24 h of incubation, a significant decrease of ATRA in the culture medium and a marked increase in the intracellular concentrations were observed. Maximal uptake by the leukemic cells was reached within minutes, with levels between 20 and 260 pmol/10(6) cells (median = 100). Interestingly, a correlation between ATRA-induced differentiation and the intracellular ATRA concentration achieved was observed. In fact, patients with intracellular levels below 60 pmol/10(6) cells defined slow uptakers, never exceeded 40% differentiated cells at day 3. On the other hand, cells with 2-4-fold higher concentration (100-250 pmol/10(6) cells) achieved 100% differentiated cells at day 3. This report suggests that intracellular ATRA concentration is a key pharmacological parameter that should be taken into account to gain further insights into ATRA sensitivity in APL patients.

Asunto(s)

Leucemia Promielocítica Aguda/metabolismo , Tretinoina/farmacocinética , Adolescente , Adulto , Anciano , Femenino , Humanos , Leucemia Promielocítica Aguda/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Tretinoina/uso terapéutico , Células Tumorales Cultivadas

RESUMEN

The retinoids are a large group of compounds structurally related to vitamin A. Retinoids elicit specific biological responses by binding to and activating nuclear receptors. Information about the metabolism and storage of vitamin A and retinoids, their plasma transport and uptake and the retinoid dose efficient on target cell had to be established because retinoic acid (RA), the natural acidic derivative of vitamin A (retinol), is likely to be a key factor during specific phases of embryonic development and maintenance of normal differentiated phenotypes in adult, so vitamin A is involved in the normal morphological differentiation of the visual system. RA appears an important agent since it induces in vitro leukemic cells from acute promyelocytic leukemia (APL) to differentiate into mature functional granulocytes which lose their self-renewal ability and die spontaneously. In vivo, APL patients treated with oral all-trans retinoic acid (all-trans RA) alone achieve complete remission in 80% of the cases. APL results from a malignant process that leads to the accumulation in the blood and in the bone marrow of myeloid precursor cells characterized by an abnormal behavior and a differentiation arrest. APL is characterized cytogenetically by a t(15;17) translocation which involves both the PML gene on chromosome 15 and the RARa gene on chromosome 17 and gives rise to the PML/RARa fusion protein. The high sensitivity of the promyelocytic blasts to all-trans RA should be related to the presence in APL blast of an abnormal protein, the PML/RAR alpha. The antineoplastic effects of retinoids suggest that these drugs could be used therapeutically for the chemoprevention of cancer.

Asunto(s)

Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Tretinoina/farmacología , Animales , Antineoplásicos/uso terapéutico , ADN de Neoplasias/efectos de los fármacos , Humanos , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/genética , Neoplasias/genética , Neoplasias Experimentales/tratamiento farmacológico , Translocación Genética/efectos de los fármacos , Tretinoina/uso terapéutico

RESUMEN

Acute promyelocytic leukemia (APL), is a homogeneous subgroup of acute myelogenous leukemias characterized by phenotypic and genetic markers. APL is associated with a reciprocal chromosomal translocation t(15,17) which has been shown to disrupt the retinoic acid receptor alpha (RAR alpha) gene. As a result, a portion of the RAR alpha gene becomes fused with a chromosome 15 locus termed PML (promyelocytic myeloid leukemia) from which chimeric PML/RAR alpha fusion mRNAs are expressed. The presence of these fusion transcripts in APL patients strongly support the hypothesis that both the t(15;17), and thus PML/RAR alpha, play a crucial role in the leukemogenesis of this disease. APL cells are specifically responsive to all-trans retinoic acid (ATRA) and this characteristic has allowed the first differentiation therapy with retinoic acid. However, failure or partial responses are observed and, though this has most frequently been reported in patients at second or third relapse. The molecular basis of the absence of ATRA response in these patients has not been determined.

Asunto(s)

Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Leucemia Promielocítica Aguda/fisiopatología , Proteínas de Neoplasias/fisiología , Proteínas de Fusión Oncogénica/fisiología , Receptores de Ácido Retinoico/fisiología , Tretinoina/farmacología , Diferenciación Celular/efectos de los fármacos , Transformación Celular Neoplásica/genética , Cromosomas Humanos Par 15/ultraestructura , Cromosomas Humanos Par 17/ultraestructura , Humanos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patología , Familia de Multigenes , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Proteínas de Fusión Oncogénica/genética , Receptores de Ácido Retinoico/efectos de los fármacos , Receptores de Ácido Retinoico/genética , Translocación Genética

RESUMEN

Recently, we described a recurrent variant translocation, t(11;17)(q23;q21), in acute promyelocytic leukemia (APL) which juxtaposes PLZF, a gene encoding a zinc finger protein, to RARA, encoding retinoic acid receptor alpha (RAR alpha). We have now cloned cDNAs encoding PLZF-RAR alpha chimeric proteins and studied their transactivating activities. In transient-expression assays, both the PLZF(A)-RAR alpha and PLZF(B)-RAR alpha fusion proteins like the PML-RAR alpha protein resulting from the well-known t(15;17) translocation in APL, antagonized endogenous and transfected wild-type RAR alpha in the presence of retinoic acid. Cotransfection assays showed that a significant repression of RAR alpha transactivation activity was obtained even with a very low PLZF-RAR alpha-expressing plasmid concentration. A "dominant negative" effect was observed when PLZF-RAR alpha fusion proteins were cotransfected with vectors expressing RAR alpha and retinoid X receptor alpha (RXR alpha). These abnormal transactivation properties observed in retinoic acid-sensitive myeloid cells strongly implicate the PLZF-RAR alpha fusion proteins in the molecular pathogenesis of APL.

Asunto(s)

Cromosomas Humanos Par 11 , Cromosomas Humanos Par 17 , Leucemia Promielocítica Aguda/genética , Receptores de Ácido Retinoico/genética , Translocación Genética , Secuencia de Aminoácidos , Animales , Línea Celular , Clonación Molecular , Variación Genética , Haplorrinos , Humanos , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/etiología , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas Recombinantes de Fusión/genética , Activación Transcripcional/efectos de los fármacos , Transfección , Tretinoina/farmacología , Dedos de Zinc/genética

RESUMEN

Vitamin A (retinol) and retinoic acid, its natural derivative, play an important role in the growth, differentiation and development of known normal tissues. Retinoids have recently become of interest to research in areas as diverse as dermatology, embryonal development and cancer research. Retinol is the major retinoid transported in the blood and tissues by its specific carrier retinol binding protein (RBP). The normal level of retinol in plasma is regulated very precisely by retinol homeostasis. RBP-retinol circulation supplies target cells, which then activate retinol into retinoic acid (RA) if they possess the NAD-dependent enzymatic oxidation system. RA, which is one of the most active metabolites of retinol, is also present in low concentration in the blood and the RA rate formation varies from tissues depending on specific need of the cell. The cellular transport and biological activity of retinoids may be mediated by their specific cytoplasmic binding proteins cellular retinol binding protein (CRBP) and the cellular retinoic acid binding protein (CRABP) which may function as shuttles targetting RA to nucleosol fraction and/or as regulator of cellular concentration of RA. The nuclear proteins RARs (retinoic acid receptors), which are members of the nuclear receptor superfamily are likely to be the final transducers of the RA signal at the gene expression. All-trans retinoic acid (ATRA) is able to specifically differentiate the malignant cells from leukemic patients with APL in short-term culture. For this reason, APL patients were successfully treated with ATRA (Chinese and French results). Acute promyelocytic leukemia M3 (French-American-British FAB classification) is a rare disease (10% of AML), characterized by a reciprocal chromosome 15-17 translocation. It has been shown that the chromosome 17 breakpoint of the translocation is localized within the RAR alpha gene. Due to the t(15;17) RAR alpha gene translocated to a gene PML on chromosome 15 resulting in synthesis of PML/RAR alpha fusion messenger RNA. Detection of PML/RAR alpha transcript is now a molecular marker of the disease. The abnormal PML/RAR alpha protein exhibits altered transcription activation properties when compared with RAR alpha. Clinical trials have demonstrated that ATRA is extremely efficient in inducing complete remission in APL patients. The morphologic finding of maturing elements in the bone marrow and peripheral blood during retinoic acid treatment indicates that the remission is obtained without hypoplasia and suggests that a differentiating mechanism is involved.(ABSTRACT TRUNCATED AT 400 WORDS)

Asunto(s)

Leucemia Promielocítica Aguda/tratamiento farmacológico , Retinoides/farmacología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Diferenciación Celular/efectos de los fármacos , Humanos , Leucemia Promielocítica Aguda/genética , Receptores de Ácido Retinoico , Retinoides/metabolismo , Retinoides/uso terapéutico , Proteínas de Unión al Retinol/metabolismo , Proteínas Celulares de Unión al Retinol , Proteínas Plasmáticas de Unión al Retinol , Translocación Genética

RESUMEN

It has been shown that patients with acute promyelocytic leukemia (AML3 subtype) treated with all-trans retinoic acid (all-trans RA), 45 mg/m2/day, achieve complete remission through differentiation of the leukemic clone to mature myeloid cells, which die spontaneously. The pharmacokinetics of all-trans RA given by mouth were studied in 15 AML3 patients. Blood samples were drawn for 24 h following a single oral dose of 45 mg/m2 and assayed for all-trans RA and 13-cis retinoic acid (13-cis RA) plasma concentrations by specific high-performance liquid chromatography. In one patient all-trans RA and 13-cis RA levels were below the detection limits at all times. In the other patients, the time to peak concentration of all-trans RA was between 60 and 210 min (median 90 min) after ingestion, with maximum concentrations between 0.03 and 2.5 micrograms/ml (median 0.4 micrograms/ml). These concentrations were within the in vitro differentiating concentration range of all-trans RA for these patients' cells. In nine patients, enterohepatic cycling was suggested by the presence on the concentration versus time curve of a secondary peak that occurred at meal times. The apparent plasma elimination half-life was between 16.8 and 77.4 min (median 30 min). Detectable plasma levels of 13-cis RA in 12 patients indicated in vivo isomerization of all-trans RA. Despite the high inter-individual variability of all-trans RA pharmacokinetics in these patients, high blast cell counts and failure to respond to differentiation treatment tended to be associated with low all-trans RA Cmax values and high clearance estimates.

Asunto(s)

Leucemia Promielocítica Aguda/tratamiento farmacológico , Tretinoina/farmacocinética , Administración Oral , Humanos , Leucemia Promielocítica Aguda/metabolismo , Estereoisomerismo , Tretinoina/administración & dosificación , Tretinoina/química