RESUMEN
The differentiation of embryonic mesenchymal cells into chondrocytes and the subsequent formation of a cartilaginous scaffold that enables the formation of long bones are hallmarks of endochondral ossification. During this process, chondrocytes undergo a remarkable sequence of events involving proliferation, differentiation, hypertrophy and eventually apoptosis. Forkhead Box O (FoxO) transcription factors (TFs) are well-known regulators of such cellular processes. Although FoxO3a was previously shown to be regulated by 1,25-dihydroxyvitamin D3 in osteoblasts, a possible role for this family of TFs in chondrocytes during endochondral ossification remains largely unstudied. By crossing Collagen2-Cre mice with FoxO1lox/lox;FoxO3alox/lox;FoxO4lox/lox mice, we generated mice in which the three main FoxO isoforms were deleted in growth plate chondrocytes (chondrocyte triple knock-out; CTKO). Intriguingly, CTKO neonates showed a distinct elongation of the hypertrophic zone of the growth plate. CTKO mice had increased overall body and tail length at eight weeks of age and suffered from severe skeletal deformities at older ages. CTKO chondrocytes displayed decreased expression of genes involved in redox homeostasis. These observations illustrate the importance of FoxO signaling in chondrocytes during endochondral ossification.
Asunto(s)
Huesos/metabolismo , Condrocitos/metabolismo , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O3/genética , Factores de Transcripción Forkhead/genética , Osteogénesis/genética , Animales , Huesos/citología , Proteínas de Ciclo Celular , Diferenciación Celular , Proliferación Celular , Condrocitos/citología , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Cruzamientos Genéticos , Femenino , Proteína Forkhead Box O1/deficiencia , Proteína Forkhead Box O3/deficiencia , Factores de Transcripción Forkhead/deficiencia , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Integrasas/genética , Integrasas/metabolismo , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Transgénicos , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Cultivo Primario de Células , Transducción de SeñalRESUMEN
1Alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active form of vitamin D3, is a pleiotropic hormone that exerts its effects on a wide range of tissues, resulting in different biological responses such as anticancer activity. It is the ligand of the vitamin D receptor (VDR), a nuclear receptor with transactivating capacity. We demonstrated in this study that 1,25(OH)2D3 induces PDZ-LIM domain-containing protein 2 (PDLIM2) expression. PDLIM2 is an adaptor molecule that links different components of the cytoskeleton, and was recently shown to be repressed in human breast cancer cells by hypermethylation of regulatory promoter regions, leading to enhanced tumorigenicity. We demonstrated that PDLIM2 was a direct target gene of 1,25(OH)2D3; its upregulation was VDR-dependent and a functional VDRE in the promoter was identified. Moreover, 1,25(OH)2D3 induced demethylation of the PDLIM2 promoter, leading to enhanced transcription. Finally, PDLIM2 was found to be crucial for 1,25(OH)2D3-induced cell adhesion and for mediating the ability of 1,25(OH)2D3 to suppress cancer cell migration and invasion. This study provides mechanistic insights into the anticancer activities of 1,25(OH)2D3 in human breast cancer cells.
Asunto(s)
Antineoplásicos/farmacología , Movimiento Celular , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas con Dominio LIM/biosíntesis , Proteínas de Microfilamentos/biosíntesis , Vitamina D/análogos & derivados , Western Blotting , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Inmunoprecipitación de Cromatina , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Proteínas con Dominio LIM/genética , Células MCF-7 , Proteínas de Microfilamentos/genética , Invasividad Neoplásica/genética , Regiones Promotoras Genéticas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Activación Transcripcional/efectos de los fármacos , Vitamina D/farmacologíaRESUMEN
Various epidemiological studies have shown an aetiological link between vitamin D deficiency and cancer incidence. The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], has potent anti-cancer activities both in vitro and in vivo. These anti-cancer effects are attained by regulating the transcription of numerous genes that are involved in different pathways to reduce tumorigenesis and are dependent on the cancer cell type. Besides reducing cell growth and inducing apoptosis, 1,25(OH)2D3 also inhibits angiogenesis and metastasis. Moreover, its potency to inhibit inflammation also contributes to its anti-tumoral activity. Here, we report the different ways in which 1,25(OH)2D3 interferes with the malignant processes that are activated in cancer cells.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Anticarcinógenos/uso terapéutico , Antineoplásicos/uso terapéutico , Calcitriol/uso terapéutico , Animales , Antiinflamatorios no Esteroideos/metabolismo , Anticarcinógenos/metabolismo , Antineoplásicos/metabolismo , Calcitriol/metabolismo , Humanos , Neoplasias/etiología , Neoplasias/prevención & control , Neoplasias/terapia , Deficiencia de Vitamina D/fisiopatologíaRESUMEN
Mutations in the BRCA1-interacting DEAH helicase Brip1 confer an increased risk of breast cancer. In the present study we aimed to unravel the transcriptional control of Brip1 and to determine its expression levels in a set of 101 primary invasive breast carcinomas. Transcription of Brip1 was found to be cell growth-related and controlled by the E2F/retinoblastoma (Rb) pathway through a conserved E2F-responsive site. Repression of Brip1 expression by the cell growth-inhibiting compound 1alpha,25-dihydroxyvitamin D3 depended on this same E2F-responsive site. In spite of its role as a tumor suppressor, both quantitative reverse transcriptase-PCR analyses and immunohistochemical stainings showed significantly elevated Brip1 expression levels in grade 3 tumors as compared to grade 1 or 2 carcinomas. Furthermore, increased Brip1 transcript levels were found in tumors with an estrogen receptor-negative, progesterone receptor-negative or HER-2-positive status. In conclusion, these data show that Brip1 is a genuine target gene for the E2F/Rb pathway and that elevated expression levels of Brip1 are detected in primary invasive breast carcinomas with unfavorable characteristics.
Asunto(s)
Neoplasias de la Mama/genética , Carcinoma/genética , Proteínas de Unión al ADN/genética , Factores de Transcripción E2F/fisiología , Regulación Neoplásica de la Expresión Génica , ARN Helicasas/genética , Animales , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Sitios de Unión , Neoplasias de la Mama/patología , Carcinoma/patología , Secuencia Conservada , Factores de Transcripción E2F/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Femenino , Humanos , Ratones , Datos de Secuencia Molecular , Invasividad Neoplásica , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , Células Tumorales CultivadasRESUMEN
1alpha,25-Dihydroxyvitamin D(3) [1,25-(OH) (2)D(3)] can exert its biological actions through binding with the nuclear vitamin D receptor (VDR), a ligand-activated transcription factor. Next to control of bone and mineral homeostasis, these actions include an immunomodulatory effect and a potent growth-inhibitory, antiproliferative or prodifferentiating action on a wide variety of cell types. The molecular mechanisms underlying this antiproliferative action form an intriguing research topic and they remain, although thoroughly studied, not completely understood. Important cell cycle regulators are involved such as cyclins, cyclin dependent kinases and their corresponding inhibitors as well as E2F transcription factors and accompanying pocket proteins. Whether 1,25-(OH)(2)D(3) influences the expression of all these proteins directly through the nuclear VDR or rather in an indirect manner is not always clear. The antiproliferative action makes 1,25-(OH) (2)D(3) a possible therapeutic tool to treat hyperproliferative disorders, among which different types of cancer. Clinical application, however, is severely hampered by calcemic effects such as hypercalcemia, hypercalciuria and increased bone resorption. Rational design of chemically modified 1,25-(OH) (2)D(3)-analogs tries to overcome this problem. As such, several thousands of analogs have been synthesized and evaluated, some of which display the desired dissociation between beneficial antiproliferative and unwanted calcemic effects. A number of those analogs are 'superagonistic' and have a several-fold stronger antiproliferative action than the parent compound. This review focuses on recent findings about the complex mechanisms behind the antiproliferative and prodifferentiating effect of 1,25-(OH) (2)D(3). Furthermore, the mode of action and possible clinical application of chemically modified 1,25-(OH) (2)D(3)-analogs will be discussed.
Asunto(s)
Inhibidores de Crecimiento , Vitamina D/análogos & derivados , Vitamina D/farmacología , Animales , Calcitriol/farmacología , Proliferación Celular/efectos de los fármacos , Humanos , Receptores de Calcitriol/efectos de los fármacos , Esteroides/síntesis química , Esteroides/farmacología , Relación Estructura-ActividadRESUMEN
This study provides a detailed and exact evaluation of the interactions between vitamin D3 receptor (VDR), retinoid X receptor (RXR), and vitamin D3 responsive elements (VDREs) mediated by two novel 14-epianalogs of 1,25-dihydroxyvitamin D [1,25(OH)2D3], 19-nor-14-epi-23-yne-1,25(OH)2D3 (TX 522) and 19-nor-14,20-bisepi-23-yne-1,25(OH)2D3 (TX 527). Both analogs were more potent (14- and 75-fold, respectively) than 1,25(OH)2D3 in inhibiting cell proliferation and inducing cell differentiation. However, DNA-independent experiments indicated that both analogs had a lower affinity to VDR and that the stability of the induced VDR conformation, as measured by limited protease digestion assays, was similar (TX 527) or even weaker (TX 522) than that induced by the parent compound. However, DNA-dependent assays such as gel shift experiments revealed that those analogs were slightly more potent (3-7 times) than 1,25(OH)2D3 in enhancing binding of VDR-RXR heterodimers to a direct repeat spaced by three nucleotides (DR3) type VDRE. The functional consequences of the ligand-VDR-RXR-VDRE interactions observed in vitro were subsequently evaluated in transfection experiments. Both 14-epianalogs enhanced transcription of VDRE containing reporter constructs more efficiently than 1,25(OH)2D3 in COS-1 and MCF-7 cells regardless of the presence of ketoconazole. Transactivation activity is suggested to be a cell-specific process because maximal transcriptional induction and the half-maximal transactivation concentration for each reporter construct were different in both cell lines. The superagonistic transactivation activity closely resembled the biological potency of these analogs on the inhibition of MCF-7 cell proliferation. These data clearly indicate that superagonistic activity starts beyond the binding of the ligand-heterodimer (VDR-RXR) complex to VDRE and thus probably involves coactivator/corepressor molecules.
Asunto(s)
Alquinos , Calcitriol/farmacología , Colecalciferol/farmacología , Receptores de Calcitriol/fisiología , Receptores de Ácido Retinoico/fisiología , Factores de Transcripción/fisiología , Activación Transcripcional/fisiología , Adenocarcinoma/patología , Animales , Neoplasias de la Mama/patología , Células COS , División Celular/efectos de los fármacos , Chlorocebus aethiops , ADN/genética , Dimerización , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Cetoconazol/farmacología , Sustancias Macromoleculares , Ratones , Conformación Proteica/efectos de los fármacos , Multimerización de Proteína , Receptores de Calcitriol/química , Receptores X Retinoide , Transfección , Células Tumorales CultivadasRESUMEN
The non-classical effects of 1alpha,25-dihydroxyvitamin D(3) (1alpha, 25(OH)(2)D(3)) create possible therapeutic applications for immune modulation (e.g., autoimmune diseases and graft rejection), inhibition of cell proliferation (e.g., psoriasis, cancer) and induction of cell differentiation (e.g., cancer). The major drawback related to the use of 1alpha,25(OH)(2)D(3) is its calcaemic effect, which prevents the application of pharmacological concentrations. Intensive research has led to the development of analogues of 1(2)D(3) characterised by a clear dissociation of the antiproliferative and prodifferentiating capacity from the calcaemic effects. Due to this dissociation, these analogues can be used not only for the treatment of bone disorders but also for non-classical applications. In the present review, a summary is given on the use of the 1alpha,25(OH)(2)D(3) analogues for the treatment of cancer, skin and immune disorders and for the prevention of graft rejection. Moreover a brief overview is given on the use of analogues for secondary hyperparathyroidism.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Antineoplásicos/uso terapéutico , Calcitriol/uso terapéutico , Vitamina D/análogos & derivados , Vitamina D/uso terapéutico , Animales , Enfermedades Autoinmunes/tratamiento farmacológico , Humanos , Hiperparatiroidismo/tratamiento farmacológico , Neoplasias/tratamiento farmacológicoRESUMEN
The biological activity of two novel 14-epi-analogues of 1,25(OH)2D3, 19-nor-14-epi-23-yne-1,25(OH)2D3 (TX 522) and 19-nor-14,20-bisepi-23-yne-1,25(OH)2D3 (TX 527), is described. Both analogues were at least 10 times more potent than 1,25(OH)2D3 in inhibiting in vitro cell proliferation and had much lower in vivo calcemic effects than 1,25(OH)2D3. Treatment with 1,25(OH)2D3, TX 522, or TX 527 in vitro was accompanied by an accumulation of cells in the G1 phase of the cell cycle. Protein levels of cyclin C and cyclin D1 in in vitro cultures of MCF-7 cells were down-regulated to 50 and 30%, respectively, of control levels at 72 and 120 h after stimulation. Protein levels of p21 and p27 at 72 h were significantly enhanced by 1,25(OH)2D3 and TX 522 but surprisingly not by TX 527. The inability of TX 527 to up-regulate p21 seemed to be cell type specific because p21 was induced in other cell types. Diminished phosphorylation of the retinoblastoma protein after treatment with 1,25(OH)2D3, TX 522, or TX 527 may ultimately contribute to the growth inhibition caused by these compounds. According to the data presented, the induction of apoptosis seemed not to be a major mechanism responsible for the growth-inhibitory effect of 1,25(OH)2D3 and analogues. Both 14-epianalogues significantly retarded tumor progression (40% reduced compared with control mice) in an in vivo model of MCF-7 breast cancer cells established in nude mice. In conclusion, these novel analogues have the eligible profile to be tested as therapeutic agents for the treatment of hyperproliferative diseases such as breast cancer.
Asunto(s)
Alquinos , Neoplasias de la Mama/tratamiento farmacológico , Colecalciferol/uso terapéutico , Proteínas Proto-Oncogénicas , Animales , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Ciclina C , Ciclina D1/metabolismo , Quinasa 4 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Femenino , Fase G1/efectos de los fármacos , Humanos , Ratones , Ratones Desnudos , Proteínas de Neoplasias/metabolismo , Receptores de Calcitriol/metabolismo , Receptores de Estrógenos/metabolismo , Proteína de Retinoblastoma/metabolismoRESUMEN
Nonsteroidal analogues of 1alpha,25(OH)2D3, lacking either the full five-membered D ring (C-ring analogues) or the full six-membered C ring (D-ring analogues) are more potent inhibitors of cell proliferation or inducers of cell differentiation than is 1alpha,25(OH)2D3. Maximal superagonistic activity was seen for the C-ring analogue with a 24(R)-hydroxyl group in the side chain [30- to 60-fold the activity of 1alpha,25(OH)2D3]. The 19-nor-16-ene-26,27-bishomo C-ring analogue showed the best ratio of antiproliferative to calcemic effects (1275-fold better than 1alpha,25(OH)2D3 and severalfold better than all vitamin D analogues so far described). The analogues are able to stimulate specific vitamin D-dependent genes and are active in transfection assays using an osteocalcin promoter VDRE. Low binding affinity to the vitamin D binding protein, differences in metabolism, or affinity for the vitamin D receptor (VDR) are not the most important explanations for the enhanced intrinsic activity. However, the analogues are able to induce conformational changes in the VDR, which makes the VDR-ligand complex more resistant against protease digestion than is 1alpha,25(OH)2D3. In contrast to 20-epimer steroidal vitamin D analogues, 20-epimer C-ring analogues were less potent than analogues with a natural C-20 configuration. In conclusion, several nonsteroidal vitamin D analogues are superagonists of 1alpha,25(OH)2D3 despite lower receptor affinity and, for the C-ring analogues, higher flexibility of the side chain; moreover, they have a better selectivity profile than all analogues yet published.
Asunto(s)
Calcitriol/análogos & derivados , Calcitriol/farmacología , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Calcitriol/metabolismo , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células HL-60 , Humanos , Receptores de Calcitriol/agonistas , Receptores de Calcitriol/metabolismoRESUMEN
The activated form of vitamin D(3), 1alpha,25(OH)(2)D(3), not only plays a central role in bone and calcium metabolism but has also potent antiproliferative and prodifferentiating effects. Moreover, the combined presence of 25(OH)D(3)-1alpha-hydroxylase as well as the vitamin D receptor in several tissues introduced the idea of a paracrine role for 1alpha,25(OH)(2)D(3). By introducing chemical modifications into the flexible parent molecule 1alpha,25(OH)(2)D(3), a whole generation of vitamin D analogs was created. Due to a clear dissociation of the antiproliferative and prodifferentiating effects from calcemic effects, these analogs can be used not only for the treatment of bone disorders but also for non-classical applications.
Asunto(s)
Calcitriol/análogos & derivados , Calcitriol/farmacología , Huesos/efectos de los fármacos , Huesos/metabolismo , Calcitriol/uso terapéutico , Calcio/sangre , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Humanos , Neoplasias/tratamiento farmacológico , Enfermedades de la Piel/tratamiento farmacológicoRESUMEN
1,25(OH)2D3 is a known growth inhibitor and differentiation inducer of several cancer cell lines. To establish the molecular mechanism of 1,25(OH)2D3 as an antiproliferating agent, its effect on proliferation and gene regulation was studied in human breast cancer MCF-7 cells. 1,25(OH)2D3 inhibited cell proliferation dose dependently through G1 arrest. Cyclin D1 transcription levels decreased rapidly in 1,25(OH)2D3-treated cells while protein levels only decreased after 72 h of treatment. Transcription levels of p21 and p27 were upregulated with chronologically consistent changes in cell cycle distribution. Experiments with TGF-beta neutralising antibodies revealed that the largest effect of 1,25(OH)2D3 on cell proliferation is likely due to a TGF-beta independent mechanism of action. The cell cycle regulatory genes, cyclin D1 and p27, are probably involved herein as their expression was not affected by the presence of neutralising antibodies. However, upregulation of p21 was completely abrogated. Therefore, the TGF-beta signalling pathway is thought to be responsible for p21 upregulation.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Calcitriol/farmacología , Ciclina D1/genética , Ciclinas/genética , Proteínas de Microfilamentos/genética , Proteínas Musculares , Northern Blotting , Western Blotting , División Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Fase G1/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta/fisiología , Células Tumorales CultivadasRESUMEN
The activated form of vitamin D3, 1 alpha,25(OH)2D3, not only plays a central role in bone and calcium metabolism, but also has potent antiproliferative and prodifferentiating effects. Moreover, the combined presence of 25(OH)D3-1 alpha-hydroxylase, as well as the vitamin D receptor in several tissues introduced the idea of a paracrine role for 1 alpha,25(OH)2D3. By introducing chemical modifications into the flexible molecule 1 alpha,25(OH)2D3, a whole generation of vitamin D analogues was created. Due to a clear dissociation of the antiproliferative and prodifferentiating effects from calcaemic effects, these analogues can be used not only for the treatment of bone disorders but also for non-classical applications. In the present review, a summary is given on the use of the 1 alpha,25(OH)2D3 analogues for the treatment of psoriasis, cancer and immune disorders together with new insights in the mechanism of action of these analogues.
Asunto(s)
Calcitriol/uso terapéutico , Vitamina D/análogos & derivados , Animales , Femenino , Humanos , Sistema Inmunológico/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Embarazo , Enfermedades de la Piel/tratamiento farmacológico , Vitamina D/uso terapéuticoRESUMEN
1alpha,25-dihydroxyvitamin D is a key calcium-regulating hormone but also displays potent differentiating and antiproliferative activities on many cell types. The structural requirements of this secosteroid hormone have been extensively studied for the A-ring and side chain, whereas relatively little is known about the requirements of the natural CD-ring structure for the vitamin D-like biological activity. We have embarked on a vast program in which derivatives were synthesized and evaluated characterized by profound structural changes in the central C/D-region. This first series of nonsteroidal analogs consists of (1R,3S)-5-((Z,2E)-4-((1S,3S)-3-(4-hydroxy-4-methylpentyl)-1,2,2-++ +trimethylcyclopentyl)-2-butenylidene)-4-methylenecyclohexan e-1,3-diol (KS 176) and derivatives thereof. These analogs are characterized by the absence of normal C- and D-rings and by the presence of an unnatural five-membered ring which we call the E-ring. KS 176 with the otherwise natural side chain structure of 1alpha,25(OH)2D3 has between 10 and 30% of the biological activity of 1alpha,25(OH)2D3 when tested in vitro (prodifferentiating effects on HL-60 and MG-63; antiproliferating activity on MCF-7 and keratinocytes) but has minimal in vivo calcemic effects. Introduction of several side chain modifications created analogs with increased intrinsic noncalcemic biological properties, whereas their calcemic potency remains very low. These data demonstrate that the full CD-rings are not mandatory for the biological activity of 1alpha,25(OH)2D3 since they can be replaced by a new ring structure which generates an appropriate spacing of the A-seco B-rings in relation to the side chain. The biological activity of these nonsteroidal analogs probably involves a classical genomic activation since they are also active in transfection assays using an osteocalcin vitamin D responsive element coupled to a human growth hormone reporter gene.
Asunto(s)
Calcitriol/análogos & derivados , Receptores de Calcitriol/efectos de los fármacos , Vitamina D/análogos & derivados , Vitamina D/farmacología , Animales , Células COS , División Celular/efectos de los fármacos , División Celular/genética , Ergocalciferoles/química , Regulación de la Expresión Génica , Células HL-60 , Humanos , Queratinocitos/efectos de los fármacos , Ratones , Receptores de Calcitriol/genética , Relación Estructura-Actividad , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas , Vitamina D/químicaRESUMEN
1,25 Dihydroxyvitamin D3 (D3), all trans retinoic acid (atRA) and the cytokine rTGF-beta2 are growth and differentiation modulators of promyelocytic leukemia. D3 gives rise to a functional monocytic cell population whereas single atRA therapy induces granulocytic cell features. rTGF-beta2 reduces HL60 cell proliferation but has no differentiating capacity. Combination treatment demonstrates additive effects between either D3 and atRA or D3 and rTGF-beta2, resulting in a cell population with mixed characteristics since individual cells exhibit both monocytic as granulocytic cell features. The capacity of single and combined treatments to induce a permanent differentiation was investigated. Therefore, cells were preincubated with the drugs during six days, test drugs were removed and cell number was monitored. The total cell count of populations treated with single agents remains constant for only a few days and then increases rapidly. rTGF-beta2 cooperated with D3 in inducing a long-lasting differentiation state (3 weeks). Addition of atRA to this combination did not significantly alter proliferation or differentiation, but some cells underwent apoptosis. Therefore, a total and permanent differentiation of leukemic cells may be achieved by repeated exposure to a combination of differentiation inducing agents.
Asunto(s)
Antígenos de Neoplasias , Calcitriol/farmacología , Moléculas de Adhesión Celular , Células HL-60/citología , Células HL-60/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Tretinoina/farmacología , Apoptosis/efectos de los fármacos , Biomarcadores , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Quimioterapia Combinada , Células HL-60/metabolismo , Humanos , Receptores de Lipopolisacáridos/efectos de los fármacos , Receptores de Lipopolisacáridos/metabolismo , Glicoproteínas de Membrana/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Monocitos/efectos de los fármacos , Nitroazul de Tetrazolio/química , Nitroazul de Tetrazolio/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
1,25(OH)2D3 and two stereoisomers of retinoic acid, all trans and 9-cis retinoic acid, are regulators of cell proliferation and differentiation. The aim of this study was to evaluate the effects of a combination of 1,25(OH)2D3 and retinoic acid (all trans or 9-cis) on proliferation and cell differentiation of the human promyelocytic leukemia cell line HL60, and to test the reversibility of the induced differentiation. Cell proliferation was inhibited as expected by 1,25(OH)2D3 and all trans retinoic acid alone (IC50 of cell survival was 4 x 10(-7) M, 9 x 10(-6) M and 9 x 10(-7) M for 1,25(OH)2D3, all trans and 9-cis retinoic acid, respectively). Combination of 1,25(OH)2D3 and either form of retinoic acid resulted in a partially additive decrease in cell proliferation. 1,25(OH)2D3 induced a monocytic differentiation (100% CD14+ cells with 10(-7) M 1,25(OH)2D3), while retinoic acid led to a predominantly granulocytic differentiation (36 and 42% CD67+ cells with 10(-6) M all trans and 9-cis retinoic acid, respectively). Additive effects on differentiation were observed upon combination of subtherapeutical doses of the drugs, achieving a mainly monocytic population, demonstrating the dominant role of 1,25(OH)2D3 in determining the direction of differentiation. The effects on proliferation and differentiation of the solitary drugs were reversible, while the proliferation arrest and differentiation induced by the combination persisted and even progressed after withdrawal of the drugs. We conclude that 1,25(OH)2D3 and retinoic acid (all trans or 9-cis) exert additive effects on inhibition of proliferation and induction of cell differentiation of HL60 cells, leading to a persistent differentiation, even after drug withdrawal.