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1.
Oncogene ; 35(36): 4752-61, 2016 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-26898757

RESUMO

Deregulation of microRNAs (miRs) contributes to progression and metastasis of prostate and other cancers. miR-23b and -27b, encoded in the same miR cluster (miR-23b/-27b), are downregulated in human metastatic prostate cancer compared with primary tumors and benign tissue. Expression of miR-23b/-27b decreases prostate cancer cell migration, invasion and results in anoikis resistance. Conversely, antagomiR-mediated miR-23b and -27b silencing produces the opposite result in a more indolent prostate cancer cell line. However, neither miR-23b/-27b expression or inhibition impacts prostate cancer cell proliferation suggesting that miR-23b/-27b selectively suppresses metastasis. To examine the effects of miR-23b/-27b on prostate cancer metastasis in vivo, orthotopic prostate xenografts were established using aggressive prostate cancer cells transduced with miR-23b/-27b or non-targeting control miRNA. Although primary tumor formation was similar between miR-23b/-27b-transduced cells and controls, miR-23b/-27b expression in prostate cancer cells decreased seminal vesicle invasion and distant metastases. Gene-expression profiling identified the endocytic adaptor, Huntingtin-interacting protein 1-related (HIP1R) as being downregulated by miR-23b/-27b. Increased HIP1R expression in prostate cancer cells inversely phenocopied the effects of miR-23b/-27b overexpression on migration, invasion and anchorage-independent growth. HIP1R rescued miR-23b/-27b-mediated repression of migration in prostate cancer cells. HIP1R mRNA levels were decreased in seminal vesicle tissue from mice bearing miR-23b/-27b-transduced prostate cancer cell xenografts compared with scrambled controls, suggesting HIP1R is a key functional target of miR-23b/-27b. In addition, depletion of HIP1R led to a more rounded, less mesenchymal-like cell morphology, consistent with decreased metastatic properties. Together, these data demonstrate that the miR-23b/-27b cluster functions as a metastasis-suppressor by decreasing HIP1R levels in pre-clinical models of prostate cancer.


Assuntos
MicroRNAs/genética , Neoplasias da Próstata/genética , Proteínas de Transporte Vesicular/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Proteínas dos Microfilamentos , Invasividade Neoplásica/genética , Metástase Neoplásica , Próstata/patologia , Neoplasias da Próstata/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Oncogene ; 31(6): 716-27, 2012 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-21765461

RESUMO

Increased androgen receptor (AR) transcriptional activity mediated by coactivator proteins may drive castration-resistant prostate cancer (CRPC) growth. Vav3, a Rho GTPase guanine nucleotide exchange factor (GEF), is overexpressed in human prostate cancers, particularly in models of CRPC progression. Vav3 coactivates AR in a Vav3 pleckstrin homology (PH) domain-dependent but GEF-independent manner. Ectopic expression of Vav3 in androgen-dependent human prostate cancer cells conferred robust castration-resistant xenograft tumor growth. Vav3 but not a Vav3 PH mutant greatly stimulated interaction between the AR amino and carboxyl termini (N-C interaction), which is required for maximal receptor transcriptional activity. Vav3 was distributed between the cytoplasm and nucleus with nuclear localization-dependent on the Vav3 PH domain. Membrane targeting of Vav3 abolished Vav3 potentiation of AR activity, whereas nuclear targeting of a Vav3 PH mutant rescued AR coactivation, suggesting that nuclear localization is an important function of the Vav3 PH domain. A nuclear role for Vav3 was further demonstrated by sequential chromatin immunoprecipitation assays, which revealed that Vav3 and AR were recruited to the same transcriptional complexes of an AR target gene enhancer. These data demonstrate the importance of Vav3 in CRPC and define a novel nuclear function of Vav3 in regulating AR activity.


Assuntos
Núcleo Celular/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Receptores Androgênicos/metabolismo , Animais , Sítios de Ligação/genética , Western Blotting , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Masculino , Metribolona/farmacologia , Camundongos , Camundongos SCID , Microscopia de Fluorescência , Mutação , Orquiectomia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-vav/genética , Receptores Androgênicos/genética , Transativadores/genética , Transativadores/metabolismo , Transplante Heterólogo , Carga Tumoral
4.
Endocrinology ; 142(3): 1107-16, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11181525

RESUMO

Androgen and glucocorticoid receptor (AR, GR), two closely related members of the nuclear receptor superfamily, can recognize a similar cis-acting DNA sequence, or hormone response element (HRE). Despite this apparent commonality, these receptors regulate distinct target genes in vivo. The AR gene itself is regulated by AR but not GR in a variety of cell types, including osteoblast-like cells, as shown here. To understand this specificity, we first identified the DNA sequences responsible for androgen-mediated up-regulation of AR messenger RNA. A 6.5-kb region encompassing exon D, intron 4, and exon E of the AR gene contains four exonic HREs and exhibits cell type-specific, AR-mediated transcriptional enhancement when placed upstream of a heterologous promoter and reporter gene. A 350-bp fragment consisting of just exons D and E exhibits the same cell- and androgen-specificity as the 6.5-kb region, as well as the native AR gene. Consistent with a role for the exonic HREs, androgen regulation via this intragenic enhancer requires the HREs as well as a functional receptor DNA binding domain. A panel of AR/GR chimeric receptors was used to test which AR domains (amino-terminal, DNA binding or ligand binding) confer androgen-specific regulation of the 350-bp enhancer. Only chimeric receptors containing the amino-terminus of AR induced reporter gene activity from the AR gene enhancer. Further, a constitutively active AR consisting of only the AR amino-terminus and DNA binding domain (AA phi) retained the capacity to activate the internal responsive region, unlike a constitutively active chimera harboring the GR amino-terminus and AR DNA binding domain (GA phi). Thus, the AR amino terminus is the sole determinant for androgen-specific regulation of the AR gene internal enhancer. These findings support a model in which the amino termini of ARs bound to HREs within the AR gene interact with an exclusive auxiliary factor(s) to elicit androgen-specific regulation of AR messenger RNA. This is the first example of androgen-specific response in which the necessary and sufficient distinguishing capacity resides within the AR amino terminus.


Assuntos
Androgênios/fisiologia , Elementos Facilitadores Genéticos/fisiologia , Éxons/fisiologia , Expressão Gênica/fisiologia , Fragmentos de Peptídeos/fisiologia , Receptores Androgênicos/genética , Receptores Androgênicos/fisiologia , Animais , Linhagem Celular , DNA/metabolismo , Hormônios/fisiologia , Ligantes , Camundongos , Osteoblastos/metabolismo , Osteoblastos/fisiologia , Estrutura Terciária de Proteína/fisiologia , RNA Mensageiro/metabolismo , Receptores Androgênicos/metabolismo , Receptores de Glucocorticoides/fisiologia , Elementos de Resposta/fisiologia , Ativação Transcricional/fisiologia , Regulação para Cima
5.
Endocrinology ; 141(7): 2567-73, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10875259

RESUMO

Expression of MET, the receptor for hepatocyte growth factor (HGF), has been associated with androgen-insensitive prostate cancer. In this study we evaluated MET activation by HGF and HGF action in prostate cancer cell lines. HGF causes phosphorylation (activation) of the MET receptor in three androgen-unresponsive cell lines (DU 145, PC-3, and ALVA-31) together with morphological change. Although HGF is known to stimulate the growth of normal epithelial cells, including those from prostate, we found that HGF inhibited ALVA-31 and DU 145 (hormone-refractory) cell lines. Moreover, HGF and vitamin D additively inhibited growth in each androgen-unresponsive cell line, with the greatest growth inhibition in ALVA-31 cells. Further studies in ALVA-31 cells revealed distinct cooperative actions of HGF and vitamin D. In contrast to the accumulation of cells in G1 seen during vitamin D inhibition of androgen-responsive cells (LNCaP), growth inhibition of the androgen-unresponsive ALVA-31 cell line with the HGF and vitamin D combination decreased, rather than increased, the fraction of cells in G1, with a corresponding increase in the later cell cycle phases. This cell cycle redistribution suggests that in androgen-unresponsive prostate cancer cells, HGF and vitamin D act together to slow cell cycle progression via control at sites beyond the G1/S checkpoint, the major regulatory locus of growth control in androgen-sensitive prostate cells.


Assuntos
Androgênios/fisiologia , Fator de Crescimento de Hepatócito/farmacologia , Neoplasias da Próstata/patologia , Vitamina D/farmacologia , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Resistência a Medicamentos , Sinergismo Farmacológico , Humanos , Masculino , Fosforilação , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Tirosina/metabolismo , Vitamina D/análogos & derivados
6.
Endocrinology ; 141(5): 1699-704, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10803579

RESUMO

Androgen-mediated growth repression of androgen-independent prostate cancer (AIPC) cells has been reported in androgen-independent PC-3 cells overexpressing the androgen receptor, and in androgen-independent derivatives of LNCaP cells that develop following prolonged culture in androgen-free media. Using two models of AIPC, PC3/AR cells and LNCaP-OM1 cells, a subclone of LNCaP cells derived by prolonged culturing in charcoal-stripped media, we investigated whether expression of neutral endopeptidase 24.11 (NEP), a cell-surface peptidase that cleaves and inactivates neuropeptides implicated in the growth of AIPC, is induced by androgen, and whether NEP contributes to the observed androgen-mediated growth repression. These cell lines each express high levels of androgen receptor. Culturing in dihyrotestosterone (DHT) resulted in a 30-56% (PC3) and 35-43% (LNCaP-OM1) decrease in cell number over 7 days concomitant with a significant increase in NEP enzyme specific activity. Northern analysis detected an increase in NEP transcripts following DHT treatment in PC3/AR cells. The addition of the NEP enzyme inhibitor phosphoramidon to PC3 and LNCaP-OM1 or the NEP competitive inhibitor CGS 24592 to LNCaP-OM1 blocked the increase in NEP enzyme activity and reversed the DHT-induced growth inhibition. Neither phosphoramidon or CGS 24592 alone inhibited cell growth. Furthermore, the reversal of growth inhibition in LNCaPOM1 cells was dose dependent on the concentration of CGS 24592. These data indicate that androgen-induced growth repression of AIPC cells PC3 and LNCaP-OM1 results in part from androgen-induced expression of NEP in these cells.


Assuntos
Androgênios/fisiologia , Neprilisina/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/biossíntese , Animais , Western Blotting , Divisão Celular/efeitos dos fármacos , Di-Hidrotestosterona/farmacologia , Masculino , Ratos , Receptores Androgênicos/genética , Transfecção , Células Tumorais Cultivadas
7.
Clin Cancer Res ; 6(3): 901-8, 2000 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10741714

RESUMO

Prostate cancer cells contain specific receptors [vitamin D receptors (VDRs)] for 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), which is known to inhibit the proliferation and invasiveness of these cells. These findings support the use of 1alpha,25(OH)2D3 for prostate cancer therapy. However, because 1alpha,25(OH)2D3 can cause hypercalcemia, analogues of 1alpha,25(OH)2D3 that are less calcemic but that exhibit potent antiproliferative activity would be attractive as therapeutic agents. We investigated the effects of two different types of less calcemic vitamin D compounds, 25-hydroxyvitamin D3 [25(OH)D3] and 19-nor-1alpha,25-dihydroxyvitamin D2 [19-nor-1,25(OH)2D2], and compared their activity to 1alpha,25(OH)2D3 on (a) the proliferation of primary cultures and cell lines of human prostate cancer cells; and (b) the transactivation of the VDRs in the androgen-insensitive PC-3 cancer cell line stably transfected with VDR (PC-3/ VDR). 19-nor-1alpha,25(OH)2D2, an analogue of 1alpha,25(OH)2D3 that was originally developed for the treatment of parathyroid disease, has been shown to be less calcemic than 1alpha,25(OH)2D3 in clinical trials. Additionally, we recently showed that human prostate cells in primary culture possess 25(OH)D3-1alpha-hydroxylase, an enzyme that hydroxylates the inactive prohormone, 25(OH)D3, to the active hormone, 1alpha,25(OH)2D3, intracellularly. We reasoned that the hormone that is formed intracellularly would inhibit prostate cell proliferation in an autocrine fashion. We found that 1alpha,25(OH)2D3 and 19-nor-1alpha,25(OH)2D2 caused similar dose-dependent inhibition in the cell lines and primary cultures in the [3H]thymidine incorporation assay and that both compounds were significantly more active in the primary cultures than in LNCaP cells. Likewise, 25(OH)D3 had inhibitory effects comparable to those of 1alpha,25(OH)2D3 in the primary cultures. In the chloramphenicol acetyltransferase (CAT) reporter gene transactivation assay in PC-3/ VDR cells, 1alpha,25(OH)2D3 and 19-nor-1alpha,25(OH)2D2 caused similar increases in CAT activity between 10(-11)and 10(-9) M. Incubation of PC-3/VDR cells with 5 x 10(-8) M 25(OH)D3 induced a 29-fold increase in CAT activity, similar to that induced by 10(-8) M 1alpha,25(OH)2D3. In conclusion, our data indicate that 25(OH)D3 and 19-nor-1alpha,25(OH)2D2 represent two different solutions to the problem of hypercalcemia associated with vitamin D-based therapies: 25(OH)D3 requires the presence of 1alpha-hydroxylase, whereas 19-nor-1alpha,25(OH)2D2 does not. Both drugs are approved for human use and may be good candidates for human clinical trials in prostate cancer.


Assuntos
Calcifediol/farmacologia , Ergocalciferóis/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Contagem de Células/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Relação Dose-Resposta a Droga , Humanos , Masculino , Neoplasias da Próstata/patologia , Receptores de Calcitriol/genética , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Timidina/metabolismo , Ativação Transcricional/efeitos dos fármacos , Trítio , Células Tumorais Cultivadas
8.
Prostate ; 42(2): 137-44, 2000 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-10617871

RESUMO

BACKGROUND: Type 1 vasoactive intestinal peptide receptor (VIP1R) is expressed in many secretory epithelial cells. We investigated VIP1R expression as a marker of prostate secretory epithelial differentiation in normal and malignant prostate tissues and in PC-3 human prostate cancer cells either lacking or expressing a functional androgen receptor. METHODS: VIP1R mRNA in rat prostate was assessed by in situ hybridization. VIP1R mRNA in human prostate tissue was identified by Northern blot hybridization. VIP1R mRNA expression in human prostate cancer cell lines in the presence or absence of androgen was determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Human prostate cell lines were treated with VIP, and changes in intracellular cAMP were measured by radioimmunoassay. RESULTS: VIP1R mRNA was expressed only in epithelial cells in normal rat prostate. VIP1R mRNA was present in both normal and malignant human prostate. Well-differentiated LNCaP cells expressed functional VIP receptors, while poorly differentiated PC-3 cells did not. PC-3 cells stably expressing the androgen receptor (PC3/AR) did express functional VIP receptors, but VIP1R mRNA levels were not androgen-regulated. CONCLUSIONS: VIP1R expression indicates epithelial differentiation in normal and malignant prostate. PC3/AR cells and LNCaP cells, both of which express VIP1R and prostate-specific antigen (PSA), are the only prostate cancer cell lines known to express these two markers of prostate epithelial differentiation.


Assuntos
Diferenciação Celular , Regulação Neoplásica da Expressão Gênica , Próstata/citologia , Neoplasias da Próstata/patologia , Receptores Androgênicos/fisiologia , Receptores de Peptídeo Intestinal Vasoativo/biossíntese , Animais , Biomarcadores Tumorais/análise , Northern Blotting , Células Epiteliais/fisiologia , Humanos , Hibridização In Situ , Masculino , Neoplasias da Próstata/química , Neoplasias da Próstata/genética , Ratos , Receptores de Peptídeo Intestinal Vasoativo/análise , Receptores de Peptídeo Intestinal Vasoativo/genética , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo , Células Tumorais Cultivadas
9.
Mol Endocrinol ; 13(11): 1896-911, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10551783

RESUMO

The androgen receptor (AR) gene is transcriptionally regulated by AR (autoregulation); however, the androgen response elements (AREs) required for this process have not been found in the AR promoter or in the 5'-flanking region. We previously showed that the AR cDNA contains AREs involved in AR mRNA autoregulation and that auto(up)regulation is reproduced in PC3 cells (a human prostate cancer cell line) expressing the human AR cDNA driven by a heterologous promoter. A 350-bp fragment of the AR cDNA contains the requisite AREs (ARE-1 and ARE-2) and, when linked upstream of a reporter gene, confers androgen inducibility in a cell-specific manner. Here we report that, although an AR cDNA harboring silent mutations of ARE-1 and ARE-2 produces a transcriptionally active AR, AR mRNA encoded by this mutant cDNA is not up-regulated in androgen-treated PC3 cells. Thus, ARE-1 and ARE-2 are essential for androgen-mediated up-regulation of AR mRNA in this model. Since ARE-1 and ARE-2 are located on separate exons (exons D and E) in the AR gene, we evaluated these AREs in their native context, a 6.5-kb AR genomic fragment. Androgen regulated the 6.5-kb AR genomic fragment and the 350-bp region of the AR cDNA at comparable levels, suggesting that sequences in exons D and E are likely to be involved in androgen-mediated up-regulation of the native AR gene. Furthermore, androgen regulated both responsive regions in U2OS cells, a human osteoblastic cell line that exhibits androgen-mediated up-regulation of native AR mRNA. DNAse I footprinting of the 350-bp region with recombinant AR (DNA- and ligand-binding domains) suggested the presence of additional AREs. Gel shift analyses and mutational studies showed that maximal androgen regulation and AR binding were dependent on the integrity of four AREs (ARE-1, ARE-1A, IVSARE, and ARE-2). While the presence of multiple, nonconsensus AREs is common among other androgen-regulated enhancers, the androgen-responsive region of the AR gene is unique because it contains exonic AREs. DNA binding studies with nuclear extracts were performed to determine whether non-AR transcription factors contribute to androgen regulation of the 350-bp region. These studies, in conjunction with mutational analysis and reporter gene assays with dominant negative Myc and Max expression vectors, showed that Myc and Max interaction with a Myc consensus site is required for androgen regulation of the 350-bp fragment. These results represent a novel interaction between AR and the Myc family of proteins and support a model of androgenic control of AR mRNA via AR and Myc family interaction with a unique internal androgen-responsive region harboring multiple exonic regulatory sequences.


Assuntos
Proteínas Proto-Oncogênicas c-myc/genética , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Elementos de Resposta/genética , Fatores de Transcrição , Androgênios/metabolismo , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Fatores de Transcrição de Zíper de Leucina Básica , Sítios de Ligação , Pegada de DNA , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Humanos , Masculino , Dados de Sequência Molecular , Mutação , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Células Tumorais Cultivadas , Regulação para Cima
10.
Cancer Epidemiol Biomarkers Prev ; 8(3): 241-8, 1999 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10090302

RESUMO

The steroid hormone 1,25-dihydroxyvitamin D [1,25(OH)2D, also known as calcitriol] is known to inhibit the proliferation and to promote the differentiation of human prostate cancer cells. Additionally, we showed that 1,25(OH)2D markedly inhibits the invasiveness of human prostate cancer cells in vitro (G. G. Schwartz et al., Cancer Epidemiol. Biomark. Prev., 6: 727-732, 1997). These properties support the use of 1,25(OH)2D as differentiation therapy in prostate cancer. However, the use of 1,25(OH)2D in vivo is limited by the risk of hypercalcemia. We therefore compared the effects of 1,25(OH)2D and of EB1089, an analogue of 1,25(OH)2D with reduced calcemic effects, in an in vivo model of androgen-insensitive metastatic prostate cancer, the rat Dunning MAT LyLu prostate cancer model. Tumor growth and metastasis were studied using Copenhagen rats given s.c. injections of MAT LyLu cells. Fifty male rats were divided into five groups of 10 rats each. Four experimental groups received i.p. injections of low and high doses of 1,25(OH)2D and EB1089 (0.5 and 1.0 microg/kg, low and high, respectively). A control group received injections of vehicle only. Tumor volumes were measured three times per week. Rats were weighed weekly. The number of metastases to the lungs and the extent of hypercalcemia were evaluated. Compared with controls, tumor volumes were significantly smaller in all experimental groups. Similarly, the number of lung metastases (number of foci/lung) was reduced markedly by both 1,25(OH)2D and EB1089. Control rats developed 22.7 (+/- 1.98 SE) tumor foci per lung. Rats treated with 1,25(OH)2D and with EB1089 (1.0 microg/kg) developed 10.4 (+/- 2.81) and 7.70 (+/- 1.29) tumor foci, respectively (P < 0.001 and P < 0.0001, respectively; drug versus control). Compared with controls (10.79 +/- 0.1 mg/dl), serum calcium levels were significantly elevated in both 1,25(OH)2D and EB1089-treated rats (P < 0.01). However, EB1089 was significantly less calcemic than 1,25(OH)2D (12.59 +/- 0.21 mg/dl versus 14.47 +/- 0.46 mg/dl; 1.0 microg/kg; P < 0.001). Rats treated with 1,25(OH)2D showed marked weight loss: 20.0 +/- 1.9% and 26.3 +/- 1.7% of their initial weight (low and high doses, respectively, P < 0.001). Weight loss was significantly lower in rats treated with EB1089 at the high dose 8.4 (+/- 2.9) %. Moreover, rats treated with low-dose EB1089 gained 5.2 (+/- 3.7) % of their initial weight. In conclusion, 1,25(OH)2D and EB1089 showed marked and equivalent inhibition of prostate cancer metastasis in vivo. EB1089 was significantly less calcemic than 1,25(OH)2D and did not induce severe weight loss. This is the first report of a vitamin D analogue that significantly inhibits prostate cancer metastasis in vivo and that does so without producing cachexia or unacceptable hypercalcemia.


Assuntos
Antineoplásicos/uso terapêutico , Calcitriol/análogos & derivados , Calcitriol/uso terapêutico , Neoplasias Pulmonares/secundário , Neoplasias da Próstata/tratamento farmacológico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Caquexia/prevenção & controle , Calcitriol/administração & dosagem , Calcitriol/efeitos adversos , Cálcio/sangue , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Hipercalcemia/prevenção & controle , Injeções Intraperitoneais , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/prevenção & controle , Masculino , Transplante de Neoplasias , Veículos Farmacêuticos , Neoplasias da Próstata/patologia , Ratos , Células Tumorais Cultivadas , Aumento de Peso , Redução de Peso
11.
Endocrinology ; 139(3): 1197-207, 1998 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9492054

RESUMO

1Alpha,25-dihydroxyvitamin D3 (1,25 D), the most active metabolite of vitamin D3, exerts antiproliferative and prodifferentiating effects on some human prostate cancer cell lines. We previously reported an inverse relationship between functional vitamin D receptor (VDR) levels and antiproliferative response to 1,25 D in two human prostate cancer cell lines, LNCaP and ALVA 31. Although LNCaP cells are far more sensitive to growth inhibition by 1,25 D than ALVA 31 cells, LNCaP express approximately half the number of VDR as ALVA 31. Two other human prostate cancer cell lines studied, PC3 and DU145, express lower levels of functional VDR and are relatively insensitive to growth inhibition by 1,25 D. In this report, we investigated potential mechanisms of the variable antiproliferative activity of 1,25 D. In PC3 cells stably expressing VDR [PC3(VDR)] at levels comparable to LNCaP, 1,25 D treatment resulted in only moderate growth inhibition. These results further support the contention that VDR expression, although required, is not sufficient for maximal growth suppression by 1,25 D, as is exhibited by LNCaP cells. We did not detect 1,25 D-mediated DNA fragmentation after 4 days of 1,25 D treatment in either LNCaP or ALVA 31 cells. This result suggests that variability in 1,25 D sensitivity does not derive from differences in the capacity of these cells to undergo apoptosis in response to 1,25 D. Flow cytometry of propidium iodine-stained cells revealed that 48 h 1,25 D treatment of LNCaP cells resulted in a 2-fold decrease of cells in G2/M plus S phases and accumulation of LNCaP cells in the G1/G0 phase. This effect persisted for 72 h after 1,25 D removal. In contrast, 1,25 D did not significantly alter the cell cycle distribution of ALVA 31 or PC3(VDR) cells. Consistent with accumulation of cells in G1/G0, 1,25 D treatment of LNCaP cells resulted in decreased retinoblastoma protein phosphorylation, repressed E2F transcriptional activity, increased levels of the cyclin-dependent kinase (CDK) inhibitor p21(WAF1, CIP1), and decreased CDK2 activity. However, p21 messenger RNA levels were not altered, suggesting translational or posttranslational regulation of p21 by 1,25 D. In contrast, p21 was not detected in ALVA 31 or PC3(VDR) and was not induced by 1,25 D, consistent with the failure of 1,25 D to influence cell cycle distribution in these cells. These results suggest that variability in sensitivity to the antiproliferative effects of 1,25 D among prostate cancer cells is dependent, at least in part, on the integrity of the retinoblastoma pathway and in particular on p21 expression and 1,25 D regulation of CDK2 activity.


Assuntos
Antineoplásicos/farmacologia , Quinases relacionadas a CDC2 e CDC28 , Calcitriol/farmacologia , Proteínas de Transporte , Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes/antagonistas & inibidores , Proteínas de Ligação a DNA , Fase G1 , Neoplasias da Próstata/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/análise , Ciclinas/genética , Fragmentação do DNA/efeitos dos fármacos , Fatores de Transcrição E2F , Humanos , Masculino , Fosforilação , Neoplasias da Próstata/patologia , Proteína do Retinoblastoma/metabolismo , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Fatores de Transcrição/genética , Células Tumorais Cultivadas
12.
Bone ; 21(1): 49-56, 1997 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9213007

RESUMO

Androgen excess and deficiency affect skeletal maturation and bone cell function. Understanding the molecular basis for these androgen effects could improve therapy/prevention of short stature and osteoporosis. Androgens act through binding to androgen receptors (ARs), which modulate gene transcription via interactions with DNA response elements on target genes. Because osteoblasts contain ARs at levels just below certain androgen-sensitive tissues, we sought to define the function of AR in a number of commonly used osteoblastic cell lines. Presence and quantification of AR protein and mRNA were evaluated by ligand binding assay, western blotting, and RNAse protection assay. AR-containing osteoblastic cell lines were exposed to nonaromatizable androgens and effects on gene expression were assessed. We found no evidence for direct effects of androgen on endogenous genes nor was androgen involved in modulation of parathyroid hormone effects on early gene activation. Androgen-sensitive reporter gene constructs were stimulated by androgen only when AR cDNA expression vectors were introduced into cells by cotransfection. We conclude that, in commonly used osteoblastic cell lines, the presence of AR at the levels described here does not guarantee androgen transcriptional activity. The effects of androgen on bone in vivo may involve direct stimulation of osteoblastic cells in a different setting or stage of differentiation. Alternatively, androgen may act on bone cells other than osteoblasts, or through metabolic conversion to estrogens.


Assuntos
Androgênios/farmacologia , Osteoblastos/efeitos dos fármacos , Receptores Androgênicos/metabolismo , Transcrição Gênica/efeitos dos fármacos , Androgênios/metabolismo , Animais , Ligação Competitiva , Northern Blotting , Western Blotting , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , DNA/genética , DNA/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Genes Reporter/efeitos dos fármacos , Genes Reporter/genética , Humanos , Masculino , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Hormônio Paratireóideo/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ensaio Radioligante , Receptores Androgênicos/efeitos dos fármacos , Receptores Androgênicos/genética , Ribonucleases/metabolismo , Transcrição Gênica/genética , Ativação Transcricional , Transfecção
13.
Mol Cell Endocrinol ; 126(1): 83-90, 1997 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-9027366

RESUMO

Prostate cancer cell lines exhibit variable growth suppression by the hormonal form of vitamin D3, 1,25-Dihydroxyvitamin D3 [1,25 (OH)2D] (1,25 D3). To understand the molecular basis for this differential sensitivity to 1,25 D3, we compared growth response to 1,25 d3, vitamin D receptor (VDR) content and VDR transcriptional activity in four well-characterized human prostate cancer cell lines: LNCaP, DU145, PC-3 and ALVA-31. In PC-3 and DU145 cells, relative lack of growth inhibition by 1,25 D3 (< 10% inhibition) correlates with very low levels of VDR (9-15 fmol/mg protein) compared to classical vitamin D3 target tissues (approximately 75-200 fmol/mg protein). Transfection of DU145 and PC-3 cells with a VDR cDNA expression vector is sufficient to establish growth sensitivity to 1,25 D3, suggesting that low VDR levels are responsible for the failure of these cell lines to respond to 1,24 D3. LNCaP cells are highly sensitive to growth inhibition by 1.25 D3 (approximately 55% inhibition) and contain approximately 2-3-fold more VDR (25 fmol/mg) than the relatively 1,25 D3-insensitive PC-3 and DU145 cell lines. However, ALVA-31 cells display less than 20% growth inhibition to 1.25 D3 although they contain the highest levels of VDR (45 fmol/mg) of the four cell lines. Thus, sensitivity to growth inhibition by 1,25 D3 does not correlate with VDR content in ALVA-31 and LNCaP cells. This lack of correlation between VDR density and growth responses to 1,25 D3 led us to investigate VDR-mediated gene transcription in these cell lines. We employed two different naturally occurring vitamin D response elements (VDREs) linked to a reporter gene. Reporter gene activation by 1,25 D3 correlated well with VDR content in all four cell lines. Therefore, compared to LNCaP cells, decreased sensitivity of ALVA-31 to growth inhibition by 1,25 D3 is not due to a decrease in the general transcriptional activity of VDR. We conclude that growth inhibition by 1,25 D3 in prostate cancer cells requires VDR but that this response is modulated by non-receptor factors that are cell line-specific.


Assuntos
Divisão Celular/efeitos dos fármacos , Neoplasias da Próstata/patologia , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Vitamina D/farmacologia , Calcitriol/farmacologia , Cloranfenicol O-Acetiltransferase/genética , Humanos , Masculino , Plasmídeos , Neoplasias da Próstata/metabolismo , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas
14.
Mol Endocrinol ; 10(12): 1582-94, 1996 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8961268

RESUMO

In most cells and tissues containing androgen receptors (ARs), androgen regulates the levels of AR messenger RNA (mRNA). As the AR concentration is correlated with androgen responsiveness, this autoregulation of AR mRNA may affect cellular sensitivity to androgens. Androgens decrease levels of AR mRNA in many cell lines and tissues; however, in some tissues and possibly also at certain developmental stages, AR mRNA is up-regulated by androgens. Sequences within the 5'-flanking region and AR promoter do not appear to be sufficient for androgen regulation of AR mRNA. We have previously shown that both down- and up-regulation of AR mRNA by androgen can be reproduced in cell lines expressing a transfected human AR complementary DNA (cDNA). Sequences within the AR cDNA confer this autoregulation in transfected cells, suggesting that sequences within the transcribed region of the AR gene are sufficient for autoregulation. In this study we have determined the mechanism of androgenic up-regulation of AR mRNA encoded by the human AR cDNA in the prostate cancer cell line, PC3, and have identified the cis-acting sequences of the AR cDNA that are required. The observations that actinomycin D blocked androgenic up-regulation of AR mRNA but cycloheximide had no effect are consistent with a model in which AR is directly involved in transcriptional up-regulation of AR cDNA expression. Nuclear run-on assays showed that androgen treatment resulted in increased transcription of the AR cDNA. Furthermore, a 350-bp AR cDNA fragment inserted 5' of a thymidine kinase promoter-chloramphenicol acetyltransferase gene conferred androgen induction of chloramphenicol acetyltransferase activity in PC3 cells. This 350-bp fragment, which is located in the AR coding region, contains two putative androgen response elements (AREs) separated by 182 bp. The 5'-most ARE (ARE-1, 5'-TGTCCT-3') resembles a half-site of the palindromic consensus hormone response element, recognized by several steroid receptors, including AR, and the 3'-sequence (ARE-2, 5'-AGTACTCC-3') is identical to a portion of an androgen-responsive region found in the rat probasin gene promoter. Analysis of either ARE-1 or ARE-2 mutants revealed that these elements function synergistically. AR protein binds to the 350-bp fragment, as demonstrated by electrophoretic mobility shift assays using a glutathione-S-transferase-AR fusion protein containing the DNA- and steroid-binding domains of AR. These results indicate that the AR coding region contains an androgen-responsive region that is involved in cell line-specific up-regulation of AR mRNA.


Assuntos
Androgênios/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Regulação para Cima , Androgênios/farmacologia , Sequência de Bases , Sítios de Ligação , Cicloeximida/farmacologia , DNA Complementar/genética , DNA Complementar/metabolismo , Dactinomicina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Cinética , Masculino , Metribolona/farmacologia , Dados de Sequência Molecular , Especificidade de Órgãos , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Receptores Androgênicos/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas
15.
Steroids ; 61(9): 531-9, 1996 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8883219

RESUMO

The expression of the androgen receptor (AR) gene is regulated by androgens. Although androgens down-regulate AR mRNA in most cell lines and tissues, including the prostate, up-regulation occurs in some tissues. Androgen-mediated reduction in AR mRNA is reproduced in COS1 cells and in the androgen-sensitive human prostate cancer cell line LNCaP when each expresses the AR cDNA. We have previously established that the AR cDNA contains the requisite sequences for this down-regulation. Here we shown that androgen promoted up-regulation of AR mRNA in two androgen-independent human prostate cancer cell lines, PC3 and DU145, when each was transfected with a human AR cDNA. This effect was due to the AR cDNA and not to the heterologous promoter driving AR expression. In addition to up-regulation of AR mRNA, androgen induced comparable increases in AR protein levels in PC3 cells stably expressing an AR cDNA (PC3/AR). Up-regulation of AR in PC3/AR cells was accompanied by failure of these cells to undergo desensitization or inactivation of AR following prolonged (96 h) androgen administration, whereas the same conditions resulted in desensitization of AR transactivation in LNCaP cells and in CVl cells that stably express the AR cDNA. Androgen treatment of PC3/AR cells resulted in induction of an androgen-regulated reporter gene (MMTV-CAT) as well as the native prostate-specific antigen gene, which is silent in untransfected PC3 but is androgen up-regulated in LNCaP and in the prostate. These results suggest that ectopic expression of AR in androgen-independent prostate cancer cell lines establishes both typical and atypical androgenic responses in a target gene-specific manner. Androgenic up-regulation of AR cDNA expression may be due to distinct signaling mechanisms that influence androgen action in androgen-independent prostate cancer cells.


Assuntos
Adenocarcinoma/genética , Androgênios/farmacologia , Neoplasias da Próstata/genética , Receptores Androgênicos/genética , Regulação para Cima/genética , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Animais , Células Cultivadas , Cloranfenicol O-Acetiltransferase/efeitos dos fármacos , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Citomegalovirus/genética , DNA Complementar/biossíntese , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Vetores Genéticos/genética , Haplorrinos , Homeostase , Humanos , Rim/citologia , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Vírus do Tumor Mamário do Camundongo/genética , Regiões Promotoras Genéticas , Antígeno Prostático Específico/efeitos dos fármacos , Antígeno Prostático Específico/genética , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , RNA Mensageiro/biossíntese , Receptores Androgênicos/metabolismo , Transfecção , Células Tumorais Cultivadas
16.
Mol Cell Endocrinol ; 115(2): 177-86, 1995 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-8824893

RESUMO

Androgen receptor (AR) levels are regulated by androgens, other steroids and non-steroidal hormones via complex, tissue-specific processes. Since alterations in receptor levels may influence cellular sensitivity to androgens, understanding AR regulation is of fundamental and potentially therapeutic significance. In most target tissues and AR-containing cell lines, AR mRNA is down-regulated in response to androgens. We have reconstituted this androgen-mediated down-regulation of AR mRNA in COS 1 cells transfected with a human AR cDNA under the control of the cytomegalovirus (CMV) promoter. The sequences mediating receptor mRNA down-regulation are represented within the AR cDNA and not within the CMV promoter. Androgenic down-regulation of AR cDNA expression was time- and dose-dependent, resembling native AR mRNA down-regulation. In addition, androgenic regulation of the receptor cDNA was not dependent on protein synthesis suggesting that AR and/or another pre-existing protein(s) is involved in this process. In COS 1 cells co-transfected with androgen and glucocorticoid receptor cDNAs, dexamethasone mimicked the action of androgen in down-regulating AR mRNA. This response depended on glucocorticoid receptors. Androgen had little effect on steady-state levels of AR protein consistent with reports that androgen down-regulates AR mRNA but increases AR protein half-life (Kemppainen et al. (1992) J. Biol. Chem. 267, 968-974; Zhou et al. (1995) Mol. Endocrinol. 9, 208-218). However, glucocorticoids decreased AR protein levels in cells that co-expressed androgen and glucocorticoid receptors. These results indicate that sequences represented in the AR cDNA mediate AR mRNA down-regulation by both androgens and glucocorticoids. Inhibition of AR mRNA and protein by glucocorticoids suggests that these steroids may modulate androgen action in tissues, such as mammary gland and prostate, which express both androgen and glucocorticoid receptors.


Assuntos
Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Metribolona/farmacologia , Receptores Androgênicos/genética , Congêneres da Testosterona/farmacologia , Linhagem Celular , Acetato de Ciproterona/farmacologia , Citomegalovirus/genética , DNA Complementar/genética , Relação Dose-Resposta a Droga , Regulação para Baixo , Flutamida/análogos & derivados , Flutamida/farmacologia , Expressão Gênica/genética , Humanos , Metribolona/antagonistas & inibidores , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Congêneres da Testosterona/antagonistas & inibidores , Fatores de Tempo , Transfecção , Células Tumorais Cultivadas
17.
J Steroid Biochem Mol Biol ; 55(2): 135-46, 1995 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-7495692

RESUMO

We have analyzed structural and functional features of the human glucocorticoid receptor (hGR) for their effects on receptor subcellular distribution. COS 1 cells transiently transfected with wild type and mutant hGR cDNAs were assessed immunocytochemically using well-characterized antipeptide antibodies to the hGR. The effect of administration of steroid hormones (and the antiglucocorticoid RU486) on receptor localization was evaluated. Unliganded wild type receptors expressed in COS 1 cells were predominately cytoplasmic. Addition of glucocorticoids or the glucocorticoid receptor antagonist, RU486, resulted in complete translocation of these receptors into the nucleus whereas non-glucocorticoid steroids or dibutyryl cAMP were not effective in promoting nuclear translocation. Thus, nuclear translocation was specific for steroids capable of high affinity binding to the hGR. To elucidate the potential role of receptor domains in receptor localization, COS 1 cells transiently transfected with various receptor cDNA mutants were analyzed in a similar manner. Translocation of an hGR deletion mutant lacking the majority of the amino terminus (deletion of amino acids 77-262) was identical to the wild type receptor despite the absence of a transactivation domain. Receptors in which the DNA binding domain was either partially or totally deleted showed an impaired capacity to undergo hormone-inducible nuclear translocation. Deletion of the hinge region of the hGR (which also contains part of the nuclear localization signal, NL1) resulted in receptor localization in the cytoplasm. Mutants in the ligand binding domain exhibited two localization phenotypes, exclusively nuclear or cytoplasmic. Receptor mutants truncated after amino acid 550 were found in the nucleus in the presence and absence of hormone consistent with the existence of nuclear localization inhibitory sequences in the ligand binding domain of the receptor. However, a linker insertion mutant (at amino acid 582) which results in a receptor deficient in ligand binding did not undergo nuclear translocation indicating that nuclear localization inhibitory sequences were intact in this mutant. The role of receptor phosphorylation on hormone induced nuclear translocation was also examined. Mouse glucocorticoid receptors which contained mutations of certain hormone inducible phosphorylation sites exhibited translocation properties similar to wild type mGR indicating that these phosphorylation sites on the receptor do not play a major role in hormone inducible nuclear translocation.


Assuntos
Núcleo Celular/metabolismo , Glucocorticoides/farmacologia , Receptores de Glucocorticoides/metabolismo , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Chlorocebus aethiops , DNA Complementar , Dexametasona/farmacologia , Humanos , Imuno-Histoquímica/métodos , Rim , Camundongos , Mifepristona/farmacologia , Mutagênese , Mutagênese Insercional , Fosforilação , Receptores de Glucocorticoides/análise , Receptores de Glucocorticoides/biossíntese , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Transfecção
18.
Mol Endocrinol ; 8(12): 1764-73, 1994 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-7708063

RESUMO

Glucocorticoid receptors (GR) are ligand-dependent transcription factors that play a critical role in the endocrine control of cell growth, differentiation, and death. These steroid receptors are widely recognized to undergo down-regulation after exposure to ligand in cell cultures and animals, including humans. This reduction in cellular receptor levels leads to insensitivity to subsequent hormone administration. The mechanisms controlling homologous down-regulation of the GR are, however, poorly understood. We have previously shown (1) that a transfected human GR (hGR) complementary DNA (cDNA) contains sequences that are sufficient to recapitulate the down-regulation of both hGR messenger RNA (mRNA) and protein seen in vivo. We have now evaluated potential mechanisms involved in the hormonal regulation of the hGR mRNA and, further, have identified an intragenic domain of the hGR cDNA that contains the down-regulatory signal. Glucocorticoid treatment of COS-1 cells expressing a transfected hGR cDNA resulted in down-regulation of the hGR mRNA in the presence of cycloheximide or actinomycin-D, suggesting that a glucocorticoid-inducible protein was not essential for down-regulation. We show that prolonged receptor occupation by ligand leads to increased GR mRNA turnover, and furthermore, that either the agonist dexamethasone or the antagonist RU486 decreased transcription of the hGR cDNA. To resolve which receptor cDNA sequences are critical in down-regulation, a cotransfection strategy was employed in which a series of hGR cDNA deletion mutants was transfected in conjunction with the full-length hGR cDNA. The effects of glucocorticoid on the regulation of receptor mRNAs encoded by the mutant receptor cDNAs were examined. Deletions within the 5' half of the receptor cDNA produced transcripts that were susceptible to glucocorticoid-mediated down-regulation, whereas deletion of sequences located in the 3'-end of the receptor-coding sequence (corresponding to amino acids 550-697) resulted in receptor transcripts that were only minimally down-regulated by glucocorticoid. Together these studies indicate that multiple mechanisms control GR mRNA abundance, and an intragenic element within the ligand-binding domain is critical for this down-regulation.


Assuntos
DNA Complementar/genética , Regulação para Baixo , RNA Mensageiro/metabolismo , Receptores de Glucocorticoides/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Cicloeximida/farmacologia , DNA Complementar/química , Dactinomicina/farmacologia , Dexametasona/farmacologia , Deleção de Genes , Humanos , Rim , Mifepristona/farmacologia , Receptores de Glucocorticoides/metabolismo , Relação Estrutura-Atividade , Transcrição Gênica/efeitos dos fármacos , Transfecção
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