RESUMO
Understanding how cancer genes are mutated in individual tumors is an important issue with potential clinical and therapeutic impact. This is especially relevant with recently developed targeted therapies since mutated genes can be targets and/or predictors. However, to date, gene mutation profiling in individual tumors is still underexplored. Breast cancer is composed of various subtypes. We presumed that this heterogeneity reflected the involvement of different molecular mechanisms including gene mutations that affect defined signaling pathways. Unlike the majority of published mutational studies, this study was aimed to draw a mutation profile in individual tumors by screening a panel of cancer genes in the same tumor. Thus, five genes frequently mutated in breast cancers: TP53, PIK3CA, PTEN, CDH1, and AKT1 were screened in each of 120 human primary breast tumors. Mutations in at least one of these genes were found in 62.5% of the tumors, of which the majority carried a single-gene mutation. Interestingly, a substantial proportion of tumors carried mutations either in TP53 or in genes of the PI3K pathway (PIK3CA or PTEN or AKT1). These two distinct mutation patterns were significantly associated to hormone receptor expression but independent of HER2 status.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Carcinoma Lobular/genética , Fosfatidilinositol 3-Quinases/genética , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Antígenos CD , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Caderinas/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/mortalidade , Carcinoma Lobular/metabolismo , Carcinoma Lobular/mortalidade , Classe I de Fosfatidilinositol 3-Quinases , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas c-akt/genética , Receptores de Esteroides/metabolismo , Transdução de Sinais/genéticaRESUMO
The proto-oncoprotein Cbl is known to control several signaling processes. It is highly expressed in the testis, and because spermatogenesis is androgen dependent, we investigated the androgen dependency expression of Cbl through its testicular sub-localization and its expression levels in rats that were exposed to the antiandrogen flutamide or were hypophysectomized. We report the androgen dependency of Cbl as it localizes in pachytene spermatocytes during androgen-dependent stages, is down-regulated upon flutamide exposure, and is up-regulated with testosterone in hypophysectomized rats. Coculture experiments showed the key control exerted by the Sertoli cell on Cbl activity. As flutamide induces germ cell apoptosis, we investigate members of the Bcl-2 family upon flutamide exposure. We show that the proapoptotic Bcl-2 family member Bim mirrored Cbl expression through a posttranscriptional process. We also show that in Cbl knockout mouse testes, the imbalance between the high expression of Bim and Smac/Diablo and anti-apoptotic factors such as cellular inhibitor of apoptosis 2 favors a survival process, which makes these mice unresponsive to androgen withdrawal and could explain their hypofertility.
Assuntos
Androgênios/metabolismo , Apoptose , Regulação da Expressão Gênica , Células Germinativas/metabolismo , Proteínas Proto-Oncogênicas c-cbl/fisiologia , Antagonistas de Androgênios/farmacologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Proteínas de Transporte/metabolismo , Técnicas de Cocultura , Flutamida/farmacologia , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Imunoprecipitação , Proteínas Inibidoras de Apoptose/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Processamento Pós-Transcricional do RNA , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células de Sertoli/metabolismo , Espermatócitos/metabolismo , Testículo/metabolismo , Testosterona/metabolismo , Timo/metabolismo , Fatores de Tempo , Transcrição Gênica , Regulação para CimaRESUMO
PURPOSE: Gastrointestinal stromal tumors (GIST) are frequently associated with gain-of-function mutations of KIT, which can be inhibited by imatinib both in vitro and in vivo. The survival of patients with GIST, following imatinib therapy, has been correlated with the nature of mutations but not with KIT expression. EXPERIMENTAL DESIGN: Subcellular localization, activation, and trafficking of the mature and the immature forms of KIT were investigated in GIST samples and in NIH3T3 cells infected with two different GIST-type exon 11-mutated human KIT cDNA. RESULTS: Paranuclear dot expression of KIT was more frequent in GISTs with homozygous KIT mutations than in those with heterozygous (P = 0.01) or no mutations (P < 0.01). Activation of the immature 125 kDa form of KIT was detected in most GISTs with KIT mutations but not in GISTs without KIT mutations. In NIH3T3 cells, mutant KIT was mainly retained within endoplasmic reticulum and Golgi compartments in an immature constitutively phosphorylated form, whereas the wild-type KIT was expressed at the plasma membrane, in a mature nonphosphorylated form. Imatinib-induced inhibition of the phosphorylation of immature and mature mutant KIT proteins resulted in the restoration of KIT expression at the cell surface. CONCLUSIONS: These results show that GIST-type KIT mutations induce an activation-dependent alteration of normal maturation and trafficking, resulting in the intracellular retention of the activated kinase within the cell. These observations likely account for the absence of correlation between response to imatinib and KIT expression using immunohistochemistry and may deserve to be investigated in other tyrosine kinase-activated tumors.
Assuntos
Tumores do Estroma Gastrointestinal/metabolismo , Mutação , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Animais , Tumores do Estroma Gastrointestinal/genética , Humanos , Camundongos , Células NIH 3T3 , Fosforilação , Fator de Células-Tronco/farmacologiaRESUMO
The purpose of this present study was to investigate the role of inhibitor of apoptosis proteins (IAPs), their inhibitor, Smac, and the focal adhesion kinase (FAK) in the laminin enhancement of cisplatin-induced apoptosis in a testicular tumor germ cell line, NCCIT. We demonstrated that specifically in laminin-adherent NCCIT cells, cisplatin-induced apoptosis followed a significant decrease of c-IAP-2 protein expression and of both XIAP mRNA and protein expression. Smac expression was not modified in any tested conditions. We also found that FAK, which mediates the ECM-integrin antiapoptotic signal was down-regulated early in cells cultured on laminin. Our results provide a possible mechanistic explanation to cisplatin-induced apoptosis in NCCIT cells adhered on laminin. Cisplatin down-regulated FAK protein expression early, which therefore failed to activate c-IAP-2 and XIAP expression, resulting in a defect in the abrogation of the activation of caspases. Thus, the laminin signaling enhances the cisplatin-induced apoptosis, at least partly, through a down-regulation of survival signal molecules. Our data raise an interesting possibility that a therapeutic strategy targeting these survival molecules would be efficient to overcome chemo-resistance in testicular germ cell tumor.
Assuntos
Antineoplásicos/farmacologia , Apoptose , Cisplatino/farmacologia , Quinase 1 de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Inibidoras de Apoptose/metabolismo , Laminina/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação para Baixo , Quinase 1 de Adesão Focal/genética , Humanos , Proteínas Inibidoras de Apoptose/genética , Masculino , Neoplasias Embrionárias de Células Germinativas , RNA Mensageiro/metabolismo , Neoplasias Testiculares , Fatores de Tempo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
In utero exposure to chemicals with antiandrogen activity induces undescended testis, hypospadias, and sub- or infertility. The hypospermatogenesis observed in the adult rat testis exposed in utero to the antiandrogen flutamide has been reported to be a result of a long-term apoptotic cell death process in mature germ cells. However, little if anything is known about the upstream signaling mechanisms controlling this apoptosis. In the present study, we have investigated the possibility that the TGF-beta signaling pathway may be at play in this control of the apoptotic germ cell death process. By using a model of adult rat exposed in utero to 0, 0.4, 2, or 10 mg/kg.d flutamide, we observed that pro-TGF-beta signaling members, such as the three isoforms of TGF-beta ligands (TGF-beta1-3), the two TGF-beta receptors (TGF-betaRI and -RII) and the R-Smads Smad 1, Smad 2, Smad 3, and Smad 5 were inhibited at the mRNA and protein levels, whereas the anti-TGF-beta signaling member Smad 7 was overexpressed. Furthermore, we report that the overexpression of Smad 7 mRNA could induce an activation of c-Jun N-terminal kinase, because of the observed c-Jun overexpression, activation, and nuclear translocation leading to an increase in the transcription of the proapoptotic factor Fas-L. Together, the alterations of TGF-beta signaling may represent upstream mechanisms underlying the adult germ cell apoptotic process evidenced in adult rat testis exposed in utero to antiandrogenic compounds such as flutamide.
Assuntos
Antagonistas de Androgênios/farmacologia , Apoptose/efeitos dos fármacos , Feto/efeitos dos fármacos , Transdução de Sinais , Espermatozoides/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Envelhecimento/metabolismo , Antagonistas de Androgênios/administração & dosagem , Animais , Relação Dose-Resposta a Droga , Proteína Ligante Fas , Feminino , Flutamida/administração & dosagem , Flutamida/farmacologia , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Smad/genética , Proteína Smad1/metabolismo , Proteína Smad7/metabolismo , Testículo/citologia , Testículo/metabolismo , Fatores de Tempo , Distribuição Tecidual , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fatores de Necrose Tumoral/metabolismoRESUMO
Although it is established that in utero exposure to antiandrogenic compounds such as flutamide induces hypospermatogenesis in adult male rat offspring, the cellular and molecular mechanisms remain to be investigated. By using adult rats exposed in utero to flutamide (0.4, 2, 10 mg/kg.d) as a model, we show that the hypospermatogenesis could be related to a chronic apoptotic cell death process associated with a long-term increase in caspase-3 and -6 expression and activation in germ cells. The number of apoptotic (terminal deoxynucleotidyl transferase-mediated deoxyuridine positive) adult germ cells was dependent on the dose of flutamide. The apoptotic germ cell death process could be related to an increased expression and activation of effector caspases-3 and -6. Procaspases-3 and -6 were immunodetected in germ cells from both untreated or flutamide-treated rats, whereas cleaved active caspase-3 was detected exclusively in germ cells from adult rat exposed in utero to flutamide. Exposure to the antiandrogen increased in a dose-dependent manner as caspase-3 and -6 mRNA (in RT-PCR approaches) as well as procaspase-3 and -6 protein (in Western blotting analyses) levels in the adult rat testis. Flutamide also activates procaspases. Indeed, whereas cleaved active caspase-3 and -6 proteins were absent in control animals, they were detected in adult rat testes exposed in utero to flutamide. Our results show that whereas the apoptotic germ cell death process associated with the increased caspase expression and activation in adult rat germ cells was chronic and nonreversible when exposure to flutamide occurred in utero, it was transient when such an exposure occurred during adulthood. Indeed, although an increase in caspase-3 and -6 mRNA and procaspase-3 and -6 protein levels was observed in germ cells after 3 d of exposure to flutamide, 1-2 wk after the cessation of the antiandrogen exposure, the caspase mRNA and procaspase protein levels were back to control. Active cleaved caspase-3 and -6 protein appeared following the exposure to the antiandrogen, whereas they disappeared at cessation of exposure to flutamide. In summary, the present findings indicate that in utero exposure to the antiandrogen induced in the adult rat testes a chronic apoptotic germ cell death associated with a long-term increase in the expression and activation in germ cells of caspases-3 and -6, two key components in the death machinery.
Assuntos
Antagonistas de Androgênios/farmacologia , Apoptose/efeitos dos fármacos , Caspases/genética , Flutamida/farmacologia , Efeitos Tardios da Exposição Pré-Natal , Testículo/citologia , Fatores Etários , Animais , Caspase 3 , Caspase 6 , Caspases/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Marcação In Situ das Extremidades Cortadas , Masculino , Gravidez , RNA Mensageiro/análise , Ratos , Testículo/efeitos dos fármacosRESUMO
Although it is established that in utero exposure to the antiandrogen flutamide induces alteration of spermatogenesis in the adult rat testis offspring, the cellular and molecular mechanisms involved in such an effect remain to be investigated. In the present paper, by using as model adult rats exposed in utero to flutamide (0, 2, 10 mg/kg per day), we have investigated the hypothesis that germ cell alterations could be related to defects of energy metabolism and particularly to defects of the production and transport of lactate. Lactate is a preferential energy substrate produced by Sertoli cells and transported to germ cells by monocarboxylate transporters (MCT). A significant decrease (60%, P<0.001) in lactate production was observed in cultured Sertoli cells from rat testes exposed in utero to flutamide from the dose of 2 mg/kg per day. Such a decrease is concurrent to a decrease in lactate dehydrogenase A (LDHA) mRNA levels (evaluated through semiquantitative RT-PCR) and LDHA4 activity. The decrease in LDHA mRNA levels (to 64 +/- 9% of the control, P<0.05) was observed with the lowest dose (2 mg/kg per day) of flutamide tested. The decrease in LDHA mRNA levels was observed in both the whole testis and in isolated Sertoli cells, suggesting that such a decrease in LDHA expression occurred also in the (Sertoli) cells producing lactate. Lactate is transported from Sertoli cells to germ cells via MCT1 and MCT2. We immunolocalized MCT1 to all the different germ cell types and MCT2 exclusively to elongated spermatids. In the adult testis exposed in utero to flutamide, MCT1 (53 +/- 8%, P<0.02) and MCT2 (52 +/- 9%, P<0.02) mRNA levels were significantly reduced indicating that lactate transport to germ cells could be also altered. Together, these data support (i) the existence of a relationship between the antiandrogen activity and the energy metabolism in the testis and (ii) the concept of an androgen-dependent programming, occurring early in the fetal life in relation to the expression of some of the key genes involved in the production and transport of lactate in the seminiferous tubules.
Assuntos
Antagonistas de Androgênios/farmacologia , Flutamida/farmacologia , Ácido Láctico/metabolismo , Efeitos Tardios da Exposição Pré-Natal , Testículo/metabolismo , Animais , Transporte Biológico , Feminino , Isoenzimas/genética , L-Lactato Desidrogenase/genética , Lactato Desidrogenase 5 , Ácido Láctico/biossíntese , Masculino , Transportadores de Ácidos Monocarboxílicos/genética , Gravidez , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/metabolismo , Simportadores/genética , Testículo/citologia , Testículo/efeitos dos fármacosRESUMO
The molecular basis of testicular germ cell tumourigenesis are not well elucidated. Growth factors regulate cell growth, differentiation and apoptosis. Major families of growth factors are present in the male gonad from early fetal development to adult life. They are involved in germ cell proliferation and differentiation. Growth signalling pathways suffer deregulation in many human malignancies. Given the importance of growth signals in normal testicular development and their acquired deregulation in most human cancers, growth factors and signalling molecules that have been implicated in the genesis of testicular germ cell tumours, are reviewed. We detected a somatic mutation of SMAD4 gene, responsible for loss of protein function in seminomas. This mutational inactivation may affect the activity of several members of TGFbeta superfamily (TGFbeta, activin, inhibin, BMP). VEGF expression has been shown to predict metastasis in seminomas. A significant association of HST-1 expression, a member of fibroblast growth factors, with the nonseminomatous phenotype and with tumour stage has been described. In contrast, C-KIT is expressed by seminomas only, from the preinvasive stage. Despite intense expression in almost all seminomas, activating mutation of C-KIT gene is seldom reported. Recently, the first animal model of classical testicular seminoma has been identified in transgenic mouse overexpressing GDNF. RET (GDNF receptor) expression is demonstrated in human seminomas, and not in nonseminomatous tumours. However, the exact molecular alterations of GDNF/RET/GFRalpha1 complex in germ cell tumours are not known. Finally, beside growth factors, other signalling molecules such as peptide hormones may be involved in testicular carcinogenesis. We have demonstrated a specific pattern of somatostatin receptors expression in each type of testicular germ cell tumours, with a loss of sst3 and sst4 in seminomas and loss of sst4 and expression of sst1 in nonseminomas only. These data suggest an antiproliferative action of somatostatin in testicular cancers. In summary, many growth factors and signalling molecules seem to represent specific markers for different histological types of germ cell tumours (seminomas versus nonseminomas) and may play a role in the differentiation of germ cell tumours. Despite a complex signalling pathway involved in the physiological functions of male gonad, little is known about the implication of this signalling network in testicular malignancies. From a practical stand-point, further studies on the role of growth factors in human germ cell tumours may offer a new therapeutical perspective with the development of specific pharmacological signalling modulators that could be used as therapeutic agents.
Assuntos
Germinoma/metabolismo , Substâncias de Crescimento/metabolismo , Neoplasias Testiculares/metabolismo , Animais , Transformação Celular Neoplásica , Proteínas de Ligação a DNA/metabolismo , Fatores de Crescimento Endotelial/metabolismo , Receptores ErbB/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Germinoma/patologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Masculino , Fatores de Crescimento Neural/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores de Somatostatina/metabolismo , Transdução de Sinais , Proteínas Smad , Fator de Células-Tronco/metabolismo , Neoplasias Testiculares/patologia , Testículo/patologia , Transativadores/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Leiomyosarcoma (LMS) represent 15 % of adult sarcomas. The aim of this work was to identify novel altered pathways in LMS, which may be of therapeutic value for patients. Thirteen fresh frozen samples of soft tissue and visceral LMS were analyzed and compared with normal smooth muscle uterine tissue (NSM) for phosphoproteomic profile. Four proteins were found differentially expressed including Tyro3. The functional role of Tyro3 and its ligand Gas6 was investigated in two LMS cell lines, SK-LMS-1 and CNIO-AA. Four proteins and phosphoproteins were differentially expressed in LMS samples vs NSM: A loss of FAK Y397 phosphorylation was observed in all LMSs, while Tyro3, MSH2 and PKC theta were consistently overexpressed. Gas6, the major ligand of Tyro3, was expressed in 8 of the 13 LMS samples, and Gas6 expression highly correlated to Akt Y473 phosphorylation and to a lesser extent to Erk1/2 phosphorylation. SK-LMS-1 and CNIO-AA LMS expressed Tyro3, Axl and Gas6 at high level in CNIO-AA while at low levels in SK-LMS-1. Exposure of both cell lines to foretinib, a tyrosine kinase inhibitor of Met, Axl and Tyro3, reduced cell viability and induced caspase 3/7 activation. Transfection of CNIO-AA with small interfering RNA directed against Tyro3 and Axl genes induced a reduction of the expression of the specific proteins and, when combined, significantly reduced CNIO-AA cell viability. Leiomyosarcomas overexpress Tyro3. Gas6, a ligand of Tyro3, exerts an autocrine activities though Tyro3 and Axl in a subgroup of LMS.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Leiomiossarcoma/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Neoplasias Uterinas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Leiomiossarcoma/genética , Leiomiossarcoma/patologia , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Fosforilação , Proteômica , Receptores Proteína Tirosina Quinases/genética , Proteínas Recombinantes/farmacologia , Transfecção , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologia , Receptor Tirosina Quinase AxlRESUMO
BACKGROUND: The extended role of vascular endothelial growth factor (VEGF) in human pathophysiology led us to evaluate pre-analytical parameters possibly influencing its levels in peripheral blood and tissues. The effects on VEGF protein levels and mRNA expression were measured after storage delay (blood and tissue), use of different types of anticoagulants (blood), and after different numbers of freeze-thaw cycles (blood). METHODS: Blood from healthy donors was sampled simultaneously in ethylene diamine tetraacetic acid (EDTA), acid citrate dextrose (ACD-A), hirudin, and serum separation tubes. For each anticoagulant, VEGF was measured by enzyme-linked immunosorbent assay (ELISA) with different conditions of delay at 4°C before centrifugation (2 h, 4 h, or 48 h) and of different numbers of freeze-thaw cycles (1, 2, and 10). The transcripts coding for the VEGF165 isoform were quantified in peripheral blood mononuclear cells by RT-PCR. Muscle biopsy samples were frozen with delays of 15, 30, or 60 min after surgery. VEGF expression was quantified on immunofluorescence stained slides. RESULTS: The period of storage and the number of freeze-thaw cycles correlated with an increase in the levels of circulating VEGF (for each anticoagulant but not for serum) and its expression in PBMCs. VEGF expression measured from muscle biopsy sections was higher with freezing delays, with a peak at 30 and 60 min as compared to 15 min. CONCLUSIONS: The most reliable conditions for measuring both circulating VEGF and its gene expression are to reduce time between blood collection and centrifugation, and to avoid multiple freeze-thaw cycles. Serum collection tubes with no additive and no separator were less sensitive to the pre-analytical variations analyzed in this study. Freezing delay had a significant influence on VEGF protein expression in tissue samples.
Assuntos
Ensaio de Imunoadsorção Enzimática , Leucócitos Mononucleares/metabolismo , Fator A de Crescimento do Endotélio Vascular/análise , Anticoagulantes/química , Ácido Cítrico/química , Ácido Edético/química , Congelamento , Expressão Gênica , Glucose/análogos & derivados , Glucose/química , Hirudinas/química , Humanos , Leucócitos Mononucleares/citologia , Músculo Esquelético/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodos , Temperatura , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
We have previously reported that ADP ribosylation factor like 2 (Arl2), a small GTPase, content influences microtubule dynamics and cell cycle distribution in breast tumor cells, as well as the degree and distribution of phosphorylated P53. Here we show, in two different human breast adenocarcinoma models, that Arl2 content has a major impact on breast tumor cell aggressivity both in vitro and in vivo. Cells with reduced content of Arl2 displayed reduced contact inhibition, increased clonogenic or cluster formation as well as a proliferative advantage over control cells in an in vitro competition assay. These cells also caused larger tumors in SCID mice, a phenotype which was mimicked by the in vivo administration of siRNA directed against Arl2. Cells with increased Arl2 content displayed reduced aggressivity, both in vitro and in vivo, with enhanced necrosis and were also found to contain increased PP2A phosphatase activity. A rt-PCR analysis of fresh human tumor breast samples suggested that low Arl2 expression was associated with larger tumor size and greater risk of lymph node involvement at diagnosis. These data underline the role of Arl2, a small GTPase, as an important regulator of breast tumor cell aggressivity, both in vitro and in vivo.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Regulação Neoplásica da Expressão Gênica , Animais , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação ao GTP/metabolismo , Humanos , Metástase Linfática , Camundongos , Camundongos SCID , Invasividade Neoplásica , Fenótipo , Fosforilação , Proteína Fosfatase 2/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Fetal androgen disruption, induced by the administration of anti-androgen flutamide (0.4, 2, and 10 mg/kg day) causes a long-term apoptosis in testicular germ cells in adult male rat offspring. One of the questions raised by this observation is the role of the Sertoli cells in the adult germ cell apoptotic process. It is shown here that Sertoli cells originating from 15-day-old rats treated in utero with the anti-androgen (10 mg/kg d) did no longer protect adult germ cells against apoptosis. Indeed, untreated spermatocytes or spermatids exhibited increased (P<0.0001) active caspase-3 levels when co-cultured with Sertoli cells isolated from rat testes exposed in utero to the anti-androgen. This alteration of Sertoli cell functions was not due to modifications in the androgen signal in the adult (90-day-old) animals, since plasma testosterone and estradiol, androgen receptor expression, and androgen-targeted cell number (e.g., Sertoli cells in the seminiferous tubules) were not affected by the fetal androgen disruption. In contrast, this inability of Sertoli cells to protect germ cells against apoptosis could be accounted for by the potential failure of Sertoli cell functions. Indeed, adult testes exposed in utero to anti-androgens displayed decreased levels of several genes mainly expressed in adult Sertoli cells (anti-Mullerian hormone receptor type II (AMHR2), Cox-1, cyclin D2, cathepsin L, and GSTalpha). In conclusion, fetal androgen disruption may induce alterations of Sertoli cell activity probably related to Sertoli cell maturation, which potentially leads to increased adult germ cell apoptosis.
Assuntos
Antagonistas de Androgênios/farmacologia , Apoptose/efeitos dos fármacos , Feto/efeitos dos fármacos , Células de Sertoli/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Testículo/embriologia , Animais , Células Cultivadas , Técnicas de Cocultura , Regulação para Baixo/efeitos dos fármacos , Feminino , Flutamida/farmacologia , Expressão Gênica/efeitos dos fármacos , Glutationa Transferase/genética , Isoenzimas/genética , Masculino , Gravidez , Efeitos Tardios da Exposição Pré-Natal , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células de Sertoli/química , Células de Sertoli/fisiologia , Testículo/efeitos dos fármacos , Testículo/metabolismoRESUMO
Our study demonstrates that laminin potentiates cisplatin-induced apoptosis in NCCIT, a testicular tumor germ cell line. When cultured on laminin, NCCIT cells displayed a significantly higher susceptibility to cisplatin-induced apoptosis than on plastic or on other ECM components including fibronectin, Type IV collagen and vitronectin. This high cisplatin sensitivity observed on NCCIT cell cultured on laminin was mediated by the alpha6-integrin signaling. The knockdown of the alpha6-integrin subunit by small interfering RNAs suppressed the higher cisplatin-sensitivity supporting the existence of a crosstalk between laminin-alpha6-integrin signaling and cisplatin-induced apoptosis. Our findings indicate that in cisplatin-treated NCCIT cells, the laminin-alpha6-integrin signaling induces the activation of executioner procaspase-3 and -6 as well as apoptosis-inducing factor (AIF) transcription and expression. The ability of integrin-mediated specific stroma-tumor cell interactions to modulate the chemosensitive phenotype of a tumor cell might provide new insights to overcome cisplatin resistance of tumor cells.
Assuntos
Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Células Germinativas/efeitos dos fármacos , Integrina alfa6/fisiologia , Laminina/farmacologia , Neoplasias Testiculares/patologia , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo IV/metabolismo , Regulação para Baixo , Ativação Enzimática/efeitos dos fármacos , Fibronectinas/metabolismo , Células Germinativas/citologia , Células Germinativas/metabolismo , Humanos , Masculino , RNA Interferente Pequeno/farmacologia , Células Estromais/citologia , Células Estromais/metabolismo , Neoplasias Testiculares/metabolismo , Células Tumorais Cultivadas , Vitronectina/metabolismoRESUMO
Glutathione S-transferases (GSTs) are a family of detoxification isoenzymes present in different tissues including the testis and that conjugate many toxic substrates to glutathione. Among these substrates are carcinogens, mutagens and products of oxidative processes. In the present report we show that GSTalpha is expressed in somatic testicular Leydig cells and Sertoli cells. GSTalpha expression in Sertoli cells is under the hormonal control of FSH, testosterone, and estradiol. In Leydig cells, immunoreactive GSTalpha was present at the neonatal, pubertal, and adult periods. In Sertoli cells, GSTalpha was predominant in pubertal and adult testes (but not in neonatal testes), suggesting that its expression is controlled by gonadotropins. The regulatory action and the mechanisms of action of FSH and testosterone on GSTalpha mRNA and protein levels were studied by using a model of primary cultures of porcine testicular Sertoli cells. FSH increased GSTalpha mRNA levels in a dose-dependent manner (ED50 = 18.5 nm/ml) with a maximal effect observed after 48 h of exposure (a 3-fold increase; P < 0.001). In addition, FSH increased GSTalpha protein, which was detected as a doublet of 28 kDa. Treatment with testosterone enhanced GSTalpha mRNA levels in a dose-dependent (ED50 = 1.4 ng/ml) and time-dependent manner with a maximal effect delayed at 8 h of exposure (a 2-fold increase; P < 0.001). Similarly, Sertoli cell treatment with testosterone metabolites, dihydrotestosterone (DHT) and estradiol, led to an increase in GSTalpha mRNA levels. Because stimulatory effects of FSH and androgens were also observed on GSTalpha protein, we therefore had to determine whether the different hormones were affecting GSTalpha gene transcriptional activity, or GSTalpha mRNA stability, or both. FSH and 8-Br-cAMP (but not testosterone) increased the stability of GSTalpha mRNA. The effects of FSH and testosterone on GSTalpha protein were additive, confirming that both hormones act through distinct mechanisms on the expression of the enzyme. Taken together, the present observations indicate that Sertoli cell GSTalpha is targeted by FSH, testosterone, and its metabolites, and they reinforce the concept that Sertoli cells exert a protective role and are under endocrine control to ward against toxic agents in the context of Sertoli-germ cell interactions during spermatogenesis.
Assuntos
Hormônio Foliculoestimulante/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glutationa Transferase/genética , Isoenzimas/genética , Células de Sertoli/enzimologia , Testosterona/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Envelhecimento , Animais , Di-Hidrotestosterona/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Estradiol/farmacologia , Cinética , Células Intersticiais do Testículo/enzimologia , Masculino , RNA Mensageiro/análise , Suínos , Testículo/crescimento & desenvolvimentoRESUMO
Breast metastases from nonmammary tumors are rare. We report here the first case of pancreatic neuroendocrine carcinoma metastatic to the breast in a patient with possible multiple endocrine neoplasia type 1. The diagnosis was supported by histological examination, immunohistochemistry, and ultrastructural analysis. This observation emphasizes the importance of clinical data for an accurate diagnosis, especially during intraoperative examination. When pathologists are faced with an unusual breast tumor larger than 2 cm, we would recommend freezing and/or saving pieces of tissue for ultrastructural analysis, which might help in the diagnosis.
RESUMO
During spermatogenesis, postmeiotic germ cells utilize lactate produced by Sertoli cells as an energy metabolite. While the hormonal regulation of lactate production in Sertoli cells has been relatively well established, the transport of this energy substrate to the germ cells, particularly via the monocarboxylate transporters (MCTs), as well as the potential endocrine control of such a process remain to be characterized. Here, we report the developmentally and hormonally regulated expression of MCT2 in the testis. At Day 18, MCT2 starts to be expressed in germ cells as detected by Northern blot. The mRNA are translated into protein (40 kDa) in elongating spermatids. Ultrastructural analysis demonstrated that MCT2 protein is localized to the outer face of the cell membrane of spermatid tails. MCT2 mRNA levels are under the control of the endocrine, specifically follicle-stimulating hormone (FSH) and testosterone, and paracrine systems. Indeed, a 35-day-old rat hypophysectomy resulted in an 8-fold increase in testicular MCT2 mRNA levels. Conversely, FSH and LH administration to the hypophysectomized rats reduced MCT2 mRNA levels to the basal levels observed in intact animals. The decrease in MCT2 mRNA levels was confirmed in vitro using isolated seminiferous tubules incubated with FSH or testosterone. FSH or testosterone inhibited in a dose-dependent manner MCT2 mRNA levels with maximal inhibitory doses of 2.2 ng/ml and 55.5 ng/ml for FSH and testosterone, respectively. In addition to the endocrine control, TNFalpha and TGFbeta also exerted an inhibitory effect on MCT2 mRNA levels with a maximal effect at 10 ng/ml and 6.6 ng/ml for TGFbeta and TNFalpha, respectively. Together with previous studies, the present data reinforce the concept that among the key functions of the endocrine/paracrine systems in the testis is the control of the energy metabolism occurring in the context of Sertoli cell-germ cell metabolic cooperation where lactate is produced in somatic cells and transported to germ cells via, at least, MCT2.
Assuntos
Transportadores de Ácidos Monocarboxílicos/metabolismo , Células de Sertoli/metabolismo , Espermatogênese/fisiologia , Espermatozoides/metabolismo , Fatores Etários , Animais , Metabolismo Energético/fisiologia , Hormônio Foliculoestimulante/fisiologia , Regulação da Expressão Gênica , Imuno-Histoquímica , Masculino , Camundongos , Transportadores de Ácidos Monocarboxílicos/genética , RNA Mensageiro/análise , Ratos , Espermatozoides/ultraestrutura , Testículo/citologia , Testículo/metabolismo , Testosterona/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Although the apoptotic cell death process in the prostate is known to be under the control of androgens, the key components targeted by the hormones remain to be investigated. In the present study, we report that the expression and the activation of the effector caspases-3 and -6 are under the control of testosterone in the adult rat ventral prostate. By using a model of adult castrated rats supplemented (or not) with androgens, we observed an increase in caspase-3 (3-fold) and -6 (4-fold) mRNA (P < 0.0001) and procaspase-3 (32 kDa) and -6 (34 kDa) protein levels by 3 days and 1 wk, respectively, after castration in the ventral prostate. Castration also induced an increase in the activation of the procaspases in the ventral prostate, since active (cleaved) caspase-3 (17 kDa) and -6 (12 kDa) forms reached maximal levels by 1 wk after castration. Testosterone administration to castrated adult rats prevented the increase in caspase-3 and -6 mRNA as well as in procaspase-3 and -6 and active caspase-3 and -6 levels in the ventral prostate lobe. In contrast, no changes were observed in the initiator caspase-8 mRNA and protein (procaspase and active) levels after castration. No changes in caspase-3 and -6 expression and activation were observed in the dorsolateral and anterior prostate lobes after castration and testosterone supplementation. Together, the present results show that testosterone inhibits apoptosis in the ventral prostate by potentially targeting the transcriptional activity of effector caspase-3 and -6 genes (but not of casapase-8 gene) as well as the cleavage of procaspase-3 and -6 into active enzymes.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Próstata/enzimologia , Testosterona/fisiologia , Análise de Variância , Animais , Caspase 3 , Caspase 6 , Caspase 8 , Caspases/genética , Castração , Ativação Enzimática/fisiologia , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Imuno-Histoquímica , Masculino , Próstata/citologia , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
Foram utilizados no presente estudo, quinze ratos albinos (Sprague Dawley) portadores de fibromas transplantados no tecido subcutâneo da parede abdominal. O tumor era constituído por fibroblastos típicos, em uma densa matriz colagênica e eram provenientes de um fibroma que apareceu espontaneamente em um rato albino da mesma linhagem.