RESUMO
PURPOSE: Assess cyclin A in nonfunctioning pituitary adenomas (NFPA) and compare its expression in non-invasive and non-proliferative tumors with invasive and proliferative tumors (12× higher risk of recurrence). METHODS: Quantitative real time polymerase chain reaction to analyze cyclin A using normal pituitary gland as reference. Fold change (FC) > 1 was considered as increased. Tumor invasion was based on Knosp criteria (grades 3-4 considered invasive) and proliferation on the presence of at least two of three criteria: Ki-67 ≥ 3%; mitoses > 2/10; positive p53. Both groups were compared with Mann-Whitney test considering p value < 0.05 as statistically significant. RESULTS: Thirty-one patients with NFPA were included. Tumors were mainly of gonadotrophic origin (74.2%), followed by corticotrophic (19.4%) and lactotrophic (3.2%) origins and null-cell adenomas (3.2%). Median tumor diameter was 3.5 cm (1.8-8.0) and Ki-67 was 3.0% (0.3-11%). Sixteen patients had tumors classified as non-invasive and non-proliferative and 15 as invasive and proliferative. Median FC was 0.31 in all tumors (0.13-1.94). Cyclin A was not related to invasion or proliferation (FC 0.41 in non-invasive and non-proliferative tumors and FC 0.30 in invasive and proliferative tumors; p = 0.968). Four (12.9%) patients had tumors that exhibited increased cyclin A [median FC of 1.04 (1.02-1.94)]-three of gonadotrophic origin and one null-cell adenoma, with two tumors classified as non-invasive and non-proliferative and two tumors classified as invasive and proliferative. Median tumor diameter in these samples was 3.4 cm (2.4-3.6) and Ki-67 was 5.1% (2-11%). CONCLUSIONS: Cyclin A was increased in a minority of NFPA and does not seem to be related to invasion or proliferation.
Assuntos
Adenoma , Neoplasias Hipofisárias , Biomarcadores Tumorais , Ciclina A , Humanos , Antígeno Ki-67 , Recidiva Local de Neoplasia , Neoplasias Hipofisárias/genéticaRESUMO
The adult mammalian cardiomyocyte has a very limited capacity to reenter the cell cycle and advance into mitosis. Therefore, diseases characterized by lost contractile tissue usually evolve into myocardial remodeling and heart failure. Analyzing the cardiac transcriptome at different developmental stages in a large mammal closer to the human than laboratory rodents may serve to disclose positive and negative cardiomyocyte cell cycle regulators potentially targetable to induce cardiac regeneration in the clinical setting. Thus we aimed at characterizing the transcriptomic profiles of the early fetal, late fetal, and adult sheep heart by employing RNA-seq technique and bioinformatic analysis to detect protein-encoding genes that in some of the stages were turned off, turned on, or differentially expressed. Genes earlier proposed as positive cell cycle regulators such as cyclin A, cdk2, meis2, meis3, and PCNA showed higher expression in fetal hearts and lower in AH, as expected. In contrast, genes previously proposed as cell cycle inhibitors, such as meis1, p16, and sav1, tended to be higher in fetal than in adult hearts, suggesting that these genes are involved in cell processes other than cell cycle regulation. Additionally, we described Gene Ontology (GO) enrichment of different sets of genes. GO analysis revealed that differentially expressed gene sets were mainly associated with metabolic and cellular processes. The cell cycle-related genes fam64a, cdc20, and cdk1, and the metabolism-related genes pitx and adipoq showed strong differential expression between fetal and adult hearts, thus being potent candidates to be targeted in human cardiac regeneration strategies.NEW & NOTEWORTHY We characterized the transcriptomic profiles of the fetal and adult sheep hearts employing RNAseq technique and bioinformatic analyses to provide sets of transcripts whose variation in expression level may link them to a specific role in cell cycle regulation. It is important to remark that this study was performed in a large mammal closer to humans than laboratory rodents. In consequence, the results can be used for further translational studies in cardiac regeneration.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Coração/fisiologia , Miocárdio/metabolismo , Regeneração , Transcriptoma , Animais , Ciclina A/genética , Ciclina A/metabolismo , Feminino , Coração/crescimento & desenvolvimento , Masculino , Ovinos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Calcium (Ca2+) signaling within the cell nucleus regulates specific cellular events such as gene transcription and cell proliferation. Nuclear and cytosolic Ca2+ levels can be independently regulated, and nuclear translocation of receptor tyrosine kinases (RTKs) is one way to locally activate signaling cascades within the nucleus. Nuclear RTKs, including the epidermal growth factor receptor (EGFR), are important for processes such as transcriptional regulation, DNA-damage repair, and cancer therapy resistance. RTKs can hydrolyze phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) within the nucleus, leading to Ca2+ release from the nucleoplasmic reticulum by inositol 1,4,5-trisphosphate receptors. PI(4,5)P2 hydrolysis is mediated by phospholipase C (PLC). However, it is unknown which nuclear PLC isoform is triggered by EGFR. Here, using subcellular fractionation, immunoblotting and fluorescence, siRNA-based gene knockdowns, and FRET-based biosensor reporter assays, we investigated the role of PLCδ4 in epidermal growth factor (EGF)-induced nuclear Ca2+ signaling and downstream events. We found that EGF-induced Ca2+ signals are inhibited when translocation of EGFR is impaired. Nuclear Ca2+ signals also were reduced by selectively buffering inositol 1,4,5-trisphosphate (InsP3) within the nucleus. EGF induced hydrolysis of nuclear PI(4,5)P2 by the intranuclear PLCδ4, rather than by PLCγ1. Moreover, protein kinase C, a downstream target of EGF, was active in the nucleus of stimulated cells. Furthermore, PLCδ4 and InsP3 modulated cell cycle progression by regulating the expression of cyclins A and B1. These results provide evidence that EGF-induced nuclear signaling is mediated by nuclear PLCδ4 and suggest new therapeutic targets to modulate the proliferative effects of this growth factor.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Núcleo Celular/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Fosfolipase C delta/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cadeias Pesadas de Clatrina/antagonistas & inibidores , Cadeias Pesadas de Clatrina/genética , Cadeias Pesadas de Clatrina/metabolismo , Ciclina A/metabolismo , Ciclina B1/metabolismo , Receptores ErbB/metabolismo , Humanos , Hidrólise , Inositol 1,4,5-Trifosfato/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipase C delta/antagonistas & inibidores , Fosfolipase C delta/genética , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Proteína Quinase C/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
Growth differentiation factor 11 (GDF11) has been characterized as a key regulator of differentiation in cells that retain stemness features, despite some controversies in age-related studies. GDF11 has been poorly investigated in cancer, particularly in those with stemness capacity, such as hepatocellular carcinoma (HCC), one of the most aggressive cancers worldwide. Here, we focused on investigating the effects of GDF11 in liver cancer cells. GDF11 treatment significantly reduced proliferation, colony and spheroid formation in HCC cell lines. Consistently, down-regulation of CDK6, cyclin D1, cyclin A, and concomitant upregulation of p27 was observed after 24â¯h of treatment. Interestingly, cell viability was unchanged, but cell functionality was compromised. These effects were potentially induced by the expression of E-cadherin and occludin, as well as Snail and N-cadherin repression, in a time-dependent manner. Furthermore, GDF11 treatment for 72â¯h induced that cells were incapable of sustaining colony and sphere capacity in the absent of GDF11, up to 5â¯days, indicating that the effect of GDF11 on self-renewal capacity is not transient. Finally, in vivo invasion studies revealed a significant decrease in cell migration of hepatocellular carcinoma cells treated with GDF11 associated to a decreased proliferation judged by Ki67 staining. Data show that exogenous GDF11 displays tumor suppressor properties in HCC cells.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Fatores de Diferenciação de Crescimento/farmacologia , Neovascularização Patológica/prevenção & controle , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Ciclina A/genética , Ciclina A/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Fatores de Diferenciação de Crescimento/genética , Fatores de Diferenciação de Crescimento/metabolismo , Células Hep G2 , Humanos , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Ocludina/genética , Ocludina/metabolismo , Transdução de Sinais , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologiaRESUMO
Tyrosine kinase inhibitors (TKI) have become a first-line treatment for chronic myeloid leuakemia (CML). TKIs efficiently target bulk CML cells; however, they are unable to eliminate the leukaemic stem cell (LSC) population that causes resistance and relapse in CML patients. In this study, we assessed the effects of parthenolide (PTL) and dimethyl amino parthenolide (DMAPT), two potent inhibitors of LSCs in acute myeloid leukaemia (AML), on CML bulk and CML primitive (CD34+ lin- ) cells. We found that both agents induced cell death in CML, while having little effect on the equivalent normal hematopoietic cells. PTL and DMAPT caused an increase in reactive oxygen species (ROS) levels and inhibited NF-κB activation. PTL and DMAPT inhibited cell proliferation and induced cell cycle arrest in G0 and G2 phases. Furthermore, we found cell cycle inhibition to correlate with down-regulation of cyclin D1 and cyclin A. In summary, our study shows that PTL and DMAPT have a strong inhibitory effect on CML cells. Given that cell cycle arrest was not dependent on ROS induction, we speculate that this effect could be a direct consequence of NF-κB inhibition and if this mechanism was to be evaded, PTL and DMAPT induced cell death would be potentiated.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Sesquiterpenos/farmacologia , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina A/genética , Ciclina D1/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , NF-kappa B/genética , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
ANKHD1 is a multiple ankyrin repeat containing protein, recently identified as a novel member of the Hippo signaling pathway. The present study aimed to investigate the role of ANKHD1 in DU145 and LNCaP prostate cancer cells. ANKHD1 and YAP1 were found to be highly expressed in prostate cancer cells, and ANKHD1 silencing decreased cell growth, delayed cell cycle progression at the S phase, and reduced tumor xenograft growth. Moreover, ANKHD1 knockdown downregulated YAP1 expression and activation, and reduced the expression of CCNA2, a YAP1 target gene. These findings indicate that ANKHD1 is a positive regulator of YAP1 and promotes cell growth and cell cycle progression through Cyclin A upregulation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Fosfoproteínas/genética , Neoplasias da Próstata/genética , Proteínas de Ligação a RNA/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular Tumoral , Ciclina A/genética , Ciclina A/metabolismo , Células HeLa , Via de Sinalização Hippo , Humanos , Células K562 , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Fosfoproteínas/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição , Ativação Transcricional , Proteínas de Sinalização YAPRESUMO
The activity of cyclin-dependent kinases (CDKs), which are key regulators of the eukaryotic cell cycle, is regulated through post-translational mechanisms, including binding of a cyclin and phosphorylation. Previously studies have shown that Leishmania mexicana CRK3 is an essential CDK that is a functional homologue of human CDK1. In this study, recombinant histidine tagged L. mexicana CRK3 and the cyclin CYCA were combined in vitro to produce an active histone H1 kinase that was inhibited by the CDK inhibitors, flavopiridol and indirubin-3'-monoxime. Protein kinase activity was observed in the absence of phosphorylation of the T-loop residue Thr178, but increased 5-fold upon phosphorylation by the CDK activating kinase Civ1 of Saccharomyces cerevisiae. Seven recombinant L. major CRKs (1, 2, 3, 4, 6, 7 and 8) were also expressed and purified, none of which were active as monomers. Moreover, only CRK3 was phosphorylated by Civ1. HA-tagged CYCA expressed in L. major procyclic promastigotes was co-precipitated with CRK3 and exhibited histone H1 kinase activity. These data indicate that in Leishmania CYCA interacts with CRK3 to form an active protein kinase, confirm the conservation of the regulatory mechanisms that control CDK activity in other eukaryotes, but identifies biochemical differences to human CDK1.
Assuntos
Proteína Quinase CDC2/metabolismo , Ciclina A/metabolismo , Leishmania mexicana/enzimologia , Proteínas Quinases/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Proteína Quinase CDC2/genética , Ciclina A/genética , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Indóis/farmacologia , Leishmania mexicana/genética , Dados de Sequência Molecular , Oximas/farmacologia , Fosforilação , Piperidinas/farmacologia , Proteínas Quinases/genética , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de SequênciaRESUMO
One of the hallmarks of carcinomas is epithelial disorganization, linked to overexpression of matrix metalloproteases (MMP) like MMP-9, loss of intercellular E-cadherin and activation of epidermal growth receptor (EGFR/erbB1). Since the p53 tumor suppressor pathway is inactivated in most human cancers due to gene mutations or defective wt p53 signaling, we now investigated in human wt p53 breast carcinoma MCF-7 cells, whether single treatment with the p53 transactivation pharmacological inhibitor pifithrin-alpha, transient p53 siRNA interference or stable insertion of a dominant-negative (DN) R175H p53 mutant increase: (i) EGFR/erbB1 activation, (ii) MMP-9 expression and (iii) loss of surface E-cadherin. Transient abrogation of wt p53 function increased phosphorylation of EGFR/erbB1 and MMP-9 expression. However, all these effects were much more pronounced in cells stably transduced with the dominant negative-Arg-175His mutant p53 (DN-R175H mutant p53), which also showed loss of epithelial cytoarchitecture and extensive E-cadherin downregulation. Collectively, these results support the notion that the DN-R175H mutant p53 exerts a gain of oncogenic function by promoting disruption of E-cadherin intercellular contacts and activation of proliferation signals. Our data suggests that epithelial shape and growth control are unequally affected depending on how wt p53 function is impaired and whether partial or full tumor suppressor activity is lost.
Assuntos
Neoplasias da Mama/patologia , Caderinas/metabolismo , Carcinoma/patologia , Proliferação de Células , Células Epiteliais/metabolismo , Mutação , Proteína Supressora de Tumor p53/metabolismo , Arginina , Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Linhagem Celular Tumoral , Ciclina A/metabolismo , Regulação para Baixo , Células Epiteliais/patologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Imunofluorescência , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Dominantes , Genes erbB-1 , Histidina , Humanos , Immunoblotting , Citometria de Varredura a Laser , Metaloproteinase 9 da Matriz/metabolismo , Fosforilação , RNA Interferente Pequeno/metabolismo , Retroviridae , Ativação Transcricional , Transdução Genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Since it is important to lower the death threshold in tumor cells and to enhance response to genotoxic damage, the mechanisms of bromodeoxyuridine (BrdU)-mediated radiosensitization were now examined in human C8161 melanoma. This revealed that anti-apoptotic Bcl-2, and cell cycle controlling hyperphosphorylated Rb and cyclin A were downregulated by BrdU, even without irradiation. BrdU pretreatment and subsequent UV irradiation (10 J/m(2)) accelerated an early increase in the ratio of pro-apoptotic Bax to that of Bcl-2, increased apoptosis-associated PARP cleavage and potentiated DNA damage compared to irradiated unsensitized cells. BrdU also synergized with radiation to increase autophagic features, such as perinuclear vacuole formation. More specifically, conversion of LC3B-I into LC3B-II by immune blotting and an increased pattern of cytoplasmic and nuclear LC3B by fluorescent immunostaining, supported induction of autophagy in BrdU-pretreated cells irradiated with 25 J/m(2). This report shows for the first time that radiation sensitizers like BrdU enhance and modify radiation-induced cell death by accelerating an increased bax/bcl-2 ratio in unirradiated cells, and subsequently increasing radiation-induced apoptosis and/or autophagy depending on radiation dosage.
Assuntos
Apoptose , Autofagia , Dano ao DNA , Melanoma/radioterapia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Neoplasias Cutâneas/radioterapia , Bromodesoxiuridina/farmacologia , Linhagem Celular Tumoral , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Humanos , Melanoma/tratamento farmacológico , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína do Retinoblastoma/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , Raios Ultravioleta , Vacúolos/metabolismoRESUMO
The NFAT (Nuclear Factor of Activated T cells) family of transcription factors plays a central role in the regulation of several genes related to the immune response. Recently, NFAT proteins have been implicated in the proliferation and differentiation of different cell types. Previous studies have shown that NFAT1-deficient mice display lymphocyte hyperproliferation, shortened cell cycle duration, and cyclin overexpression. Here, we demonstrate that cyclin A2 expression is upregulated in the absence of NFAT1 in lymphocytes. Ectopic expression of NFAT1 in CHO cells decreases cyclin A2 levels. Indeed, NFAT1 binds to a consensus binding site found at the mouse cyclin A2 promoter in vitro and in vivo. Luciferase reporter assays show that NFAT1 downregulates cyclin A2 expression by directly binding to the cyclin A2 promoter. Together, these results indicate that the NFAT1 transcription factor represses cyclin A2 expression in lymphocytes, and may act as a silencer of gene transcription during the cell cycle.
Assuntos
Ciclina A/metabolismo , Regulação da Expressão Gênica , Linfócitos/fisiologia , Fatores de Transcrição NFATC/metabolismo , Proteínas Repressoras/metabolismo , Animais , Células CHO , Ciclo Celular/fisiologia , Cricetinae , Cricetulus , Ciclina A/genética , Ciclina A2 , Humanos , Linfócitos/citologia , Camundongos , Camundongos Knockout , Fatores de Transcrição NFATC/genética , Regiões Promotoras Genéticas , Elementos Reguladores de Transcrição , Proteínas Repressoras/genéticaRESUMO
MEK1/2 inhibitors like U0126 can potentiate or antagonize the antitumor activity of cytotoxic agents such as cisplatin, paclitaxel or vinblastine, depending on the drug or the target cells. We now investigated whether U0126, differentially regulates melanoma signaling in response to UV radiation or betulinic acid, a drug lethal against melanoma. This report shows that U0126 inhibits early response (ERK) kinase activation and cyclin A expression in wt p53 C8161 melanoma exposed to either UV radiation or betulinic acid. However, U0126 does not protect from UV damage, but counteracts betulinic acid-mediated apoptosis in the same cells. Protection from the latter drug by joint treatment with U0126 was also evident in wt p53 MelJuso melanoma and mutant p53 WM164 melanoma. The latter cells were the most responsive to betulinic acid, showing a selective decline in the cdk4 protein, without a comparable change in other key cell cycle proteins like cdc2, cdk2, cdk7 or cyclin A, prior to apoptosis-associated PARP fragmentation. Laser scanning cytometry also showed that betulinic acid induced a significant increase in chromatin condensation in WM164 melanoma irrespective of whether they were in adherent form or as multicellular spheroids. All these betulinic acid-induced changes were counteracted by U0126. Our data show for the first time that (a) cdk4 protein is an early target of betulinic acid-induced apoptosis and (b) unrestricted ERK signaling favours betulinic acid-induced apoptosis, but this is counteracted by U0126, partly through counteracting chromatin condensation and restoring Akt activation decreased by betulinic acid treatment.
Assuntos
Apoptose/efeitos dos fármacos , Butadienos/farmacologia , Melanoma/metabolismo , Melanoma/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Nitrilas/farmacologia , Triterpenos/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Ciclina A/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Dano ao DNA/efeitos dos fármacos , Regulação para Baixo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Triterpenos Pentacíclicos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Ácido BetulínicoRESUMO
OBJECTIVE: Although the genetic program for reinitiating DNA synthesis exists in post-mitotic cardiomyocytes, and it was reported that in human acute myocardial infarction (AMI) a significant proportion of myocytes enter mitosis, the rule is that the lost tissue is replaced by a collagen scar. The purpose of this study was to search for the basis of this discordance in order to devise future strategies to induce division of myocytes into daughter cells that may replace the lost tissue with contractile cells. METHODS: In 15 human hearts with 1- to 21-day-old infarcts, the expression of the cell cycle proteins Ki67 antigen, cyclins D, A, and B1, the presence of mitotic bodies, and the ploidy status were investigated with immunoenzymatic methods, light and laser confocal microscopy, and densitometry in the myocytes surrounding the infarct area. RESULTS: In 7- to 13-day-old infarcts, 11.61+/-6.94% of the myocytes presented Ki67+ nuclei, and a lower proportion presented cyclins D, A, and B. At earlier and later times, the proportion of Ki67+ myocytes was significantly lower. Although under confocal microscopy and fluorescent labels, some of the Ki67+ myocytes appeared to be in different stages of mitosis, with Nomarski optics and hematoxylin counterstaining, the condensed chromosomes, although arranged in metaphase and anaphase plates or split in sister chromatids, were always located within a preserved nuclear envelope, indicating the presence of endomitosis. Conventional mitosis was exceptionally observed. In the 14- and 21-day-old infarcts, the ploidy of the myocytes adjacent to the infarct was significantly higher than in distant zones. CONCLUSION: These observations indicate that in human infarcts, entrance of cardiomyocytes into the cell cycle is transient and that endomitosis, leading to polyploidy, rather than mitosis, leading to karyokinesis, is the final fate of cycling cells. Both observations may account for the discordance between the regenerative ability of myocytes and the lack of an efficient reparative process in human AMI.
Assuntos
Proteínas de Ciclo Celular/análise , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/química , Adulto , Idoso , Biomarcadores/análise , Estudos de Casos e Controles , Ciclina A/análise , Ciclina B/análise , Ciclina B1 , Ciclina D , Ciclinas/análise , Feminino , Humanos , Antígeno Ki-67/análise , Masculino , Microscopia Confocal , Microscopia de Fluorescência , Pessoa de Meia-Idade , Mitose , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Miócitos Cardíacos/patologia , PoliploidiaRESUMO
Introdução: O carcinoma epidermóide do esôfago (CEE) é o tipo histológico mais comum de câncer do órgão, sendo a sexta neoplasia em freqüência em nosso país. Geralmente, o CEE está associado a um prognóstico ruim, por se tratar de um tumor em estadio avançado no momento do diagnóstico, apresentando alta taxa de mortalidade. As alterações moleculares do CEE são pouco estudadas e os trabalhos existentes na literatura mostram resultados controversos e casuística reduzida. O objetivo desse estudo foi analisar expressão de proteínas envolvidas na regulação da proliferação celular em CEE e sua correlação com variáveis clínico-patológicas e sobrevida dos pacientes. Métodos: Foi realizada imunoistoquímica para análise de expressão de c-erbB-2, EGFR, VEGF e ciclinas A, B1 e D1 em 613 casos de CEE. Os casos representados por peças cirúrgicas foram distribuídos em três blocos de parafina de Tissue Microarray, em duplicata; os casos com biópsias foram analisados em corte convencional. Os casos foram classificados de acordo com a freqüência, intensidade e padrão de marcação das células, dependendo do anticorpo avaliado. As análises estatísticas de correlação entre as variáveis consideradas foram realizadas pelos métodos de χ2 ou teste exato de Fisher. Para as análises de sobrevida global, o cálculo da estimativa de sobrevida cumulativa foi realizado pela técnica de Kaplan-Meier e as comparações entre as curvas de sobrevida, em relação as variáveis estudadas, pelo teste de Log-rank. A análise multivariada foi feita pelo modelo de riscos proporcionais de Cox. Foi considerado o nível de significância de 5%. Resultados: Nossos resultados mostraram correlação estatisticamente significativa entre a expressão de cerbB-2 (p=0,04) e EGFR (p=0,01) e grau histológico. Ambos os marcadores foram significativamente mais expressos em casos bem/moderadamente diferenciados do que nos pouco diferenciados/ indiferenciados. Embora não tenha sido significativo, houve uma tendência de associação entre a superexpressão de c-erbB-2 e sítio do tumor, em que casos positivos ocorreram com mais freqüência no terço médio do esôfago. Nenhuma associação significativa foi verificada entre a expressão dessas proteínas e sobrevida global. Em relação a ciclina D1, foi observada correlação significativa entre a superexpressão dessa proteína e cor (p=0,026), sendo mais expressa em pacientes de cor branca. Foi observada também uma tendência à significância para correlação entre a superexpressão dessa proteína e grau de diferenciação (p=0,070), em que a ciclina D1 esteve mais expressa em casos bem/moderadamente diferenciados. Quanto a ciclina B1, foi verificado que casos negativos para essa proteína foram significativamente mais freqüentes em tumores que atingiram níveis mais profundos de infiltração (p=0,033) e com presença de invasão linfática (p=0,006). Não foi observada nenhuma correlação significativa entre a superexpressão de VEGF e ciclina A e aspectos clínico-patológicos. As análises de sobrevida global mostraram a existência de associação estatisticamente significativa entre a superexpressão de ciclinas A (p<0,0001), B1 (p=0,014) e D1 (p<0,0001) e VEGF (p=0,002) e menores estimativas de sobrevida cumulativa. Foi observada correlação significativa entre a coexpressão da maioria das proteínas estudadas, exceto entre as expressões de c-erbB-2/ciclina A, c-erbB-2/ciclina B1, c-erbB-2/VEGF, EGFR/ciclina A e EGFR/VEGF. Em relação as variáveis clínico-patológicas, gênero (p=0,044), cor (p=0,0005) e pT (p=0,021) mostraram-se como indicadores de pior prognóstico. Pela análise multivariada, pT (p=0,030) e gênero (p=0,004) permaneceram como fatores prognósticos independentes. No entanto, quando desconsideramos pT da análise multivariada, ciclina D1 (p<0,0001), cor (p=0,003) e ciclina A (p=0,023) persistiram como marcadores prognósticos independentes. Conclusão: No presente trabalho, verificamos correlação significativa entre a superexpressão de c-erbB-2, de EGFR e de ciclina D1 e grau histológico, conferindo um comportamento menos agressivo aos tumores, sugerindo que esses marcadores podem estar envolvidos na indução de outra via de sinalização, como diferenciação celular. Casos negativos para expressão de ciclina B1 foram mais freqüentes entre os tumores com invasão linfática e níveis mais profundos de infiltração, não permitindo esclarecer sua relação com a progressão tumoral nesse tumor. Não foi constatada nenhuma correlação com significância estatística entre a superexpressão de VEGF e de ciclina A e as variáveis clínicopatológicas avaliadas. As variáveis gênero, cor, pT e expressão de VEGF, ciclinas A, B1 e D1 foram consideradas indicadores de pior prognóstico pela análise univariada. No entanto, na análise multivariada, apenas pT e gênero permaneceram como marcadores prognósticos, mas quando pT foi desconsiderado dessa análises, ciclina D1, cor e ciclina A permaneceram como fatores prognósticos independentes em CEE.
Background: Esophageal squamous cell carcinoma (ESCC) is the most commom histologic type of cancer in this organ, being the sixth most common tumor in Brazil. ESCC is associated with poor prognosis, because it is usually a advanced stage disease at the moment of diagnostic, showing high mortality rates. There are few studies involving molecular alterations in ESCC and the reports show controversial results and small number of cases. The aim of this study was to analyze expression of proteins involved in cellular proliferation regulation in ESCC and the correlation with clinicopathological variables and patient survival. Methods: Immunohistochemistry was performed to analyze the c-erbB-2, EGFR, VEGF and cyclins A, B1 and D1 expression in 613 cases of ESCC. Cases represented by surgical resections were performed in three tissue microarray paraffin blocks spotted in duplicate and cases with small biopsies were performed in conventional slides. The cases were scored according the frequency, intensity and staining pattern of tumoral cells, depending on the antibody evaluated. Statistical analysis between the variables considered in this study were performed using chi-square or Fisher's exact tests. For the overall survival analysis, the cumulative survival rates was calculated by Kaplan-Meier method and the comparison between the survival curves, according the variables studied, by the Log-rank test. Multivariate analysis was performed using Cox regression method. A p value <0,05 was considered statistically significant. Results: Our results showed significant correlation between c-erbB-2 (p=0,04) and EGFR (p=0,01) expression and histological grade. Both markers were significantly more expressed in well/moderatelly differentiated than in poorly differentiated/undifferentiated cases. Despite it were not statistically significant, there was a tendency to association between c-erbB-2 overexpression and tumor site, which positive cases were more frequently in the middle third of the esophagus. No significant association was verified between these proteins expression and overall survival. It was observed a tendecy to significance for the correlation between cyclin D1 overexpression and histological grade (p=0,070), in which this protein was more expressed in well/moderately differentiated cases. Regarding cyclin B1, it was verified that negative cases for this protein was significantly more frequent in deeper infiltration lesions (p=0,033) and in cases with lymphatic invasion (p=0,006). It was not observed any significant correlation between VEGF and cyclin A overexpression and clinicopathological aspects. Overall survival analysis showed statistically significant association between cyclins A (p<0,0001), B1 (p=0,014), D1 (p<0,0001) and VEGF (p=0,002) and lower cumulative survival rates. It was verified significant correlation between the majority of proteins studied, except c-erbB-2/cyclin A, c-erbB-2/cyclin B1, c-erbB-2/VEGF, EGFR/cyclin A and EGFR/VEGF. Regarding the clinicopathological features, gender (p=0,044), ethnia (p=0,0005) and pT (p=0,021) were considered worse prognostic factors. Multivariate analysis showed that pT (p=0,030) and gender (=0,004) persisted as independent prognostic factors. However, when T is not considered in the multivariate analysis, cyclin D1 (<0,0001), ethnia (p=0,003) and cyclin A (p=0,023) remain as independent prognostic markers. Conclusion: In the present work, we verified significant correlation between c-erbB-2, EGFR and cyclin D1 overexpression and histological grade, giving a less agressive behaviour to the tumors, suggesting that these markers can be involved in induction of other signaling pathway, as cellular differentiation. Negative cases for cyclin B1 expression occurred more frequent in cases with lymhtic invasion and deeper infiltration, not being possible elucidate cyclin B1 relation with tumoral progression in ESCC. It was not verified any correlation with statistically significance between VEGF and cyclin A overexpression and the clinicopathological features considered. The variables gender, ethnia, pT and VEGF, cyclins A, B1 nd D1 expression were considered worse prognostic factores by univariate analysis. However, in multivariate analysis only pT and gender remained as prognostic markers, but when pT was not considered in this analysis, cyclin D1, ethnia and cyclin A persisted as independent prognostic factores in ESCC.
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Sobrevida , Neoplasias Esofágicas , Carcinoma de Células Escamosas , Carcinoma de Células Escamosas do Esôfago , Proteínas , Ciclinas , Receptor ErbB-2 , Ciclina A , Ciclina D1 , Fator A de Crescimento do Endotélio Vascular , Proliferação de Células , Ciclina B1 , Receptores ErbBRESUMO
The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) inhibits normal keratinocytes proliferation. However, many human papillomavirus (HPV)-immortalized or transformed cell lines are resistant to TNF-alpha antiproliferative effect. The present study analyzes the effects of TNF-alpha on organotypic cultures of primary human keratinocytes (PHKs) that express HPV-18 oncogenes. Raft cultures prepared with PHKs acutely transfected with HPV-18 whole genome or infected with recombinant retroviruses containing only E6/E7 or E7 were treated with 2 nM TNF-alpha. While BrdU incorporation into basal/parabasal cells of normal PHKs cultures was markedly inhibited by TNF-alpha cultures transfected with HPV-18 whole genome showed proliferation in all cell strata. Furthermore, BrdU incorporation into cultures expressing E6/E7 or E7 was not significantly reduced, indicating that E7 alone confers partial resistance to TNF-alpha. Besides, TNF-alpha treatment did not alter p16ink4a, p21cip1, p27kip1, or cyclin E levels, but did reduce cyclin A and PCNA levels in sensitive cells.
Assuntos
Transformação Celular Viral/efeitos dos fármacos , Queratinócitos/virologia , Papillomaviridae/patogenicidade , Fator de Necrose Tumoral alfa/farmacologia , Ciclina A/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Humanos , Queratinócitos/patologia , Proteínas Oncogênicas Virais/biossíntese , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fase SRESUMO
Full length Mcl-1 is an anti-apoptotic protein consisting of two closely migrating 42/40kDa species. We now investigated the relationship of these isoforms to the expression of cell cycle stimulatory (cyclin A) and inhibitory (p21WAF1) proteins and to the induction of apoptosis in wt p53 MCF-7 and mutant p53 SKBR3 human breast carcinomas. The latter cells exhibited lower 42kDa Mcl-1, higher expression of cyclin A relative to that of p21WAF1, and apoptosis in response to okadaic acid, a phosphatase 1/2A inhibitor. The proteasome inhibitor MG-115 selectively increased expression of the 40kDa Mcl-1 isoform and induced p21WAF1, but also promoted preferential apoptosis in SKBR3 cells. Neither okadaic acid nor MG-115 caused comparable effects in MCF-7 cells. However, vanadate or acetyl furanonaphthoquinone induced the 40kDa Mcl-1 and greater Jun kinase (JNK) phosphorylation without apoptosis-associated PARP fragmentation in MCF-7 cells. Our data suggest that the higher susceptibility of SKBR3 cells to undergo apoptosis may be partly due to their greater proliferative potential (cyclin A), low expression of the anti-apoptotic 42kDa Mcl-1 isoform, and suboptimal JNK activation in response to stress.
Assuntos
Apoptose/fisiologia , Neoplasias da Mama/metabolismo , Ciclina A/metabolismo , Ciclinas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/patologia , Cromatina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Inibidores Enzimáticos/metabolismo , Feminino , Genes p53 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Leupeptinas/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides , Ácido Okadáico/farmacologia , Fosforilação , Inibidores de Proteases/farmacologia , Isoformas de Proteínas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
TGF-beta1 modulation of cell cycle components was assessed in an experimental model in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary tumors in Balb/c mice. TGF-beta1 inhibited both MPA-induced proliferation of progestin-dependent C4HD epithelial cells and proliferation of the progestin-independent variant cell type C4HI, arresting cells in G(1) phase of the cell cycle. Progestin-independent 60 epithelial cells evidenced reduced response to TGF-beta1 antiproliferative effects. TGF-beta1 inhibition of cyclins D1 and A expression and up-regulation of p21(CIP1) levels were the common findings in all three cell types. In addition, a significant content reduction of cyclin D1/cdk4 and cyclin A/cdk2 complexes was found after TGF-beta1 inhibition of MPA-dependent and -independent proliferation. TGF-beta1 inhibited cyclin D2 expression and up-regulated p27(KIP1) levels only when acting as inhibitor of MPA-induced proliferation of C4HD cells. Regulation of these two cell cycle components resulted in decreased cyclin D2/cdk2 complex and in increased p27(KIP1) association with cdk2 in C4HD cells treated with TGF-beta1. These two molecular mechanisms, unobserved in progestin-independent growth of C4HI or 60 cells, were associated with a significantly higher degree of inhibition of cdk2 kinase activity in C4HD cells compared to that found in TGF-beta-treated C4HI or 60 cells. Reduced sensitivity of 60 cells to the growth-inhibitory effects of TGF-beta1 correlated with significantly lower levels of p15(INK4B), p21(CIP1), and p27(KIP1) expressed in these cells, compared to the levels present in C4HD or C4HI cells, and correlated as well with lack of expression of p16(INK4). Thus, common targets were found to exist in TGF-beta1 inhibitory action on breast cancer cells, but regulation of specific targets was found when TGF-beta1-inhibited proliferation driven by the progesterone receptor.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Congêneres da Progesterona/metabolismo , Proteínas Proto-Oncogênicas , Fator de Crescimento Transformador beta/metabolismo , Proteínas Supressoras de Tumor , Animais , Ciclo Celular , Divisão Celular , Ciclina A/biossíntese , Ciclina D1/biossíntese , Ciclina D2 , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/biossíntese , Ciclinas/biossíntese , Ciclinas/metabolismo , Ciclinas/fisiologia , Regulação para Baixo , Feminino , Neoplasias Mamárias Experimentais/induzido quimicamente , Acetato de Medroxiprogesterona/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Associadas aos Microtúbulos/metabolismo , Congêneres da Progesterona/farmacologia , Proteínas Serina-Treonina Quinases/biossíntese , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1 , Células Tumorais Cultivadas , Regulação para CimaRESUMO
DFMO is an irreversible inhibitor of ornithine decarboxilase (ODC), the key enzyme in mammalian polyamine biosynthesis. The goal of this study was to determine the effects of DFMO on the expression of cyclin A at different stages of the cell cycle of Hep-2 cells. The cell cycle analysis, done by measuring the incorporation of thymidine in the cell DNA, revealed that DFMO produced a lower and constant level of that incorporation; this effect is probably due to the incapacity of the cells to culminate the phase S of the cell cycle. The expression of cyclin A increased in the phases S and G2 in control cells, almost disappearing in phase M. However, in DFMO treated cultures, the expression of cyclin A was increased in M and this effect remained still after 48 h treatment. We conclude that polyamines could exert an effect on the cyclin destruction mechanism, and the depletion caused by DFMO would alter this mechanism.
Assuntos
Ciclina A/biossíntese , Eflornitina/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores da Ornitina Descarboxilase , Humanos , Poliaminas/metabolismo , Células Tumorais CultivadasRESUMO
DFMO is an irreversible inhibitor of ornithine decarboxilase (ODC), the key enzyme in mammalian polyamine biosynthesis. The goal of this study was to determine the effects of DFMO on the expression of cyclin A at different stages of the cell cycle of Hep-2 cells. The cell cycle analysis, done by measuring the incorporation of thymidine in the cell DNA, revealed that DFMO produced a lower and constant level of that incorporation; this effect is probably due to the incapacity of the cells to culminate the phase S of the cell cycle. The expression of cyclin A increased in the phases S and G2 in control cells, almost disappearing in phase M. However, in DFMO treated cultures, the expression of cyclin A was increased in M and this effect remained still after 48 h treatment. We conclude that polyamines could exert an effect on the cyclin destruction mechanism, and the depletion caused by DFMO would alter this mechanism.
Assuntos
Humanos , Ciclina A , Eflornitina , Inibidores Enzimáticos/farmacologia , Ornitina Descarboxilase , Poliaminas , Células Tumorais CultivadasRESUMO
DFMO is an irreversible inhibitor of ornithine decarboxilase (ODC), the key enzyme in mammalian polyamine biosynthesis. The goal of this study was to determine the effects of DFMO on the expression of cyclin A at different stages of the cell cycle of Hep-2 cells. The cell cycle analysis, done by measuring the incorporation of thymidine in the cell DNA, revealed that DFMO produced a lower and constant level of that incorporation; this effect is probably due to the incapacity of the cells to culminate the phase S of the cell cycle. The expression of cyclin A increased in the phases S and G2 in control cells, almost disappearing in phase M. However, in DFMO treated cultures, the expression of cyclin A was increased in M and this effect remained still after 48 h treatment. We conclude that polyamines could exert an effect on the cyclin destruction mechanism, and the depletion caused by DFMO would alter this mechanism.(AU)
Assuntos
Humanos , RESEARCH SUPPORT, NON-U.S. GOVT , Ciclina A/biossíntese , Eflornitina/farmacologia , Inibidores Enzimáticos/farmacologia , Ornitina Descarboxilase/antagonistas & inibidores , Poliaminas/metabolismo , Células Tumorais CultivadasRESUMO
In mammalian cells, terminal differentiation is mutually exclusive with proliferation. However, resistance to differentiation-inducing therapy requires alternative strategies to control poorly responsive tumors. We now show that retroviral transfer of the antisense cyclin D1 gene to differentiation-refractory K1735 melanoma leads to loss of in vivo tumorigenicity, shortened replicative ability, induction of the tumor suppressor p53 protein and of the cdk-inhibitor p21WAF1, increased beta-galactosidase pH 6.0 activity, and elevation in the ratio of superoxide dismutases to peroxidases, all properties associated with replicative senescence. However, pigmentation and tyrosinase expression, characteristic of differentiated melanocytic cells or apoptosis-associated PARP cleavage, were not increased by antisense cyclin D1 transduction. Our data suggests that targetting cyclin D1 inhibition suppresses melanoma tumorigenicity by promoting a cytostatic differentiation-independent pathway, mediated by activation of p53 and anti-oxidant functions.