RESUMO
Coactosin-like protein (CLP, or Cotl1), is an F-actin-binding protein, whose role in cancer is largely unknown. Here we show that CLP/Cotl1 is highly expressed in a rat epithelial breast cancer cell line (FE1.3) compared with its mesenchymal counterpart (FE1.2). Knockdown of CLP/Cotl1 in FE1.3 cells increased cell proliferation, whereas its overexpression in FE1.2 cells inhibited proliferation in culture and reduced tumor growth in xenograft assays in mice. Mechanistically, we identified two major pathways through which CLP/Cotl1 exerts its suppressive effects. First, CLP/Cotl1 re-expression in FE1.2 and in human MCF7 breast cancer cells induced expression of the growth-suppressor gene interleukin-24 (IL-24), which independently of p53 upregulates the tumor-suppressor genes p53 apoptosis effector related to PMP-22 (PERP) and p21cip1. Second, overexpression of CLP/Cotl1 potentiated the growth-suppressive effect of transforming growth factor-ß1 (TGFß1), leading to downregulation of TGFß-responsive genes vascular growth factor A/B (VEGFA/VEGFB), hypoxia inducing factor 1α (HIF-1α) and trombospondin 1 (TSP1), which mediate various hallmarks of cancer progression including angiogenesis, invasion and metastasis. CLP/Cotl1 inhibited TGFß signaling via a non-canonical signaling involving IL-24-instigated inhibition of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) phosphorylation and subsequent post-transcriptional downregulation of SMAD2 and SMAD4. We also showed that CLP/COTL1 expression sensitizes breast cancer cells to chemotherapeutic drugs, and this was further enhanced by addition of exogenous TGFß1. CLP/Cotl1 expression is lost in many human malignancies including prostate, uterine and breast cancers. Thus, our results uncover a novel tumor-suppressor role for CLP/Cotl1 and identify the downstream effectors interleukin 24 (IL-24)/PERP and IL-24/MAPK/ERK/TGFß as potential targets for precision therapy.
Assuntos
Neoplasias da Mama/patologia , Proteínas de Transporte/metabolismo , Proteínas dos Microfilamentos/metabolismo , Animais , Proteínas de Transporte/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Genes Supressores de Tumor , Células HEK293 , Humanos , Interleucinas/metabolismo , Sistema de Sinalização das MAP Quinases , Células MCF-7 , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Proteínas dos Microfilamentos/genética , RNA Interferente Pequeno/metabolismo , Ratos , Fator de Crescimento Transformador beta1/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Multiplatform genomic analyses have identified 93 frequently altered genes in breast cancer. Of these, as many as 49 genes are directly or indirectly involved in transcription. These include constitutive and inducible DNA-binding transcription factors (DB-TFs, 13 genes), corepressors/coactivators (14 genes), epigenetic (10), and mediator/splicing/rRNA (3) factors. At least nine additional genes are immediate upstream regulators of transcriptional cofactors. G:profiler analysis reveals that these alterations affect cell cycle, development/differentiation, steroid hormone, and chromatin modification pathways. A notable observation is that DB-TFs that mediate major oncogenic signaling (e.g., WNT, receptor tyrosine kinase (RTK), NOTCH, and HIPPO), which switch from default repression (signal OFF) to transcriptional activation (signal ON), are not altered in breast cancer. Instead, corepressors (e.g., pRb for E2F1 downstream of various proliferation signals) or upstream factors (e.g., APC and AXIN for TCF, downstream of canonical WNT signaling) are lost, or coactivators (e.g., NOTCH1/2 for CSL/RBPJk) are induced. In contrast, constitutive (MYC, TBX3) and signal-induced (TP53, FOXA1) DB-TFs that do not mediate default repression are directly altered in breast cancer. Some of these TFs have been implicated in the establishment of super-enhancers and positive transcriptional elongation. In addition, oncogenic transcription is induced by mutations affecting regulatory elements or chromatin conformation that create new TF-binding sites in promoters and enhancers of oncogenic genes to promote tumorigenesis. Here we review these diverse oncogenic alterations in TFs in BC and discuss implications for therapy.
Assuntos
Neoplasias da Mama/metabolismo , Genoma , Fatores de Transcrição/metabolismo , Neoplasias da Mama/genética , Feminino , Humanos , Oncogenes , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Friend leukemia virus-induced erythroleukemia-1 (Fli-1), an E26 transformation specific (ETS) transcription factor, was isolated a quarter century ago through a retrovirus mutagenesis screen. Fli-1 has since been recognized to play critical roles in normal development and homeostasis. For example, it transcriptionally regulates genes that drive normal hematopoiesis and vasculogenesis. Indeed, Fli-1 is one of 10 key regulators of hematopoietic stem/progenitor cell maintenance and differentiation. Aberrant expression of Fli-1 also underlies a number of virally induced leukemias, including Friend virus-induced erythroleukemia and various types of human cancers, and it is the target of chromosomal translocations in childhood Ewing's sarcoma. Abnormal expression of Fli-1 is important in the etiology of autoimmune diseases such as systemic lupus erythematosus and systemic sclerosis. These studies establish Fli-1 as a strong candidate for drug development. Despite difficulties in targeting transcription factors, recent studies identified small-molecule inhibitors for Fli-1. Here we review past and ongoing research on Fli-1 with emphasis on its mechanistic function in autoimmune disease and malignant transformation. The significance of identifying Fli-1 inhibitors and their clinical applications for treatment of disease and cancer with deregulated Fli-1 expression are discussed.
Assuntos
Transformação Celular Neoplásica/genética , Hematopoese/genética , Leucemia/genética , Proteína Proto-Oncogênica c-fli-1/fisiologia , Sarcoma de Ewing/genética , Animais , Células-Tronco Hematopoéticas/citologia , Humanos , Lúpus Eritematoso Sistêmico/genética , Camundongos , Neovascularização Patológica/genética , Proteína Proto-Oncogênica c-fli-1/genética , Escleroderma Sistêmico/genéticaRESUMO
The Ets transcription factor, Fli-1 is activated in murine erythroleukemia and overexpressed in various human malignancies including Ewing's sarcoma, induced by the oncogenic fusion protein EWS/Fli-1. Recent studies by our group and others have demonstrated that Fli-1 plays a key role in tumorigenesis, and disrupting its oncogenic function may serve as a potential treatment option for malignancies associated with its overexpression. Herein, we describe the discovery of 30 anti-Fli-1 compounds, characterized into six functional groups. Treatment of murine and human leukemic cell lines with select compounds inhibits Fli-1 protein or mRNA expression, resulting in proliferation arrest and apoptosis. This anti-cancer effect was mediated, at least in part through direct inhibition of Fli-1 function, as anti-Fli-1 drug treatment inhibited Fli-1 DNA binding to target genes, such as SHIP-1 and gata-1, governing hematopoietic differentiation and proliferation. Furthermore, treatment with select Fli-1 inhibitors revealed a positive relationship between the loss of DNA-binding activity and Fli-1 phosphorylation. Accordingly, anti-Fli-1 drug treatment significantly inhibited leukemogenesis in a murine erythroleukemia model overexpressing Fli-1. This study demonstrates the ability of this drug-screening strategy to isolate effective anti-Fli-1 inhibitors and highlights their potential use for the treatment of malignancies overexpressing this oncogene.
RESUMO
Erythroleukemia induced by Friend Murine Leukemia Virus (F-MuLV) serves as a powerful tool for the study of multistage carcinogenesis and hematological malignancies in mice. Fli-1, a proto-oncogene and member of the Ets family, is activated through viral integration in F-MuLV-induced erythroleukemia, and is the most critical event in the induction of this disease. Fli-1 aberrant regulation is also observed in human malignancies, including Ewing's sarcoma, which is often linked to expression of the EWS/Fli-1 fusion oncoprotein. Here we examined the effects of Fli-1 inhibition to further elucidate its role in these pathological occurrences. The constitutive suppression of Fli-1, through RNA interference (RNAi), inhibits growth and induces death in F-MuLV-induced erythroleukemia cells. Expression of a dominant negative protein Engrailed (En)/Fli-1 reduces proliferation of EWS/Fli-1-transformed NIH-3T3 cells, and both F-MuLV-induced and human erythroleukemia cells. F-MuLV-induced erythroleukemia cells also display increased apoptosis, associated with reduced expression of bcl-2, a known fli-1 target gene. Introduction of En/Fli-1 into an F-MuLV-infected erythroblastic cell line induces differentiation, as shown by increased alpha-globin expression. These results suggest, for the first time, an essential role for continuous Fli-1 overexpression in the maintenance and survival of the malignant phenotype in murine and human erythroleukemias.
Assuntos
Proliferação de Células , Sobrevivência Celular , Vírus da Leucemia Murina/patogenicidade , Leucemia Eritroblástica Aguda/patologia , Proteína Proto-Oncogênica c-fli-1/metabolismo , Animais , Sequência de Bases , Diferenciação Celular , Linhagem Celular Tumoral , Primers do DNA , Regulação para Baixo , Humanos , Imuno-Histoquímica , Camundongos , Proto-Oncogene Mas , Proteína Proto-Oncogênica c-fli-1/genética , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: To study the role of BRCA mutations in ovarian cancer survival. PATIENTS AND METHODS: Blood samples and specimens of ovarian tumors (whenever blood samples were not available) at the time of the primary surgery were obtained in the course of a nationwide case-control study of women with ovarian cancer in Israel. The three common BRCA mutations in Israel (185delAG, 5382insC, and 6174delT) were analyzed with a multiplex polymerase chain reaction to amplify the exons containing the three mutations using fluor-labeled primers in a single reaction. Because each mutation is a small insertion or deletion, they can be detected as length polymorphisms. Patients were followed for up to 5 years (range, 20 to 64 months). Statistical analysis was performed using the Kaplan-Meier method and the log-rank test. Stepwise Cox regression analysis was used for determination of independent prognostic factors. RESULTS: This report is based on 896 blood or tumor specimens analyzed for the presence of the BRCA mutations. Of these, 234 women (26.1%) were found to be positive. A significant difference in survival pattern was found between BRCA1/BRCA2 carriers and noncarriers among the women with invasive ovarian cancer (median survival, 53.4 months v. 37.8 months; 3-year survival, 65.8% v. 51.9%, respectively). These differences were independent of age at diagnosis or stage of the disease. CONCLUSION: Our data indicate that the survival of patients with ovarian cancer is affected by BRCA germline mutation, at least in the early years after diagnosis.
Assuntos
DNA de Neoplasias/genética , Genes BRCA1 , Genes BRCA2 , Mutação em Linhagem Germinativa/genética , Neoplasias Ovarianas/genética , Estudos de Casos e Controles , Análise Mutacional de DNA , Feminino , Humanos , Pessoa de Meia-Idade , Invasividade Neoplásica , Neoplasias Ovarianas/patologia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Prognóstico , Estudos Retrospectivos , Análise de SobrevidaRESUMO
Erythroleukemias induced by various strains of Friend virus are multistage malignancies that result from the accumulation of genetic mutations, including the activation of proto-oncogenes and the inactivation of tumor suppressor genes. In this study, we demonstrate that Bcl-2 expression is activated in the majority of F-MuLV-induced erythroleukemia cell lines. In contrast, Bcl-2 was not expressed in any of the FV-P-induced erythroleukemia cell lines and protein levels were low or negligible in FV-A-induced erythroleukemia cell lines examined. In vivo, Bcl-2 expression levels gradually increased in F-MuLV-induced erythroleukemic cells prior to adaptation to culture. High expression of Bcl-2 in F-MuLV-induced erythroleukemic cells was shown to proceed the emergence of p53 mutation suggesting that Bcl-2 expression may delay p53 mutation in the leukemic cells. This is further supported by the demonstration that the majority of F-MuLV-induced erythroleukemia cell lines established from primary tumors induced in p53 mutant mice express low to negligible levels of Bcl-2. We have shown that the high levels of Bcl-2 expression in FV-P-induced erythroleukemic cells inhibited apoptosis induced by etoposide, low serum and p53 expression. Similarly, ectopic Bcl-2 expression within these cells also provided protection from apoptosis induced by etoposide and growth in low serum. These results suggest that the anti-apoptotic action of Bcl-2 may confer a selective in vivo and in vitro growth advantage to F-MuLV-induced erythroleukemic cells, which is not shared by FV-P/FV-A-induced erythroleukemic cells. The observed induction of Bcl-2 expression in vivo constitutes a novel but late oncogenic event associated with the progression of F-MuLV-induced erythroleukemias.
Assuntos
Apoptose/fisiologia , Vírus da Leucemia Murina de Friend , Leucemia Eritroblástica Aguda/patologia , Leucemia Eritroblástica Aguda/virologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Animais , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologiaRESUMO
INTRODUCTION: Surgery is the treatment of choice in most early stages of cervical cancer and advanced stages of ovarian cancer. Failing to preoperatively diagnose para-aortic and parametrial metastases in cervical cancer or a non-resectable, ovarian cancer, may results in a superfluous laparotomy. AIM: To evaluate the advantage of using laparoscopy in cervical and ovarian cancer. PATIENTS AND METHODS: Study population includes patients with ovarian or cervical cancer referred between 1997-1999. A CT scan and a trans-vaginal sonography were used to detect involvement of pelvic and para-aortic lymph nodes, parametrium and other metastases. In patients with cervical cancer, para-aortic lymph node dissection was laparoscopically performed. When para-aortic nodes were negative and parametrium was clear, radical hysterectomy and pelvic lymph nodes dissection was conducted through laparotomy. When para-aortic lymph nodes or parametrium were positive, patients were referred for radiation therapy. In ovarian cancer patients, the upper abdomen and the pelvis were examined laparoscopically to evaluate the possibility of optimal debulking surgery. Staging was done for patients who were not candidates for optimal debulking surgery and second debulking surgery was considered. RESULTS: Nine patients with stage 1b-11a cervical cancer were included. Preoperatively, 2 of them were suspected for lymph node involvement. Following laparoscopy one was confirmed to have para-aortic lymph node involvement and the other did not. In the remaining 8 patients, one was found to have parametrial involvement and laparotomy was avoided, while the rest were treated surgically. Eighteen ovarian cancer patients were included in this study. Laparoscopy revealed an extensive disease in 7 patients and therefore staging laparoscopy was completed. In the remaining 11 patients, laparotomy was performed, operable disease was found and complete debulking surgery was conducted. In only one patient of the 11 complete debulking surgeries was not possible. CONCLUSIONS: In accordance with the experience and skills of the surgical team, we propose utilizing laparoscopy in cases where laparotomy may be avoided.
Assuntos
Laparoscopia , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/cirurgia , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/cirurgia , Feminino , Humanos , Histerectomia , Estadiamento de Neoplasias , Neoplasias Ovarianas/patologia , Reprodutibilidade dos Testes , Tomografia Computadorizada por Raios X , Neoplasias do Colo do Útero/patologiaRESUMO
We studied the relative RNA expression of clock genes throughout one 24-hour period in biopsies obtained from the oral mucosa and skin from eight healthy diurnally active male study participants. We found that the human clock genes hClock, hTim, hPer1, hCry1, and hBmal1 are expressed in oral mucosa and skin, with a circadian profile consistent with that found in the suprachiasmatic nuclei and the peripheral tissues of rodents. hPer1, hCry1, and hBmal1 have a rhythmic expression, peaking early in the morning, in late afternoon, and at night, respectively, whereas hClock and hTim are not rhythmic. This is the first human study to show a circadian profile of expression for all five clock genes as documented in rodents, suggesting their functional importance in man. In concurrent oral mucosa biopsies, thymidylate synthase enzyme activity, a marker for DNA synthesis, had a circadian variation with peak activity in early afternoon, coinciding with the timing of S phase in our previous study on cell-cycle timing in human oral mucosa. The major peak in hPer1 expression occurs at the same time of day as the peak in G(1) phase in oral mucosa, suggesting a possible link between the circadian clock and the mammalian cell cycle.
Assuntos
Ritmo Circadiano/fisiologia , Proteínas de Drosophila , Proteínas do Olho , Mucosa Bucal/metabolismo , Células Fotorreceptoras de Invertebrados , Pele/metabolismo , Transativadores/genética , Fatores de Transcrição ARNTL , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteínas CLOCK , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular , Ritmo Circadiano/genética , Criptocromos , Flavoproteínas/genética , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Mucosa Bucal/enzimologia , Proteínas Nucleares/genética , Proteínas Circadianas Period , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G , Timidilato Sintase/genética , Timidilato Sintase/metabolismo , Fatores de Transcrição/genéticaRESUMO
In previous studies, we identified a common site of retroviral integration designated Fli-2 in Friend murine leukemia virus (F-MuLV)-induced erythroleukemia cell lines. Insertion of F-MuLV at the Fli-2 locus, which was associated with the loss of the second allele, resulted in the inactivation of the erythroid cell- and megakaryocyte-specific gene p45(NFE2). Frequent disruption of p45(NFE2) due to proviral insertion suggests a role for this transcription factor in the progression of Friend virus-induced erythroleukemias. To assess this possibility, erythroleukemia was induced by F-MuLV in p45(NFE2) mutant mice. Since p45(NFE2) homozygous mice mostly die at birth, erythroleukemia was induced in +/- and +/+ mice. We demonstrate that +/- mice succumb to the disease moderately but significantly faster than +/+ mice. In addition, the spleens of +/- mice were significantly larger than those of +/+ mice. Of the 37 tumors generated from the +/- and +/+ mice, 10 gave rise to cell lines, all of which were derived from +/- mice. Establishment in culture was associated with the loss of the remaining wild-type p45(NFE2) allele in 9 of 10 of these cell lines. The loss of a functional p45(NFE2) in these cell lines was associated with a marked reduction in globin gene expression. Expression of wild-type p45(NFE2) in the nonproducer erythroleukemic cells resulted in reduced cell growth and restored the expression of globin genes. Similarly, the expression of p45(NFE2) in these cells also slows tumor growth in vivo. These results indicate that p45(NFE2) functions as an inhibitor of erythroid cell growth and that perturbation of its expression contributes to the progression of Friend erythroleukemia.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Vírus da Leucemia Murina de Friend/fisiologia , Leucemia Eritroblástica Aguda/patologia , Leucemia Eritroblástica Aguda/virologia , Fatores de Transcrição/metabolismo , Animais , Animais Recém-Nascidos , Divisão Celular , Células Clonais/metabolismo , Células Clonais/patologia , Células Clonais/virologia , Proteínas de Ligação a DNA/genética , Progressão da Doença , Fatores de Ligação de DNA Eritroide Específicos , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Genótipo , Globinas/genética , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Subunidade p45 do Fator de Transcrição NF-E2 , Fatores de Transcrição/genética , Transfecção , Células Tumorais CultivadasRESUMO
A major obstacle in the clinical management of malignant melanoma is its intrinsic resistance to chemotherapy and radiation therapy. Consequently, most patients with melanoma often do not respond to conventional anticancer therapy in a clinically significant manner. Recent advances in cancer research have provided new insights into the mechanisms of intrinsic resistance in melanomas. We have recently reported that the over-expression of tyrosinase-related protein 2 (TYRP2), an enzyme that is well characterized for its function in melanin synthesis, is associated specifically with resistance to DNA damaging drugs and radiation treatment. This review will summarize our findings as well as discuss the possible mechanisms by which TYRP2 over-expression contributes to intrinsic resistance in human malignant melanoma.
Assuntos
Antineoplásicos/uso terapêutico , Linhagem da Célula/fisiologia , Resistencia a Medicamentos Antineoplásicos , Oxirredutases Intramoleculares/metabolismo , Melanoma/enzimologia , Tolerância a Radiação , Humanos , Melanoma/tratamento farmacológico , Melanoma/radioterapiaRESUMO
Fli-2 is a common site of proviral integration in multistage erythroleukemia cells induced by Friend murine leukemia virus (F-MuLV) or the polycythemia strain of Friend leukemia virus (FV-P). Previously, we reported that integration of Friend virus into the Fli-2 locus in CB3, an erythroleukemia cell line that harbors a homozygous inactivation of the Fli-2 locus, results in the loss of expression of two genes encoding the 45-kDa subunit of the erythroid-specific nuclear factor p45 NFE2 and the splicing factor HnRNP A1. Here, we report the identification of a third gene, Heterochromatin protein 1 (HP1alpha, also known as CBX5), which is located downstream of HnRNP A1, and p45 NFE2. Northern blot analysis revealed that the expression of HP1alpha, along with p45 NFE2 and HnRNP A1, is either undetectable or substantially reduced in CB3 cells, suggesting that HP1alpha expression is also regulated by proviral insertion within the Fli-2 locus in CB3 cells. Because p45 NFE2 was previously mapped to mouse chromosome 15, our results demonstrate that HP1alpha and HnRNP A1 are also located on mouse chromosome 15 and that the p45 NFE2, HnRNP A1, and HP1alpha genes are arranged contiguously. Contiguous arrangement of these three genes was also detected in man; this consequently localizes HP1alpha to human chromosome band 12q13.
Assuntos
Proteínas Cromossômicas não Histona/genética , Cromossomos Humanos Par 12/genética , Proteínas de Ligação a DNA/genética , Heterocromatina/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , Proteínas Proto-Oncogênicas , Ribonucleoproteínas/genética , Supressão Genética , Transativadores/genética , Fatores de Transcrição/genética , Animais , Homólogo 5 da Proteína Cromobox , Fatores de Ligação de DNA Eritroide Específicos , Ordem dos Genes/genética , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Subunidade p45 do Fator de Transcrição NF-E2 , Proteína Proto-Oncogênica c-fli-1 , RNA Nuclear Heterogêneo/genética , Proteínas de Ligação a RNA/genética , Mapeamento por RestriçãoRESUMO
Tyrosinase-related protein-2 (TYRP2) is a melanocyte-specific enzyme that catalyses the non-decarboxylative tautomerization of L-dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) in the melanin biosynthetic pathway. We have recently demonstrated that the constitutive expression of TYRP2 in human melanoma cells positively correlates with cis-diamminedichloroplatinum (II) (CDDP) resistance, and that the ectopic expression of TYRP2 in CDDP-sensitive cells rendered them more resistant to CDDP treatment. Here, we demonstrate that this correlation between constitutive TYRP2 expression and CDDP resistance applies to a panel of distinct human melanoma cell lines obtained from patients with melanoma at various stages of disease progression. We further show that CDDP resistance correlates only with TYRP2 expression and is associated neither with tyrosinase and TYRP1 expression, nor with cellular melanin content. Together, these results further support the notion that TYRP2 is a novel mediator of CDDP resistance in melanoma cells and suggest that this function of TYRP2 is independent of cellular melanin content and of the other regulatory enzymes of the melanogenic pathway.
Assuntos
Cisplatino/toxicidade , Resistencia a Medicamentos Antineoplásicos , Oxirredutases Intramoleculares/genética , Melaninas/metabolismo , Glicoproteínas de Membrana , Oxirredutases , Sobrevivência Celular/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Oxirredutases Intramoleculares/metabolismo , Melanoma , Monofenol Mono-Oxigenase/genética , Proteínas/genética , Células Tumorais CultivadasRESUMO
Terminal erythroid differentiation is accompanied by decreased expression of c-Kit and decreased proliferation of erythroid progenitor cells. Using a newly established erythroleukemia cell line HB60-5, which proliferates in response to erythropoietin (Epo) and stem cell factor (SCF) and differentiates when stimulated with Epo alone, we characterized several events associated with the cell cycle during erythroid differentiation. Forty-eight h after SCF withdrawal and Epo stimulation, there was strong inhibition of cyclin-dependent kinase (cdk) 4 and cdk6 activities, associated with an increase in the binding of p27 and p15 to cdk6. A significant increase in the binding of p27 to cyclin E- and cyclin A-associated cdk2 correlated with the inhibition of these kinases. In addition, the expression of c-Myc and its downstream transcriptional target Cdc25A were found to be down-regulated during Epo-induced terminal differentiation of HB60-5 cells. The loss of Cdc25A was associated with an increase in the phosphotyrosylation of cyclin E-associated cdk2, which may contribute to cell cycle arrest during differentiation. Although overexpression of p27 in HB60-5 cells caused G1 arrest, it did not promote terminal erythroid differentiation. Thus, the cell cycle arrest that involves p27 is part of a broader molecular program during HB60-5 erythroid differentiation. Moreover, we suggest that SCF stimulation of erythroblasts, in addition to inhibiting erythroid differentiation, activates parallel or sequential signals responsible for maintaining cyclin/cdk activity.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Eritrócitos/citologia , Fase G1/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas , Fator de Células-Tronco/farmacologia , Proteínas Supressoras de Tumor , Animais , Diferenciação Celular/efeitos dos fármacos , Ciclina A/metabolismo , Ciclina D2 , Ciclina D3 , Ciclina E/metabolismo , Ciclina G , Ciclina G1 , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Regulação para Baixo/fisiologia , Eritrócitos/enzimologia , Leucemia Eritroblástica Aguda , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Associadas aos Microtúbulos/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fase S/fisiologia , Transfecção , Células Tumorais Cultivadas , Fosfatases cdc25/metabolismoRESUMO
We have recently isolated the erythroleukemic cell line, HB60-5, that proliferates in the presence of erythropoietin (Epo) and stem cell factor (SCF), but undergoes terminal differentiation in the presence of Epo alone. Ectopic expression of the ets related transcription factor Fli-1 in these cells resulted in the establishment of the Epo-dependent cell line HB60-ED that proliferates in the presence of Epo. In this study, we utilized these two cell lines to examine the signal transduction pathways that are activated in response to Epo and SCF stimulation. We demonstrate that Epo, but not SCF, phosphorylates STAT-5 in both HB60-5 and HB60-ED cells. Interestingly, SCF activates the Shc/ras pathway in HB60-5 cells while Epo does not. However, both Epo and SCF are capable of activating the Shc/ras pathway in HB60ED cells. Furthermore, enforced expression of gp55 in HB60-5 cells by means of infection with the Spleen Focus Forming virus-P (SFFV-P), confers Epo independent growth, which is associated with the up-regulation of Fli-1. Activation of the Shc/ras pathway is readily detected in gp55 expressing cells in response to both Epo and SCF, and is associated with a block in STAT-5B tyrosine phosphorylation. These results suggest that STAT-5 activation, in the absence of Shc/ras activation, plays a role in erythroid differentiation. Moreover, Fli-1 is capable of switching Epo-induced differentiation to Epo-induced proliferation, suggesting that this ets factor regulated genes whose products modulate the Epo-Epo-R signal transduction pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Eritropoetina/metabolismo , Vírus da Leucemia Murina de Friend , Leucemia Eritroblástica Aguda/metabolismo , Proteínas do Leite , Proteínas Proto-Oncogênicas , Transdução de Sinais , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Eritropoese/fisiologia , Eritropoetina/farmacologia , Humanos , Leucemia Eritroblástica Aguda/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas/metabolismo , Proteína Proto-Oncogênica c-fli-1 , Receptores da Eritropoetina/metabolismo , Fator de Transcrição STAT5 , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Fator de Células-Tronco/metabolismo , Fator de Células-Tronco/farmacologia , Transativadores/metabolismo , Células Tumorais Cultivadas , Proteínas do Envelope Viral/metabolismo , Proteínas ras/metabolismoRESUMO
Ovarian cancer is a component of the autosomal-dominant hereditary breast-ovarian cancer syndrome and may be due to a mutation in either the BRCA1 or BRCA2 genes. Two mutations in BRCA1 (185delAG and 5382insC) and one mutation in BRCA2 (6174delT) are common in the Ashkenazi Jewish population. One of these three mutations is present in approximately 2% of the Jewish population. Each mutation is associated with an increased risk of ovarian cancer, and it is expected that a significant proportion of Jewish women with ovarian cancer will carry one of these mutations. To estimate the proportion of ovarian cancers attributable to founding mutations in BRCA1 and BRCA2 in the Jewish population and the familial cancer risks associated with each, we interviewed 213 Jewish women with ovarian cancer at 11 medical centers in North America and Israel and offered these women genetic testing for the three founder mutations. To establish the presence of nonfounder mutations in this population, we also completed the protein-truncation test on exon 11 of BRCA1 and exons 10 and 11 of BRCA2. We obtained a detailed family history on all women we studied who had cancer and on a control population of 386 Ashkenazi Jewish women without ovarian or breast cancer. A founder mutation was present in 41.3% of the women we studied. The cumulative incidence of ovarian cancer to age 75 years was found to be 6.3% for female first-degree relatives of the patients with ovarian cancer, compared with 2.0% for the female relatives of healthy controls (relative risk 3.2; 95% CI 1.5-6.8; P=.002). The relative risk to age 75 years for breast cancer among the female first-degree relatives was 2.0 (95% CI 1.4-3.0; P=.0001). Only one nonfounder mutation was identified (in this instance, in a woman of mixed ancestry), and the three founding mutations accounted for most of the observed excess risk of ovarian and breast cancer in relatives.
Assuntos
Efeito Fundador , Genes BRCA1/genética , Judeus/genética , Mutação/genética , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genética , Fatores de Transcrição/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteína BRCA2 , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/genética , Análise Mutacional de DNA , Feminino , Frequência do Gene/genética , Predisposição Genética para Doença/genética , Humanos , Incidência , Israel/epidemiologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , América do Norte/epidemiologia , Neoplasias Ovarianas/epidemiologia , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , LinhagemRESUMO
Gene amplification is frequently present in human tumors, although specific target genes relevant to many amplified loci remain unidentified. An expression cloning assay enabled identification of a candidate oncogene derived from human chromosome 3p14.1. The cDNA retrieved from morphologically transformed cells contained the full-length protein coding region and detected an abundant transcript in the same cells. Sequence analysis revealed identity with the wild-type sequence of p44S10, a highly conserved subunit of the 26S proteasome that exhibits similarity to the Arabidopsis fus6/cop11 family of signaling molecules. p44S10 gene copy number and mRNA expression were increased in association with segmental 1.8 - 11-fold chromosomal gains in cutaneous malignant melanoma cell lines (5/13; 40%) and tumors (2/40; 5%), and in breast cancer MCF-7 cells. Likewise, malignant progression of human radial growth phase WM35 melanoma cells was associated with amplification and increased expression of endogenous p44S10, and increased expression of p44S10 was sufficient to induce proliferation of WM35 cells in vivo. The results demonstrate segmental copy number gains within chromosome 3p in cutaneous malignant melanoma and suggest that deregulation of a proteasome regulatory particle subunit may contribute to the malignant phenotype.
Assuntos
Adenosina Trifosfatases/genética , Cisteína Endopeptidases/genética , Amplificação de Genes , Melanoma/enzimologia , Melanoma/genética , Complexos Multienzimáticos/genética , Proteínas Oncogênicas/genética , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/genética , Adenosina Trifosfatases/isolamento & purificação , Animais , Linhagem Celular Transformada , Cromossomos Humanos Par 17 , Cromossomos Humanos Par 20 , Cromossomos Humanos Par 3 , Cisteína Endopeptidases/isolamento & purificação , Cisteína Endopeptidases/metabolismo , Progressão da Doença , Ativação Enzimática/genética , Humanos , Melanoma/patologia , Melanoma Experimental/enzimologia , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , Proteínas Oncogênicas/isolamento & purificação , Proteínas Oncogênicas/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/isolamento & purificação , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma , Ratos , Análise de Sequência de DNA , Neoplasias Cutâneas/patologia , Células Tumorais CultivadasRESUMO
A major obstacle in the systemic treatment of advanced malignant melanoma is its intrinsic resistance to conventionally used chemotherapeutic agents. In order to investigate the mechanisms of this intrinsic resistance, we have previously utilized retroviral insertional mutagenesis on an early-stage, drug sensitive human melanoma cell line (WM35) to establish mutated cell lines that exhibited increased resistance to cis-diammi-nedichloroplatinum(II) (CDDP). Here, we demonstrate that this increased resistance to CDDP is mediated by the over-expression of tyrosinase-related protein-2 (TYRP2), an enzyme that normally functions in the biosynthesis of the pigment, melanin. Northern and Western blot analyses revealed that the expression of TYRP2 in the virally-derived cell lines as well as in a panel of human melanoma cell lines positively correlated with their levels of resistance to CDDP. Furthermore, enforced expression of TYRP2 in WM35 cells by transfection elevated their resistance to CDDP. The increased CDDP resistance in the virally-derived clones and TYRP2 transfectants was accompanied by a reduction in CDDP-induced apoptosis. Interestingly, the virally-derived CDDP-resistant clones also showed cross resistance to carboplatin and methotrexate, but not taxol, suggesting that TYRP2 over-expression may confer resistance specifically to DNA damaging agents. Overall, these results demonstrate a novel mechanism of drug resistance in human melanoma cells that is mediated by the over-expression of TYRP2. Since TYRP2 is expressed only in cells of melanocytic lineage, this may represent the first report of a lineage-specific mechanism of drug resistance. In summary, these findings suggest a significant role for TYRP2 in the intrinsic drug resistance phenotype of human melanoma cells and may have important implications in the development of chemosensitization strategies for the clinical management of this disease.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Oxirredutases Intramoleculares/fisiologia , Melanoma/tratamento farmacológico , Apoptose/efeitos dos fármacos , Carboplatina/farmacologia , Cisplatino/uso terapêutico , Humanos , Metotrexato/farmacologia , Paclitaxel/farmacologia , Células Tumorais CultivadasRESUMO
Aberrant expression of the Fli-1 transcription factor following genetic mutation has been recognized as a seminal event in the initiation of certain types of malignant transformation. Indeed, the etiology of a number of virally induced leukemias, including Friend virus-induced erythroleukemia, has been associated with Fli-1 overexpression. The clinical relevance of Fli-1 becomes apparent in human Ewing's sarcoma in which Fli-1 is the target of a characteristic chromosomal translocation. As such, Fli-1 has generated considerable interest over the past several years for its role in malignant transformation and tumor progression. This review will present a synopsis of the current research on Fli-1 with emphasis on its function in malignant transformation. Moreover, the possible role of Fli-1 in cellular proliferation, differentiation and survival, as well as the recent development of transgenic and knock-out mice to investigate the function of Fli-1 will be discussed. Finally, the significance of identifying target genes that are regulated by Fli-1 and their role in cellular function will be reviewed.