RESUMEN
Immunotherapy targeting leukemia-associated antigens has shown promising results. Because of the heterogeneity of leukemia, vaccines with a single peptide have elicited only a limited immune response. Targeting several peptides together elicited peptide-specific cytotoxic T lymphocytes (CTLs) in leukemia patients, and this was associated with clinical responses. Thus, the discovery of novel antigens is essential. In the current study, we investigated cyclin E as a novel target for immunotherapy. Cyclin E1 and cyclin E2 were found to be highly expressed in hematologic malignancies, according to reverse transcription polymerase chain reaction and western blot analysis. We identified two HLA-A*0201 binding nonameric peptides, CCNE1M from cyclin E1 and CCNE2L from cyclin E2, which both elicited the peptide-specific CTLs. The peptide-specific CTLs specifically kill leukemia cells. Furthermore, CCNE1M and CCNE2L CTLs were increased in leukemia patients who underwent allogeneic hematopoietic stem cell transplantation, and this was associated with desired clinical outcomes. Our findings suggest that cyclin E1 and cyclin E2 are potential targets for immunotherapy in leukemia.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Vacunas contra el Cáncer/inmunología , Ciclina E/inmunología , Ciclinas/inmunología , Antígeno HLA-A2/inmunología , Leucemia/inmunología , Proteínas Oncogénicas/inmunología , Adulto , Anciano , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/farmacología , Epítopos de Linfocito T/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Linfocitos T Citotóxicos/inmunología , Adulto JovenRESUMEN
Trace elements play important roles in human health, but little is known about their functions in humoral immunity. Here, we show an important role for iron in inducing cyclin E and B cell proliferation. We find that iron-deficient individuals exhibit a significantly reduced antibody response to the measles vaccine when compared to iron-normal controls. Mice with iron deficiency also exhibit attenuated T-dependent or T-independent antigen-specific antibody responses. We show that iron is essential for B cell proliferation; both iron deficiency and α-ketoglutarate inhibition could suppress cyclin E1 induction and S phase entry of B cells upon activation. Finally, we demonstrate that three demethylases, KDM2B, KDM3B and KDM4C, are responsible for histone 3 lysine 9 (H3K9) demethylation at the cyclin E1 promoter, cyclin E1 induction and B cell proliferation. Thus, our data reveal a crucial role of H3K9 demethylation in B cell proliferation, and the importance of iron in humoral immunity.
Asunto(s)
Linfocitos B/inmunología , Proliferación Celular , Histonas/química , Histonas/inmunología , Inmunidad Humoral , Lisina/inmunología , Animales , Linfocitos B/química , Linfocitos B/citología , Ciclo Celular , Células Cultivadas , Ciclina E/genética , Ciclina E/inmunología , Desmetilación , Proteínas F-Box/genética , Proteínas F-Box/inmunología , Histonas/genética , Hierro/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/inmunología , Activación de Linfocitos , Lisina/genética , Ratones , Ratones Endogámicos C57BL , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/inmunología , Regiones Promotoras Genéticas , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
The present study aimed to evaluate the inhibitory effects of an irinotecan derivative, ZBH1208, on brain tumors, and to explore the underlying molecular mechanisms. To determine the effects of ZBH1208, a brain tumor mouse model was established by transplanting B22 cells. Subsequently, the visceral indices of immune organs and white blood cell counts were determined, and the effects of ZBH1208 on the expression levels of cell cyclerelated proteins were assessed by western blotting. The tumor inhibition rates of 20 and 40 mg/kg ZBH1208 were 11.7 and 54.1%, respectively. Compared with the negative control group, ZBH1208 barely affected visceral indices or white blood cell count. Furthermore, the expression levels of p53, p21, cyclindependent kinase 7 (CDK7), Wee1, phosphorylated (p)cell division cycle 2 (CDC2) (Tyr15), pCDC2 (Thr161) and cyclin B1 proteins were upregulated, whereas the expression levels of cyclin E were downregulated, and those of CDC2, CDK2 and CDC25C were barely altered. In conclusion, the present study demonstrated that ZBH1208 suppressed the growth of B22 mouse brain tumor xenografts, but did not affect their visceral indices or white blood cell counts. It was suggested that ZBH1208 exerted its effects by regulating the expression of p53, p21, Wee1, pCDC2 (Tyr15) and cyclin E proteins.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Camptotecina/análogos & derivados , Regulación Neoplásica de la Expresión Génica , Inmunidad Innata/efectos de los fármacos , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/patología , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/inmunología , Camptotecina/farmacología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/inmunología , Línea Celular Tumoral , Ciclina B1/genética , Ciclina B1/inmunología , Ciclina E/genética , Ciclina E/inmunología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/inmunología , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/inmunología , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Vida Libre de Gérmenes , Irinotecán , Masculino , Ratones , Ratones Endogámicos ICR , Trasplante de Neoplasias , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/inmunología , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/inmunología , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
Coinfection of hepatitis B virus (HBV) with hepatitis C virus (HCV) is quite common, leading to an increase in morbidity and mortality. As such, HBV vaccination is recommended in HCV-infected individuals. However, HBV vaccine responses in HCV-infected individuals are often blunted compared with uninfected populations. The mechanism for this failure of vaccine response in HCV-infected subjects remains unclear. In this study, we investigated the expression and function of an inhibitory receptor, killer cell lectin-like receptor subfamily G member 1 (KLRG1), in the regulation of CD4(+) T cells and HBV vaccine responses during HCV infection. We demonstrated that KLRG1 was overexpressed on CD4(+) T cells from HCV-infected, HBV vaccine nonresponders compared with HBV vaccine responders. The capacity of CD4(+) T cells to proliferate and secrete IL-2 cytokine was inversely associated with the level of KLRG1 expression. Importantly, blocking KLRG1 signaling resulted in a significant improvement in CD4(+) T cell proliferation and IL-2 production in HCV-infected, HBV vaccine nonresponders in response to TCR stimulation. Moreover, blockade of KLRG1 increased the phosphorylation of Akt (Ser(473)) and decreased the expression of cell cycle inhibitors p16(ink4a) and p27(kip1), which subsequently enhanced the expression of cyclin-dependent kinase 2 and cyclin E. These results suggest that the KLRG1 pathway impairs CD4(+) T cell responses to neoantigen and induces a state of immune senescence in individuals with HCV infection, raising the possibility that blocking this negative-signaling pathway might improve HBV vaccine responses in the setting of chronic viral infection.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Vacunas contra Hepatitis B/inmunología , Hepatitis B/inmunología , Hepatitis C/inmunología , Lectinas Tipo C/genética , Transactivadores/genética , Envejecimiento/genética , Envejecimiento/inmunología , Linfocitos T CD4-Positivos/virología , Proliferación Celular , Células Cultivadas , Coinfección/genética , Coinfección/inmunología , Ciclina E/genética , Ciclina E/inmunología , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/inmunología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/inmunología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/inmunología , Hepacivirus/inmunología , Hepatitis B/genética , Hepatitis B/prevención & control , Virus de la Hepatitis B/inmunología , Hepatitis C/genética , Hepatitis C/virología , Humanos , Interleucina-2/genética , Interleucina-2/inmunología , Lectinas Tipo C/inmunología , Fosforilación/genética , Fosforilación/inmunología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Receptores Inmunológicos , Transducción de Señal/genética , Transducción de Señal/inmunología , Transactivadores/inmunologíaRESUMEN
AIMS: To clarify which immunohistochemical markers could be helpful in distinguishing between classical Hodgkin's lymphoma (cHL) and primary mediastinal B-cell lymphoma (PMBCL) to more narrowly define 'B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and cHL'. METHODS AND RESULTS: Two hundred and 83 cHLs and 51 PMBCLs were analysed on validated tissue microarray platforms with antibodies to BOB.1, CD15, CD20, CD23, CD30, CD79a, cyclin E, LMP-1, MUM1p, p63 and Oct2. The marker cut-off scores were calculated using receiver-operating characteristic curves. Markers with the highest positive predictive value for cHL were: CD15, cyclin E, LMP-1 (all 100%), MUM1p (93%) and CD30 (83%). High sensitivity was achieved only by CD30 (92%) and cyclin E (79%). Nineteen percent of PMBCLs were also positive for CD30, which led to a lower specificity of CD30 as regards cHL (81%) compared with cyclin E (100%). The antibodies with the highest positive predictive value for PMBCL were: CD23 (98%), p63 (96%), BOB.1 (94%) and CD79a (90%), with high sensitivity for BOB.1 (100%), CD79a (89%) and p63 (82%). CONCLUSIONS: The use of at least three of the most accurate immunohistochemical markers, cyclin E, CD79a and BOB.1, may be helpful in the differential diagnosis of cHL and PMBCL.
Asunto(s)
Biomarcadores de Tumor , Antígenos CD79/análisis , Ciclina E/análisis , Enfermedad de Hodgkin/diagnóstico , Linfoma de Células B Grandes Difuso/diagnóstico , Transactivadores/análisis , Anticuerpos Antineoplásicos/inmunología , Antígenos CD79/inmunología , Ciclina E/inmunología , Diagnóstico Diferencial , Enfermedad de Hodgkin/inmunología , Enfermedad de Hodgkin/patología , Humanos , Inmunohistoquímica , Linfoma de Células B Grandes Difuso/inmunología , Linfoma de Células B Grandes Difuso/patología , Valor Predictivo de las Pruebas , Curva ROC , Transactivadores/inmunologíaRESUMEN
BACKGROUND: Lung cancer is the leading cause of cancer death in the majority of developed countries. Cyclin E regulates the the G(1)-S phase transition of the cell cycle. Cyclin A increases during the S- and G(2)-phases, and is a regulator of the transition to mitosis.The aim of this study was to evaluate the prognostic significance of cyclin A and cyclin E expression in primary, resected stage I-IIIA non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: The expression of cyclin A and E was investigated in the paraffin-embedded tumor tissue of 71 patients (53 men and 18 women; age 59.27+/-8.50 years), using a monoclonal antibodies to cyclin A and to cyclin E. RESULTS: Forty-seven out of 71 (66%) tumor tissue specimens were positive for cyclin A and twenty-six (37%) were positive for cyclin E. In the majority of cases, nuclear staining was apparent. Cyclin A and cyclin E expression was significantly higher in squamous cell carcinoma than in adenocarcinoma (cyclin A: Chi(2) Yates'a 4.6; p=0.032; cyclin E: Chi(2) Yates'a 5.12: p=0.023). The prognostic value of cyclin A and E expression was examinated in all patients and in patients with squamous cell lung cancer and adenocarcinoma and separately for every stage, but no correlations were found. CONCLUSION: No prognostic value of cyclin A and E expression was found in NSCLC, but significantly higher cyclin A and E expression was found in squamous cell carcinomas than in adenocarcinomas.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Ciclina A/metabolismo , Ciclina E/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenocarcinoma/cirugía , Anciano , Anticuerpos Monoclonales/farmacología , Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/cirugía , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/cirugía , Ciclina A/inmunología , Ciclina E/inmunología , Femenino , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/cirugía , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , PronósticoRESUMEN
B-cell receptor (BCR) ligation induces proliferation and survival in mature B-cells but conversely, can lead to apoptosis in immature B-cells. We have previously shown that c-Rel, a member of the NF-kappaB transcription factor family, is essential for mature B-cell survival and proliferation via regulation of the anti-apoptotic molecule Bcl-X and cell cycle genes E2F3a and cyclin E. Here, we report that c-Rel-deficient mature B-cells are rendered sensitive to BCR-induced growth arrest and apoptosis in a manner that strongly resembles the phenotypic response of immature B-cells to BCR signaling. We further demonstrate that BCR-stimulated immature B-cells are defective in NF-kappaB activation, but that introduction of two downstream c-Rel target genes, Bcl-X and cyclin E, can restore survival and proliferation to these cells. Our studies therefore suggest that specific blockade of NF-kappaB activation may be responsible for the growth arrest and apoptosis of BCR-activated immature B-cells.
Asunto(s)
Linfocitos B/inmunología , Ciclina E/genética , Ciclina E/inmunología , FN-kappa B/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Animales , Linfocitos B/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Cruzamientos Genéticos , Ciclina E/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Genes rel/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Receptores de Antígenos de Linfocitos B/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos B/inmunología , Transducción de Señal/inmunología , Proteína bcl-XRESUMEN
Cell cycle analysis has become increasingly important in verifying the effect of anti-tumor drugs and cytokinetic research. In the early methods of cell cycle analysis, the flow cytometry relied on DNA content, and therefore, the cell cycle could be only broken into three stages: G(0)/G(1), S, and G(2)/M phase. It could not distinguish the G(0), G(1), G(2), and M phase cells, let alone the sub-phases in G(1) phase. In cell cycle, expression of cyclin E living up to the maximal level in the cells undergoing transition from G(1) to S phase, and G(2) + M cells are cyclin E negative. Expression of cyclin A is progressively increasing during S phase and is maximal in G(2) phase cells. Therefore, in the current study we established a cyclin E + A/DNA multiparameter flow cytometric technique by using a mixture of cyclin E and cyclin A antibodies, which can identify six stages in the whole cell cycle: G(0), early G(1), late G(1), S, G(2), and M phase. Furthermore, we found that cyclin E + A/DNA multiparameter flow cytometry could also be used for stathmokinetic analysis of lymphocyte leukemia MOLT-4 cells after addition of the stathmokinetic agent vinblastine to cultures of exponentially growing MOLT-4 cells. We believe that this new technique will provide a much better tool for molecular cell biology research and especially for cell proliferation kinetics investigations.
Asunto(s)
Separación Celular/métodos , Ciclina A/metabolismo , Ciclina E/metabolismo , Antineoplásicos Fitogénicos/farmacología , Ciclo Celular , División Celular , Ciclina A/inmunología , Ciclina E/inmunología , Fase G1 , Fase G2 , Humanos , Inmunohistoquímica , Cinética , Leucemia Linfoide/metabolismo , Mitosis , Fase S , Factores de Tiempo , Células Tumorales Cultivadas , Vinblastina/farmacologíaRESUMEN
We studied the cytotoxic effects of recombinant TNF-alpha and supernate of phytohemagglutinin stimulated peripheral blood mononuclear cells individually and in combination against a cholangiocarcinoma cell line. Levels of cyclins D1, E and A in the cell line were detected by immunoblotting, and the cell cycle stage was assayed by propidium iodide staining followed by flow cytometry analysis. Viable and apoptotic cells were assessed by trypan blue dye exclusion, DAPI staining, agarose DNA laddering and propidium iodide staining. At the beginning of each experiment, the majority of cholangiocarcinoma cells expressed cyclin A and were in S phase as determined by propidium iodide staining. Treatment of such cells with recombinant TNF-alpha resulted in cytotoxic effects clearly evident at 36 hours post exposure. There was a synergistic killing effect when recombinant TNF-alpha was combined with PHA supernate and this effect could be partly neutralized by monoclonal anti TNF-alpha, interleukin (IL)-2, IL-12 and IFN-gamma.
Asunto(s)
Neoplasias de los Conductos Biliares/tratamiento farmacológico , Neoplasias de los Conductos Biliares/metabolismo , Conductos Biliares Intrahepáticos/efectos de los fármacos , Conductos Biliares Intrahepáticos/metabolismo , Colangiocarcinoma/tratamiento farmacológico , Colangiocarcinoma/metabolismo , Ciclina A/biosíntesis , Ciclina A/efectos de los fármacos , Ciclina D1/biosíntesis , Ciclina D1/efectos de los fármacos , Ciclina E/biosíntesis , Ciclina E/efectos de los fármacos , Citotoxicidad Inmunológica/efectos de los fármacos , Fitohemaglutininas/uso terapéutico , Proteínas Recombinantes/uso terapéutico , Células Tumorales Cultivadas/efectos de los fármacos , Factor de Necrosis Tumoral alfa/uso terapéutico , Adulto , Neoplasias de los Conductos Biliares/inmunología , Conductos Biliares Intrahepáticos/inmunología , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/inmunología , Biomarcadores de Tumor/metabolismo , Colangiocarcinoma/inmunología , Ciclina A/inmunología , Ciclina D1/inmunología , Ciclina E/inmunología , Citotoxicidad Inmunológica/inmunología , Susceptibilidad a Enfermedades , Quimioterapia Combinada , Femenino , Humanos , Immunoblotting , Masculino , Fitohemaglutininas/inmunología , Proteínas Recombinantes/inmunología , Factores de Tiempo , Células Tumorales Cultivadas/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Cyclin E is a critical cell cycle protein in the regulated progression of normal cells to replicate their DNA. Ectopic overexpression of cyclin E results in accelerated G(1) progression, chromosome instability, and a reduced requirement for growth factors. Dysregulated cyclin E expression is found in nearly all breast cancers examined. Toward the goal of developing a system to block cyclin E function in normal and breast cancer cells, we have developed anti-cyclin E single-chain antibodies (sFvs) for use as intrabodies. We have cloned the variable region genes from two hybridoma cell lines that produce anti-human cyclin E antibodies, linked them into sFvs, and showed their ability to bind cyclin E when expressed as sFv-F(c) fusion proteins. Engineering of the sFvs as sFv-F(c) intrabodies resulted in a dramatic increase in the sFv half-life as analyzed by pulse-chase and immunofluorescence, and these fusion intrabodies can be expressed in the cytosol or retargeted to the nucleus of breast cancer cell lines. Stable expression of a nuclear-targeted anti-cyclin E intrabody appears to inhibit the growth of the breast cancer cell line SKBR3. This work sets the stage for the development of intrabody-based inducible or tissue-specific cyclin E knockouts and for identifying cyclin E and its vital cell cycle functions as a potential gene therapy target in breast and other cancers.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Ciclina E/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/aislamiento & purificación , Secuencia de Bases , Neoplasias de la Mama/inmunología , Células COS , División Celular , Chlorocebus aethiops , Clonación Molecular , ADN Complementario , Expresión Génica , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/aislamiento & purificación , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/aislamiento & purificación , Líquido Intracelular/inmunología , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Células Tumorales CultivadasRESUMEN
In somatic cells, cyclin E-cdk2 activity oscillates during the cell cycle and is required for the regulation of the G1/S transition. Cyclin E and its associated kinase activity remain constant throughout early sea urchin embryogenesis, consistent with reports from studies using several other embryonic systems. Here we have expanded these studies and show that cyclin E rapidly and selectively enters the sperm head after fertilization and remains concentrated in the male pronucleus until pronuclear fusion, at which time it disperses throughout the zygotic nucleus. We also show that cyclin E is not concentrated at the centrosomes but is associated with condensed chromosomes throughout mitosis for at least the first four cell cycles. Isolated mitotic spindles are enriched for cyclin E and cdk2, which are localized to the chromosomes. The chromosomal cyclin E is associated with active kinase during mitosis. We propose that cyclin E may play a role in the remodeling of the sperm head and re-licensing of the paternal genome after fertilization. Furthermore, cyclin E does not need to be degraded or dissociated from the chromosomes during mitosis; instead, it may be required on chromosomes during mitosis to immediately initiate the next round of DNA replication.
Asunto(s)
Quinasas CDC2-CDC28 , Ciclina E/análisis , Ciclina E/metabolismo , Erizos de Mar/embriología , Espermatozoides/química , Animales , Western Blotting , Cromatina/química , Cromosomas/química , Ciclina E/inmunología , Quinasa 2 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/inmunología , Quinasas Ciclina-Dependientes/metabolismo , Electroforesis en Gel de Poliacrilamida , Femenino , Fertilización , Técnicas In Vitro , Masculino , Microscopía Confocal , Mitosis , Óvulo/metabolismo , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Erizos de Mar/química , Erizos de Mar/metabolismo , Espermatozoides/citología , Huso Acromático/metabolismoRESUMEN
Our aim was to assess the prognostic implications of the expression of p27(KIP1) and cyclin E in gastric lymphoma. We investigated the prognostic value of the immunoreactivity of these molecules in 92 cases of primary gastric lymphoma: 34 LGMLs, 24 DLCLMLs and 34 DLCLs. p27 was positive in 88% of LGMLs, 71% of DLCLMLs and 32% of DLCLs (p = 0.004); cyclin E was positive in 9%, 33% and 59% of cases, respectively (p < 0.00001). p27/cyclin E immunoreactivity significantly correlated with histologic category, stage and LDH serum level. p27 immunoreactivity was significantly associated with better survival, whereas cyclin E reactivity was significantly related to worse outcome. Five-year CSS was 94% for patients with p27(+)/cyclin E(-) phenotype (n = 42), 79% for p27(+)/cyclin E(+) (n = 14) or p27(-)/cyclin E(-) (n = 16) phenotype and 60% for p27(-)/cyclin E(+) phenotype (n = 16) (p = 0.02). The prognostic role of p27/cyclin E expression was confirmed when analyzed separately within LGMLs and large-cell lymphomas. Immunoreactivity for p27 and cyclin E is an independent predictor of survival in PGLs that may be an adjunctive tool in identifying high-risk patients. It correlates with histologic category, stage and LDH serum level. p27(-)/cyclin E(+) phenotype is associated with worse survival, probably due to a synergistic effect of both cell-cycle defects. The predictive role of these molecules within each histologic group of PGLs deserves to be confirmed in larger series.
Asunto(s)
Proteínas de Ciclo Celular/inmunología , Ciclina E/inmunología , Linfoma no Hodgkin/inmunología , Linfoma no Hodgkin/mortalidad , Proteínas Supresoras de Tumor/inmunología , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Proteínas de Ciclo Celular/biosíntesis , Núcleo Celular/metabolismo , Ciclina E/biosíntesis , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Femenino , Humanos , Inmunohistoquímica , L-Lactato Deshidrogenasa/sangre , Linfoma no Hodgkin/diagnóstico , Masculino , Persona de Mediana Edad , Análisis Multivariante , Fenotipo , Pronóstico , Factores de Riesgo , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/inmunología , Neoplasias Gástricas/mortalidad , Proteínas Supresoras de Tumor/biosíntesisRESUMEN
The expression of cyclin E and cyclin-dependent kinases 2 (CDK2) in metastatic foci, the relationship of their expression with some clinicopathologic characteristics, and the correlation of their expression with prognosis remain unclear. To examine the roles of their expression in the progression of colorectal carcinoma, 21 normal mucosa, 9 hyperplastic polyps, 58 adenomas, 17 adenocarcinoma in adenomas, 203 primary cancers, 21 lymph node metastases, and 10 hepatic metastases were immunohistochemically stained with anti-cyclin E, anti-CDK2, and anti-Ki67 antibodies. In the carcinogenic process, both cyclin E and CDK2 expressions increased significantly. From the primary to the lymph node-metastatic foci, cyclin E protein remained unchanged, but CDK2 increased significantly. From the primary to the liver-metastatic foci, cyclin E apparently decreased, and CDK2 was reduced almost to zero. In primary carcinomas, the reduction of cyclin E was significantly associated with large tumor size, mucinous type, venous invasion, deep infiltration, lymph nodal metastasis, peritoneal metastasis, advanced stage, and poor prognosis. Decreased CDK2 was obviously correlated with large tumor size, venous invasion, deep infiltration, hepatic metastasis, advanced stage, and poor prognosis. Increased cyclin E protein was related to elevated CDK2, which was further linked to higher Ki67. Thus, CDK2 overexpression could facilitate lymph node metastasis. The overexpression of cyclin E and CDK2 may mainly promote the progression of early cancer. Anti-cyclin E or anti-CDK2 chemotherapy should be targeted to the cancers with such overexpression.
Asunto(s)
Adenocarcinoma/metabolismo , Quinasas CDC2-CDC28 , Neoplasias Colorrectales/metabolismo , Ciclina E/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Adenocarcinoma/mortalidad , Adenocarcinoma/secundario , Adenocarcinoma/cirugía , Adenoma/metabolismo , Adenoma/patología , Anciano , Antígenos de Neoplasias/análisis , Biomarcadores de Tumor/análisis , Colon/metabolismo , Colon/patología , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/cirugía , Ciclina E/inmunología , Quinasa 2 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/inmunología , Supervivencia sin Enfermedad , Humanos , Hiperplasia , Técnicas para Inmunoenzimas , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Pólipos Intestinales/metabolismo , Pólipos Intestinales/patología , Antígeno Ki-67/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundario , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Metástasis Linfática , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Proteínas Serina-Treonina Quinasas/inmunología , Tasa de SupervivenciaRESUMEN
OBJECTIVE: To investigate cyclin E expression as a possible marker for early cervical neoplasia using ThinPrep gynecologic specimens from premenopausal women. STUDY DESIGN: Archived ThinPrep liquid-based cervical/endocervical specimens (Cytyc Corporation, Boxborough, Massachusetts, U.S.A.) diagnosed as human papillomavirus infection (HPV) (20), atypical squamous cells of undetermined significance (ASCUS) (48) and within normal limits (WNL)/benign cellular changes (BCC) (21) were resampled in duplicate, fixed in 95% ethanol, subjected to immunocytochemical staining with the cyclin E antibody (clone 13A3, Novocastra Laboratories Ltd., Newcastle upon Tyne, U.K.) and HPV antibody (clone K1H8, Dako Corporation, Carpinteria, California, U.S.A.) and the expression scored by two pathologists and correlated with the cytologic diagnosis. A case was scored as positive if it contained > 10 abnormal squamous cells with nuclear immunocytochemical staining. RESULTS: The cylin E antibody assay was positive in 20 (100%) cases cytologically diagnosed as HPV. These cases were also anti-HPV antibody positive. Four cases (19%) cytologically diagnosed as WNL/BCC were cyclin E positive. Of these, two were anti-HPV antibody positive. Thirty-four (73%) cases cytologically diagnosed as ASCUS were positive for the cyclin E assay and for anti-HPV antibody staining. CONCLUSION: Cyclin E expression correlates strongly with morphologic features of HPV in ThinPrep specimens and may serve as a surrogate marker for HPV infection and early cervical preneoplastic lesions.
Asunto(s)
Ciclina E/biosíntesis , Neoplasias de Células Escamosas/inmunología , Papillomaviridae/inmunología , Infecciones por Papillomavirus/inmunología , Infecciones Tumorales por Virus/inmunología , Displasia del Cuello del Útero/inmunología , Neoplasias del Cuello Uterino/inmunología , Adulto , Anticuerpos Antivirales/análisis , Biomarcadores , Ciclina E/inmunología , Estudios de Factibilidad , Femenino , Humanos , Neoplasias de Células Escamosas/patología , Infecciones por Papillomavirus/patología , Factores de Tiempo , Infecciones Tumorales por Virus/patología , Neoplasias del Cuello Uterino/patología , Frotis Vaginal , Displasia del Cuello del Útero/patologíaRESUMEN
Atherosclerosis is a 'response-to-injury' process associated with chronic inflammation, tissue repair and a considerable cell turnover. These growth-related processes are controlled by the 'cell cycle clock' which is composed of cyclin-dependent kinases (Cdks), their activating subunits, the cyclins, and by inhibitors of Cdks (Ckis). P27 is a Cki which associates with cyclin A-Cdk2, cyclin D-Cdk4 and with cyclin E (CE)-Cdk2 complexes thereby abrogating their catalytic activity leading to potent inhibition of late G1 to S-phase transition. Furthermore, TGF-beta1 mRNA and immunoreactivity are locally increased in atherosclerotic lesions. Since TGF-beta1 growth suppressive function in the late G1 phase may be mediated by p27, blocking the catalytic activity of CE-Cdk2 complexes, via the stimulation of TGF-beta-RI and TGF-beta-RII, we investigated the topographical association between TGF-beta-RI, TGF-beta-RII, P27Kip1 and CE by immunohistochemistry in coronary artery segments without atherosclerosis and carotid atheromatous plaques of 11 patients undergoing carotid endarterectomy. P27-immunoreactivity was present in 11/11 atherosclerotic (92.7 +/- 3.3% of the cells) and 5/5 control (80.9 +/- 3.7% of the cells; P < 0.002 versus control) specimens and localized to nuclei of macrophages (CD68-positive), vascular smooth muscle cells (alpha-actin positive), T-lymphocytes (CD3-positive) as well as to the nuclei of endothelial cells. In the atherosclerotic tissue, TGF-beta-RI and TGF-beta-RII-immunoreactivity was present in 11/11 specimens and localized to inflammatory cells and to cells with VSMC-like-morphology. TGF-beta-RI-immunoreactivity was present in 87.4 +/- 5.3% (controls 75.3 +/- 7.48%; n.s.) and TGF-beta-RII-immunoreactivity was present in 83.7 +/- 6.8% (controls 39.5 +/- 7.3%; P < 0.002) of the cells. Double immunolabeling, and investigation of serial sections revealed co-expression of TGF-beta-RI and TGF-beta-RII in virtually all cells positive for P27. In the atherosclerotic specimens, CE-immunoreactivity was present in all specimens in macrophages (CD68-positive), vascular smooth muscle cells (alpha-actin positive) and in endothelial cells in 12.58 +/- 13.58% of the nuclei whereas in the controls CE staining was restricted to 0.19 +/- 0.43% of the cells (P < 0.001). Importantly, as shown by immunofluorescent double-labeling, we found cells expressing P27 that were simultaneously positive for CE. In summary, the present study provides evidence that TGF-beta1 present in human atherosclerotic tissue may mediate its growth suppressive activity also by p27, blocking the activity of CE-Cdk2 complexes. Quantitative analysis revealed that TGF-beta-RII, p27 and CE are concordantly upregulated in the atherosclerotic tissue with chronic inflammation, supporting the view that TGF-beta1, p27 and CE may play an important role in the processes associated with chronic inflammation and cell turnover in advanced human atherosclerotic plaques. Taken together, these results provide a possible link between the chronic inflammation associated with advanced atherosclerosis, the effects of extracellular growth factors and cell cycle control.
Asunto(s)
Arteriosclerosis/metabolismo , Arteriosclerosis/patología , Proteínas de Ciclo Celular , Ciclina E/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Supresoras de Tumor , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , División Celular/fisiología , Técnicas de Cultivo , Ciclina E/inmunología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Humanos , Inmunohistoquímica , Valores de Referencia , Sensibilidad y Especificidad , Factor de Crecimiento Transformador beta/inmunología , Regulación hacia ArribaRESUMEN
Genetic alterations in the MMAC1 tumor suppressor gene (also referred to as PTEN or TEP1) occur in several types of human cancers including glioblastoma. Growth suppression induced by overexpression of MMAC1 in cells with mutant MMAC1 alleles is thought to be mediated by the inhibition of signaling through the phosphatidylinositol 3-kinase pathway. However, the exact biochemical mechanisms by which MMAC1 exerts its growth-inhibitory effects are still unknown. Here we report that recombinant adenovirus-mediated overexpression of MMAC1 in three different MMAC1-mutant glioblastoma cell lines blocked progression from G0/G1 to S phase of the cell cycle. Cell cycle arrest correlated with the recruitment of the cyclin-dependent kinase (CDK) inhibitor, p27Kip1, to cyclin E immunocomplexes, which resulted in a reduction in CDK2 kinase activities and a decrease in levels of endogenous phosphorylated retinoblastoma protein. CDK4 kinase activities were unaffected, as were the levels of the CDK inhibitor p21Cip1 present in cyclin E immunocomplexes. Therefore, overexpression of MMAC1 via adenovirus-mediated gene transfer suppresses tumor cell growth through cell cycle inhibitory mechanisms, and as such, represents a potential therapeutic approach to treating glioblastomas.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas de Ciclo Celular , Ciclina E/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Glioblastoma/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiología , Monoéster Fosfórico Hidrolasas/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Fase S/fisiología , Proteínas Supresoras de Tumor , Adenovirus Humanos/genética , Complejo Antígeno-Anticuerpo/metabolismo , Ciclo Celular/fisiología , División Celular/efectos de los fármacos , Ciclina E/inmunología , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Ciclinas/análisis , Vectores Genéticos/genética , Humanos , Sustancias Macromoleculares , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/genética , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/fisiología , Proteína de Retinoblastoma/metabolismo , Transducción de Señal , Transfección , Células Tumorales CultivadasRESUMEN
Mast cells are widely distributed in human tissues, including the human uterus. However, the function of mast cells in uterine smooth muscle has not been clearly established. Mast cells possess secretory granules containing such substances as heparin, serotonin, histamine and many cytokines. To help establish the role of mast cells in the human myometrium, the action of heparin was investigated using smooth muscle cells (SMC) from normal myometrium and from leiomyoma. The proliferation of cultured myometrial and leiomyomal SMC was inhibited by heparin treatment. Flow cytometric analysis showed that the population in the G1 phase of the cell cycle increased under heparin treatment. Western blotting analysis showed that markers of SMC differentiation such as alpha-smooth muscle actin (alpha-SMA), calponin h1 and cyclin-dependent kinase inhibitor p27 were induced by heparin, whereas cell-cycle-related gene products from the G1 phase of the cell cycle, such as cyclin E and cdk2, were not changed. Taken together, these results indicate that heparin inhibits the proliferation of myometrial and leiomyomal SMC through the induction of alpha-SMA, calponin h1 and p27. We suggest that heparin from mast cells may induce differentiation in uterine SMC and may influence tissue remodelling and reconstruction during physiological and pathophysiological events.
Asunto(s)
Actinas/metabolismo , Quinasas CDC2-CDC28 , Proteínas de Ciclo Celular , Heparina/fisiología , Leiomioma/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Miometrio/metabolismo , Proteínas Supresoras de Tumor , Neoplasias Uterinas/metabolismo , Actinas/efectos de los fármacos , Western Blotting , Proteínas de Unión al Calcio/efectos de los fármacos , Proteínas de Unión al Calcio/inmunología , Proteínas de Unión al Calcio/metabolismo , Ciclo Celular , División Celular/efectos de los fármacos , Colorantes/química , Ciclina E/inmunología , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/inmunología , Quinasas Ciclina-Dependientes/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Heparina/farmacología , Humanos , Leiomioma/tratamiento farmacológico , Leiomioma/patología , Proteínas de Microfilamentos , Proteínas Asociadas a Microtúbulos/efectos de los fármacos , Proteínas Musculares/efectos de los fármacos , Proteínas Musculares/metabolismo , Miometrio/citología , Miometrio/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Sales de Tetrazolio/química , Tiazoles/química , Neoplasias Uterinas/tratamiento farmacológico , Neoplasias Uterinas/patología , CalponinasRESUMEN
p202 is an IFN-inducible, primarily nuclear, phosphoprotein (52-kDa) whose constitutive overexpression in transfected cells inhibits colony formation. To investigate the molecular mechanism(s) by which expression of p202 protein impairs colony formation, we established stable cell lines that inducibly express p202. Using this cell model, we demonstrate that the induced expression of p202 in asynchronous cultures of these cells was accompanied by: (a) an increase in steady-state levels of p21(WAF1/CIP1/SDI1) (p21) mRNA and protein; (b) a decrease in Cdk2 protein kinase activity; and (c) an increase in the functional form of retinoblastoma protein (pRb). Transient transfection of a p202-encoding plasmid in Saos-2 cells, which do not harbor a wild-type p53 protein, resulted in an increase in p21 protein, which indicated that p202 could regulate expression of p21 protein independent of p53 protein. Moreover, we demonstrate that expression of p202 in these cells increased cell doubling time without accumulation of cells in a particular phase of the cell cycle. Taken together, these results are consistent with the possibility that p202 protein contributes to the cell growth retardation activity of the IFNs, at least in part, by modulating p21 protein levels.
Asunto(s)
División Celular , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ciclinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Fosfoproteínas/metabolismo , Animales , Northern Blotting , Proteínas Portadoras , Ciclo Celular/genética , Línea Celular , Proteínas Cromosómicas no Histona , Ciclina A/inmunología , Ciclina D1/inmunología , Ciclina E/inmunología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Proteínas de Unión al ADN , Fibroblastos/metabolismo , Citometría de Flujo , Histonas/metabolismo , Humanos , Interferón-alfa/metabolismo , Ratones , Microscopía Fluorescente , Proteína de Retinoblastoma/inmunología , Proteína de Retinoblastoma/metabolismo , Tetraciclina/farmacología , Factores de Tiempo , Transfección , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
Cyclin E is a G1 cyclin that is essential for the transition from G1 to S phase in the cell cycle. Alterations to cyclin E expression or regulation could be important in tumorigenesis. Previous immunohistochemical and immunoblotting studies have investigated the expression of cyclin E in breast carcinomas. In this study, cyclin E has been investigated in a range of non-malignant and malignant breast using immunohistochemistry. Normal and benign tissue from pre- and post-menopausal women (39 cases), non-involved tissue from cancer-containing breasts (47 cases), ductal carcinoma in situ (22 cases) and invasive breast carcinomas (109 cases) have been examined. There was no reactivity in any of the non-malignant breast. Only one ductal carcinoma in situ contained more than 5% reactive cells. A total of 28% of invasive carcinomas had > 5% of reactive cells (range 0-88% positive cells, mean 12.59%, median 1.0%). A significant association was found with poorer differentiation (P < 0.001), high MIB1 index (P < 0.001), lack of oestrogen receptor (0.05 > P > 0.025) and the presence of p53 protein (0.05 > P > 0.025). Virtually all cases with cyclin E and p53 were poorly differentiated. The presence of cyclin E is therefore only found in breast malignancies and is associated with more aggressive features, including high proliferation.