RESUMEN
Smoking is strongly associated with abnormalities in histone-to-protamine transition and with alteration of protamine expression in human spermatozoa. A proper protamine to histone ratio is, however, essential for sperm chromatin maturity and DNA integrity. Alterations in these sperm nuclear proteins were observed in infertile men. The present prospective study is aimed at evaluating the possible relationship among smoking, semen quality and the histone-to-protamine transition ratio in mature spermatozoa. Histone H2B and protamine 1 (P1) and 2 (P2) were quantified using acid-urea polyacrylamide gel electrophoresis in the spermatozoa of 35 smokers and 19 non-smokers. Levels of lipid peroxidation marker malondialdehyde (MDA) were measured in seminal plasma by thiobarbituric acid assay. Cotinine concentrations were determined in seminal plasma using an enzyme-linked immunosorbent assay. Histone H2B levels in smokers (292.27 ± 58.24 ng/10(6)) were significantly higher (p = 0.001) than that of non-smokers (109.1 ± 43.70 ng/10(6)), besides, a significant difference (p > 0.0001) was found for the P1 and P2 ratio between smokers (1.71 ± 0.071) and non-smokers (1.05 ± 0.033). The H2B/(H2B+P1 + P2) ratio (0.29 ± 0.71) of smokers were significantly higher (p = <0.0001) than that of non-smokers (0.12 ± 0.01). The concentrations of MDA (µm) (7.13 ± 1.15) and cotinine (ng/mL) (60.44 ± 31.32) in seminal plasma of smokers were significantly higher (p = 0.001) than those in the samples of the non-smoker group (4.42 ± 1.16 and 2.01 ± 2.84 respectively). In addition, smokers showed significantly (p ≤ 0.002) lower sperm count, motility (p = 0.018), vitality (p = 0.009) and membrane integrity (p = 0.0001) than non-smokers. These results reveal that patients who smoke possess a higher proportion of spermatozoa with an alteration of the histone to protamine ratio than patients who do not smoke, and suggest that cigarette smoking may inversely affect male fertility.
Asunto(s)
Fertilidad/efectos de los fármacos , Histonas/metabolismo , Protaminas/metabolismo , Fumar/efectos adversos , Recuento de Espermatozoides , Motilidad Espermática/efectos de los fármacos , Adulto , Supervivencia Celular , Cromatina/genética , Cotinina/metabolismo , Humanos , Infertilidad Masculina , Masculino , Malondialdehído/metabolismo , Estudios Prospectivos , Semen/química , Análisis de Semen , Espermatozoides/efectos de los fármacosRESUMEN
Numerous signalling pathways in cells are influenced by the ubiquitous Ser/Thr protein kinase CK2. Protein kinase CK2 is composed of two regulatory beta-subunits and two catalytic alpha- or alpha'-subunits. Several of the known CK2 substrates are proteins known to regulate transcriptional events. Here, we describe that protein kinase CK2 interacts with the splicing factor hPrp3p, which is important for the assembly of the spliceosome. In a two-hybrid screen hPrp3p is exclusively bound to the catalytic alpha- or alpha'-subunits of CK2 but not to the regulatory beta-subunit. The interaction was confirmed by coimmunoprecipitation experiments in vitro and in vivo. Moreover, both proteins colocalized in nuclear speckles which is typical for splicing factor compartments within the nucleus. Phosphorylation experiments revealed that hPrp3p is also a substrate of protein kinase CK2. The main phosphorylation site was mapped to C-terminal residues. In vitro and in vivo splicing assays showed that the splicing activity is significantly influenced by the CK2-hPrp3p interaction. Thus, these data showed that CK2 is involved in the regulation of RNA processing.
Asunto(s)
Quinasa de la Caseína II/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Secuencia de Aminoácidos , Quinasa de la Caseína II/genética , Dominio Catalítico , Ciclo Celular , Bases de Datos de Proteínas , Células HeLa , Holoenzimas/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilación , Unión Proteica , Empalme del ARN/genética , Ribonucleoproteína Nuclear Pequeña U4-U6/química , Ribonucleoproteína Nuclear Pequeña U4-U6/genética , Empalmosomas/metabolismoRESUMEN
Cdk-activating kinase (CAK) is a trimeric complex consisting of cdk7, cyclin H, and MAT1, which activates the cell-cycle-regulating cdks through T loop phosphorylation. In addition, other substrates of the CAK complex have been identified when CAK is assembled with the TFIIH core proteins, thereby regulating transcription and nucleotide excision repair. Little is known about the regulation of the CAK complex through cyclin H. In this study we further analyzed cyclin H regulation and identified two basic clusters in the C terminus of the protein as putative nuclear localization sequences (NLSs). Fusion constructs of full-length and truncated cyclin H sequences demonstrated the functionality of the NLSs. A peptide-binding assay revealed that at least one NLS interacts with the nuclear import receptors importin alpha/beta. Phosphorylation in the vicinity of the NLSs by cyclin C/cdk8 or protein kinase CK2, however, does not influence the nuclear translocation of cyclin H.
Asunto(s)
Núcleo Celular/metabolismo , Ciclinas/metabolismo , Señales de Localización Nuclear , alfa Carioferinas/metabolismo , beta Carioferinas/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Quinasa de la Caseína II/metabolismo , Chlorocebus aethiops , Ciclina C , Ciclina H , Ciclinas/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Fosforilación , Estructura Terciaria de Proteína , Eliminación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Protein kinase CK2 is a ubiquitous serine/threonine kinase which is involved in many proliferation-related processes in the cell. It is composed of two regulatory beta-subunits and two catalytic alpha-subunits. Its regulation still remains mysterious in spite of many years of intense research. One of its regulators is the cdk inhibitory molecule p21(WAF1)-a protein which is expressed in situations of genotoxic stress. p21(WAF1) binds to the beta-subunit of CK2 and inhibits the activity of CK2. Using deletion mutants of CK2 beta as well as a peptide library consisting of 15-amino-acid-long peptides derived from the polypeptide chain of CK2 beta we mapped the binding region for p21(WAF1) on the polypeptide chain of CK2 beta. We localized an amino-terminal and a carboxy-terminal binding domain. Binding of p21(WAF1) to both regions of the CK2 beta-subunit interferes with the phosphotransferase activity of the CK2 holoenzyme.
Asunto(s)
Ciclinas/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Quinasa de la Caseína II , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Biblioteca de Péptidos , Mapeo Peptídico , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eliminación de SecuenciaRESUMEN
Mdm2 is a cellular oncoprotein the most obvious function of which is the down-regulation of the growth suppressor protein p53. It represents a highly phosphorylated protein but only little is yet known about the sites phosphorylated in vivo, the kinases that are responsible for the phosphorylation or the functional relevance of the phosphorylation status. Recently, we have shown that mdm2 is a good substrate for protein kinase CK2 at least in vitro. Computer analysis of the primary amino acid sequence of mdm2 revealed 19 putative CK2 phosphorylation sites. By using deletion mutants of mdm2 and a peptide library we identified the serine residue at position 269 which lies within a canonical CK2 consensus sequence (EGQELSDEDDE) as the most important CK2 phosphorylation site. Moreover, by using the mdm2 S269A mutant for in vitro phosphorylation assays this site was shown to be phosphorylated by CK2. Binding studies revealed that phosphorylation of mdm2 at S269 does not have any influence on the binding of p53 to mdm2.
Asunto(s)
Proteínas Nucleares , Proteínas Serina-Treonina Quinasas/química , Proteínas Proto-Oncogénicas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Western Blotting , Quinasa de la Caseína II , Línea Celular , Codón , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Humanos , Insectos , Datos de Secuencia Molecular , Mutación , Péptidos/química , Fosforilación , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
We analyzed the subcellular localization of p53 in prostate and bladder carcinoma cells. Using laser scanning microscopy and PAb1620, a monoclonal antibody recognizing the wildtype conformation of p53, and another monoclonal antibody directed against the mutant conformation of the protein (PAb240), we found two different subsets of p53 within the same cell. The wildtype subgroup was found in the nucleolus, whereas the mutant protein was confined to the nucleus. The results obtained by immunofluorescence were verified by Western blot analysis and immunoprecipitation. Thus, our findings demonstrate an unusual subcellular localization pattern of p53 in prostate and bladder cancer cells which may indicate another mechanism of inactivation of p53.
Asunto(s)
Neoplasias de la Próstata/química , Proteína p53 Supresora de Tumor/análisis , Neoplasias de la Vejiga Urinaria/química , Western Blotting , Nucléolo Celular/química , Núcleo Celular/química , Humanos , Inmunohistoquímica , Cariotipificación , Masculino , Microscopía Confocal , Mutación , Neoplasias de la Próstata/genética , Células Tumorales CultivadasRESUMEN
OBJECTIVE: To determine the presence of p53 serum antibodies in patients with clinically well-defined urological cancer using a new enzyme-linked immunosorbent assay (ELISA). PATIENTS AND METHODS: The study included 73 patients with prostatic cancer, 72 with transitional cell carcinoma of the urinary tract, 37 with renal cell cancer and 16 controls with a benign disease, all of whom were tested using the ELISA for p53 autoantibodies. The specific reaction of the ELISA (positive p53 antibody titre) was confirmed by Western Blot analysis. RESULTS: Thirteen patients with cancer and one control patient (7.6% overall) were positive for p53 autoantibodies. The sensitivity of the test was low, whereas the specificity was remarkably high. Surprisingly, 9 of the 13 p53-positive patients died within a median of 3.7 months (range 2-6) and the one positive control patient died of undetected lung cancer. There was no significant correlation of p53 antibody positivity with clinical stage or tumour-specific differences. CONCLUSIONS: The expression of p53 autoantibody seems to be a very late but significant event in urological tumour development, with the worst outcome (tumour-specific death) within a few weeks of developing positivity. In histopathologically heterogeneous tumour entities, p53 autoantibodies might be independent prognostic factors in patients with urological cancers.
Asunto(s)
Autoanticuerpos/análisis , Carcinoma de Células Renales/inmunología , Carcinoma de Células Transicionales/inmunología , Genes p53/inmunología , Neoplasias Renales/inmunología , Neoplasias de la Próstata/inmunología , Proteína p53 Supresora de Tumor/inmunología , Neoplasias Urológicas/inmunología , Western Blotting , Carcinoma de Células Renales/genética , Carcinoma de Células Transicionales/genética , Ensayo de Inmunoadsorción Enzimática , Humanos , Neoplasias Renales/genética , Masculino , Pronóstico , Neoplasias de la Próstata/genética , Proteína p53 Supresora de Tumor/genética , Neoplasias Urológicas/genéticaRESUMEN
The presence of p53 autoantibodies and p53 protein overexpression in ovarian carcinoma patients were determined and compared. p53 antibodies were detected in sera samples, cyst and/or ascitic fluids of individual patients by two separate techniques (ELISA assay and immunoblot). p53 protein accumulation was assessed immunohistochemically in tissue sections and corresponding tumor effusion cells. The relations between p53 overexpression, the presence of p53 autoantibodies and histology of tumors, grade of differentiation and clinical stage of the disease were considered. p53 expression was found in 20 of 46 (43.5%) ovarian carcinomas and significant relationship between p53 reactivity in tumor tissue and effusion cells in individual patients was evident. In the subset of carcinomas with detectable p53 accumulation only two cases (one serous, one endometrioid) were associated with the presence of p53 autoantibodies (10%). Among 26 p53- negative carcinomas also two cases (7.6%) were seropositive. The strong correlation between the presence of p53 autoantibodies in the sera and respective cyst or ascitic fluids were revealed with no exception of this coincidence. There was no association between the detection of antibodies against p53 and FIGO stage and tumor grade. Our results clearly indicate that p53 overexpression is not sufficient to elicit p53 humoral response in ovarian carcinoma patients. The presence of p53 autoantibodies in this type of cancer is not a frequent event and their importance as independent prognostic factor seems to be very limited.
Asunto(s)
Líquido Ascítico/metabolismo , Autoanticuerpos/metabolismo , Quistes Ováricos/metabolismo , Neoplasias Ováricas/metabolismo , Proteína p53 Supresora de Tumor/inmunología , Autoanticuerpos/sangre , Western Blotting , Diferenciación Celular/fisiología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunohistoquímica , Estadificación de Neoplasias , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/patología , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
Somatic mutation of the gene encoding the cellular p53 protein is the most common event in the development of human cancer. Patients with various types of cancer have circulating antibodies against p53. We screened sera of patients with different types of tumors. Some of the sera was used to precipitate p53 from different human tumor cell lines. Serum from a patient with breast cancer recognized wild-type and mutant form of p53 from an in vitro translation reaction. p53 recognized by this serum was found to be metabolically stable and phosphorylated in vivo. Furthermore, this serum recognized p53 in a complex with a protein kinase, which phosphorylated p53 in an in vitro phosphorylation reaction.