Pyrocarbon versus cobalt-chromium in the context of spherical interposition implants: an in vitro study on cultured chondrocytes.

In the context of shoulder surgical replacement, a new generation of spherical interposition implants has been developed, with the implant being a mobile spacer rubbing against the glenoid cartilage and humeral bone cavity. The aim of the present study was to compare pyrocarbon (PyC) versus cobalt-chromium (CoCr) implants, regarding preservation and regeneration of the surrounding tissues. The effect of the biomaterials on chondrocytes was analysed in vitro. Murine primary chondrocytes were grown on discs made of PyC or CoCr using two culture media to mimic either cartilage-like or bone-like conditions (CLC or BLC). Chondrocytes did grow on PyC and CoCr without alteration in cell viability or manifestation of cytotoxicity. The tissue-like cell membranes grown under BLC were examined for the chondrocyte's ability to mineralise (by alizarin red matrix staining, calcium deposit and alkaline phosphatase activity) and for their mechanical properties (by rheological tests). For the chondrocytes grown under CLC and BLC, extracellular matrix components were analysed by histological staining and immunolabelling. Under CLC, PyC promoted type II collagen expression in chondrocytes, suggesting that they may generate a more cartilage-like matrix than samples grown on both CoCr and plastic control. In BLC, the tissue-like cell membranes grown on PyC were more mineralised and homogenous. The mechanical results corroborated the biological data, since the elastic modulus of the tissue-like cell membranes developed on the PyC surface was higher, indicating more stiffness. Overall, the results suggested that PyC might be a suitable biomaterial for spherical interposition implants.

Multi-scale mechanical characterization of prostate cancer cell lines: Relevant biological markers to evaluate the cell metastatic potential.

BACKGROUND: Considering the importance of cellular mechanics in the birth and evolution of cancer towards increasingly aggressive stages, we compared nano-mechanical properties of non-tumoral (WPMY-1) and highly aggressive metastatic (PC-3) prostate cell lines both on cell aggregates, single cells, and membrane lipids. METHODS: Cell aggregate rheological properties were analyzed during dynamic compression stress performed on a homemade rheometer. Single cell visco-elasticity measurements were performed by Atomic Force Microscopy using a cantilever with round tip on surface-attached cells. At a molecular level, the lateral diffusion coefficient of total extracted lipids deposited as a Langmuir monolayer on an air-water interface was measured by the FRAP technique. RESULTS: At cellular pellet scale, and at single cell scale, PC-3 cells were less stiff, less viscous, and thus more prone to deformation than the WPMY-1 control. Interestingly, stress-relaxation curves indicated a two-step response, which we attributed to a differential response coming from two cell elements, successively stressed. Both responses are faster for PC-3 cells. At a molecular scale, the dynamics of the PC-3 lipid extracts are also faster than that of WPMY-1 lipid extracts. CONCLUSIONS: As the evolution of cancer towards increasingly aggressive stages is accompanied by alterations both in membrane composition and in cytoskeleton dynamical properties, we attribute differences in viscoelasticity between PC-3 and WPMY-1 cells to modifications of both elements. GENERAL SIGNIFICANCE: A decrease in stiffness and a less viscous behavior may be one of the diverse mechanisms that cancer cells adopt to cope with the various physiological conditions that they encounter.

Targeted resequencing of a genomic region influencing tameness and aggression reveals multiple signals of positive selection.

The identification of the causative genetic variants in quantitative trait loci (QTL) influencing phenotypic traits is challenging, especially in crosses between outbred strains. We have previously identified several QTL influencing tameness and aggression in a cross between two lines of wild-derived, outbred rats (Rattus norvegicus) selected for their behavior towards humans. Here, we use targeted sequence capture and massively parallel sequencing of all genes in the strongest QTL in the founder animals of the cross. We identify many novel sequence variants, several of which are potentially functionally relevant. The QTL contains several regions where either the tame or the aggressive founders contain no sequence variation, and two regions where alternative haplotypes are fixed between the founders. A re-analysis of the QTL signal showed that the causative site is likely to be fixed among the tame founder animals, but that several causative alleles may segregate among the aggressive founder animals. Using a formal test for the detection of positive selection, we find 10 putative positively selected regions, some of which are close to genes known to influence behavior. Together, these results show that the QTL is probably not caused by a single selected site, but may instead represent the joint effects of several sites that were targets of polygenic selection.

Antifungal properties of the immunosuppressant FK-506: identification of an FK-506-responsive yeast gene distinct from FKB1.

FK-506 is a novel and potent antagonist of T-cell activation and an inhibitor of fungal growth. Its immunosuppressive activity can be antagonized by the structurally related antibiotic rapamycin, and both compounds interact with cytoplasmic FK-506-binding proteins (FKBPs) in T cells and yeast cells. In this paper, we show that FK-506 and two analogs inhibit vegetative growth of Saccharomyces cerevisiae in a fashion that parallels the immunosuppressive activity of these compounds. Yeast mutants resistant to FK-506 were isolated, and at least three complementation groups (fkr1, fkr2, and fkr3) were defined. These fkr mutants show no alteration in their levels of FK-506-binding activity. Likewise, strains carrying null alleles of FKB1 (the yeast gene coding for the FKBP) remain FK-506 sensitive, indicating that depletion of yeast FKBP is not sufficient to confer an FK-506 resistance phenotype, although fkb1 null mutants are resistant to rapamycin. FKB1 does not map to the three fkr loci defined here. These results suggest that yeast FKBP mediates the inhibitory effect of rapamycin but that at least one other protein is directly involved in mediating the activity of FK-506. Interestingly, the ability of FK-506 to rescue a temperature-sensitive growth defect of the fkr3 mutant suggests that the FKR3 gene may define such a protein.

Flavopiridol induces G1 arrest with inhibition of cyclin-dependent kinase (CDK) 2 and CDK4 in human breast carcinoma cells.

Flavopiridol (L86-8275), a N-methylpiperidinyl, chlorophenyl flavone, can inhibit cell cycle progression in either G1 or G2 and is a potent cyclin-dependent kinase (CDK) 1 inhibitor. In this study, we used MCF-7 breast carcinoma cells that are wild type for p53 and pRb positive and contain CDK4-cyclin D1 and MDA-MB-468 breast carcinoma cells that are mutant p53, pRb negative, and lack CDK4-cyclin D1 to investigate the G1 arrest produced by Flavopiridol. Recombinant CDK4-cyclin D1 was inhibited potently by Flavopiridol (Kiapp, 65 nM), competitive with respect to ATP. Surprisingly, CDK4 immunoprecipitates derived from Flavopiridol-treated MCF-7 cells (3 h, 300 nM Flavonolpiridol) had an approximately 3-fold increased kinase activity compared with untreated cells. Cyclin D and CDK4 levels were not different at 3 hr, but cyclin D levels and CDK4 kinase activity decreased thereafter. The phosphorylation state of pRb was shifted from hypercoincident to hypocoincident with the development of G1 arrest. Asynchronous MDA-MB-468 cells were inhibited in cell cycle progression at both G1 and G2 by Flavopiridol. Flavopiridol inhibited the in vitro kinase activity of CDK2 using an immune complex kinase assay (IC50, 100 nM at 400 microM ATP). Immunoprecipitated CDK2 kinase activity from either MCF-7 or MDA-MB-468 cells exposed to Flavopiridol (300 nM) for increasing time showed an initial increased activity (approximately 1.5-fold at 3 h) compared with untreated cells, followed by a loss of kinase activity to immeasurable levels by 24 h. This increased immunoprecipitated kinase activity was dependent on the Flavopiridol concentration added to intact cells and was associated with a reduction of CDK2 tyrosine phosphorylation. Cyclin E and A levels were not altered to the same extent as cyclin D, and neither CDK4 nor CDK2 levels were changed in response to Flavopiridol. Inhibition of the CDK4 and/or CDK2 kinase activity by Flavopiridol can therefore account for the G1 arrest observed after exposure to Flavopiridol.

Essential role for SphK1/S1P signaling to regulate hypoxia-inducible factor 2α expression and activity in cancer.

The sphingosine kinase-1/sphingosine 1-phosphate (SphK1/S1P) signaling pathway has been reported to modulate the expression of the canonical transcription factor hypoxia-inducible HIF-1α in multiple cell lineages. HIF-2α is also frequently overexpressed in solid tumors but its role has been mostly studied in clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, where HIF-2α has been established as a driver of a more aggressive disease. In this study, the role of SphK1/S1P signaling with regard to HIF-2α was investigated in various cancer cell models including ccRCC cells. Under hypoxic conditions or in ccRCC lacking a functional von Hippel-Lindau (VHL) gene and expressing high levels of HIF-2α, SphK1 activity controls HIF-2α expression and transcriptional activity through a phospholipase D (PLD)-driven mechanism. SphK1 silencing promotes a VHL-independent HIF-2α loss of expression and activity and reduces cell proliferation in ccRCC. Importantly, downregulation of SphK1 is associated with impaired Akt and mTOR signaling in ccRCC. Taking advantage of a monoclonal antibody neutralizing extracellular S1P, we show that inhibition of S1P extracellular signaling blocks HIF-2α accumulation in ccRCC cell lines, an effect mimicked when the S1P transporter Spns2 or the S1P receptor 1 (S1P1) is silenced. Here, we report the first evidence that the SphK1/S1P signaling pathway regulates the transcription factor hypoxia-inducible HIF-2α in diverse cancer cell lineages notably ccRCC, where HIF-2α has been established as a driver of a more aggressive disease. These findings demonstrate that SphK1/S1P signaling may act as a canonical regulator of HIF-2α expression in ccRCC, giving support to its inhibition as a therapeutic strategy that could contribute to reduce HIF-2 activity in ccRCC.

Biochemical and mutagenic analysis of the melanoma tumor suppressor gene product/p16.

P16 was originally discovered by its ability to interact with CDK4 and to specifically inhibit the catalytic activity of the CDK4/D1 kinase. Increased attention has focused on the p16 gene because of its location on chromosome 9p21, a region involved in chromosomal rearrangements in a large number of tumor types. The p16 gene is also mutated in a large number of tumor cell lines and primary tumor cells. Furthermore, linkage analysis studies suggest that the p16 gene is involved in familial melanoma susceptibility. Due to the oncogenic potential of mutations in this tumor suppressor, it is important to identify and characterize those mutations which alter p16 activity. We have performed a systematic analysis of melanoma associated p16 mutants and of mutants generated in charge to Ala mutagenesis. Using microtiter plate assays to measure both p16-cdk4 binding and cdk4/D1 kinase activity, we show here that the melanoma associated mutants are defective, as are some of the Ala mutants. These results support the idea that p16 mutation, via its deregulation of the cdk4/D1 pathway, is of biological significance in the development of melanoma. Furthermore, we have defined a region within the p16 molecule in which changes are likely to result in a defective protein.

Interaction between Cdc37 and Cdk4 in human cells.

Using the yeast two-hybrid system we have identified novel potential Cdk4 interacting proteins. Here we described the interaction of Cdk4 with a human homologue of the yeast Drosophila CDC37 gene products. Cdc37 protein specifically interacts with Cdk4 and Cdk6, but not with Cdc2, Cdk2, Cdk3, Cdk5 and any of a number of cyclins tested. Cdc37 is not an inhibitor nor an activator of the Cdk4/cyclin D1 kinase, while it appears to facilitate complex assembly between Cdk4, and cyclin D1 in vitro. Cdc37 competes with p16 for binding to Cdk4, suggesting that p16 might exert part of its inhibitory function by affecting the formation of Cdk4/cyclin D1 complexes via Cdc37.

SCF(beta-TRCP) and phosphorylation dependent ubiquitinationof I kappa B alpha catalyzed by Ubc3 and Ubc4.

NF kappa B is an important transcriptional regulator of multiple pro-inflammatory genes. In non-stimulated cells NF kappa B is anchored in the cytoplasm via the inhibitory protein I kappa B alpha. Following exposure to diverse pro-inflammatory signals (e.g. TNF alpha, IL1, LPS) various signal transduction cascades are initiated converging on the I kappa B kinase (IKK). IKK phosphorylates I kappa B alpha on serines 32 and 36 signaling the inhibitory protein for ubiquitin-mediated degradation. The SCF beta-TRCP complex is the ubiquitin ligase responsible for mediating phosphorylation dependent ubiquitination of I kappa B alpha. Here we reconstitute phosphorylation dependent ubiquitination of I kappa B alpha using recombinant components. Our results suggest that the cullin specificity of the SCF complex may reflect its ability to associate with Rbx1. We demonstrate specific ubiquitination of I kappa B alpha by Ubc3 and Ubc4 in a phosphorylation and SCF beta-TRCP dependent manner and that both are capable of associating with the SCF beta-TRCP complex isolated from human cells. Finally, we show that Ubc4 is in excess to Ubc3 in THP.1 cells and 19 times more efficient in catalyzing the reaction, suggesting that Ubc4 is the preferentially used Ubc in this reaction in vivo. Our results also suggest that ubiquitin is transferred directly from the Ubc to phospho-I kappa B alpha in a SCF beta-TRCP dependent reaction. Oncogene (2000) 19, 3529 - 3536

FLEXGene repository: from sequenced genomes to gene repositories for high-throughput functional biology and proteomics.

The vast amount of information generated by the human genome sequencing project and related projects has given rise to a new paradigm in experimental biology. This new paradigm invokes the experimentation and data analysis at genome-wide scales, as well as the generation of new technologies and resources that take full advantage of the available sequence information. The Institute of Proteomics at Harvard Medical School is building a comprehensive, characterized, arrayed and flexible gene repository that will allow full exploitation of the genomic information by enabling functional genomics as well as protein expression, purification and analysis at genome wide scale. The FLEXGene repository (Full Length EXpression-ready) will contain clones representing the complete set of open reading frames (ORFs) of different organisms including H. sapiens and several pathogens and model organisms. The clones are constructed using recombination-based cloning technology so that hundreds or thousands of coding regions can be transferred into any expression vector in a parallel and timely mode, allowing the broadest variety of experiments to be carried out.

Consumo de tabaco y alcohol en pacientes con cáncer esofágico y su relación con el tipo histológico: Hospital Vargas de Caracas período 2004 - 2009 / Consumption of snuff and alcohol use in patients with esophageal cancer and its relation to histological type: Hospital Vargas de Caracas period 2004 - 2009

El Cáncer de Esófago ocupa el noveno lugar entre las neoplasias malignas a nivel mundial y está asociado al hábito tabáquico y alcohólico. En el Hospital Vargas de Caracas ocupa el lugar número 11 entre las primeras 15 (2.5%) causas de egresos por cáncer al año. Determinar el consumo de tabaco y alcohol en pacientes ingresados por Cáncer de esófago en el "Hospital Vargas de Caracas" durante el período 2004 - 2009. Se realizó estudio retrospectivo y descriptivo, luego de la revisión de historias de 24 pacientes ingresados con diagnóstico de cáncer de esófago. Se utilizaron medidas de tendencia central para interpretación de resultados. 21 (87.5%) de los pacientes fueron masculinos y 3 (12.5%) femeninos. La edad promedio fue de 61 años. El 83.3% (20) refirieron hábito tabáquico de más de 24 paquetes/año y hábito alcohólico mayor a 120 gr/día. Histológicamente 87.5% (21) correspondió a Carcinoma Epidermoide localizado en 1/3 medio de esófago en 58.3% (14). Adenocarcinoma fue diagnosticado en 3 pacientes (12.5%). El hallazgo histopatológico más frecuente fue el carcinoma epidermoide en el 87.5% de las historias revisadas. El 83% de los pacientes tenía una asociación importante al hábito tabáquico y alcohólico, por lo que recomendamos implementación de Programa conjunto de promocióny prevención en salud entre la Sociedad Venezolana de Gastroenterología y la Sociedad Anticancerosa de Venezuela, de lucha contra los factores de riesgo de esta enfermedad...

Esophageal Cancer ranks ninth among malignant neoplasias worldwide and is associated to smoking and alcohol consumption. In "HospitalVargas de Caracas" its ranked number 11 among the top 15 (2.5%) cases of cancer annually discharged. Determine the tobacco and alcohol consumption in patients admitted for esophageal cancer in "Hospital Vargas de Caracas" during the period 2004 to 2009. We performeda retrospective, descriptive study, after reviewing the charts of 24 patients admitted for esophageal cancer diagnosis. We used central tendency measures for interpretation of results. 21 (87.5%) patients were male and 3 (12.5%) female. The average age was 61 years. 83.3% (20) reported smoking over 24 packs-years and alcohol consumption greater than 120 g / day. Histologically, 87.5% (21) corresponded to squamous cell carcinoma located in 1 / 3 of the esophagus in 58.3% (14). 3 patients (12.5%) were diagnosed with Adenocarcinoma. Squamous cell carcinoma in 87.5% was the most common histopathological finding among the reviewed charts. 83% of patients had a significant association to smoking and alcohol, thus, we recommend the implementation of a joint prevention and health promotion program about the management of risk factors of this disease between the Venezuelan Society of Gastroenterology and Venezuela's Anti-Cancer Society, the control of risk factors of this disease...

Consumo de tabaco y alcohol en pacientes con cáncer esofágico en el Hospital Vargas de Caracas durante el período 2004-2009 / Tobacco and alcohol consumption in patients with esophageal cancer at

El Cáncer de Esófago ocupa el noveno lugar entre las neoplasias malignas a nivel mundial y está asociado al hábito tabáquico y alcohólico. En el Hospital Vargas de Caracas ocupa el lugar número 11 entre las primeras 15 (2.5%) causas de egresos por cáncer al año. Determinar el consumo de tabaco y alcohol en pacientes ingresados por Cáncer de esófago en el Hospital Vargas de Caracas durante el período 2004 - 2009. Se realizó estudio retrospectivo y descriptivo, luego de la revisión de historias de 24 pacientes ingresados por cáncer de esófago con diagnóstico de cáncer de esófago. Se utilizaron medidas de tendencia central para interpretación de resultados. 21(87.5%) de los pacientes fueron masculinos y 3(12.5%), femeninos. La edad promedio fue de 61 años. El 83.3%(20) refirieron hábito tabáquico de más de 24 paquetes/año y hábito alcohólico mayor a 120 gr/día. Histológicamente 87.5% (21) correspondió a Carcinoma Epidermoide localizados en 1/3 medio de esófago en 58.3% (14). Adenocarcinoma fue diagnosticado en 3 pacientes (12.5%) El hallazgo histopatológico más frecuente fue el carcinoma epidermoide en el 87.5% de las historias revisadas. El 83% de los pacientes tenían una asociación importante al hábito tabáquico y alcohólico, por lo que recomendamos implementación de Programa conjunto de promoción y prevención en salud entre la Sociedad Venezolana de Gastroenterología y la Sociedad Anticancerosa de Venezuela, de lucha contra los factores de riesgo de esta enfermedad...

Esophageal cancer ranks ninth among malignancies worldwide and is associated with smoking and alcoholism. In Hospital Vargas de Caracas is ranked number 11 among the top 15 (2.5%) causes of cancer discharges per year. To determine the consumption of tobacco and alcohol in patients admitted due to esophageal cancer in Hospital Vargas de Caracas during 2004-2009. A retrospective descriptive study was performed after reviewing the charts of 24 patients admitted with esophageal cancer. We used central tendency measures for interpretation of results. 21 (87.5%) patients were male and three (12.5%) female. The average age was 61 years-old. 83.3% (20) reported smoking over 24 pack/years and become an alcoholic greater than 120 g/day. Histologically, 87.5% (21) corresponded to epidermoid carcinoma located in 1/3 of the esophagus in 58.3% (14). Adenocarcinoma was diagnosed in 3 patients (12.5%). The most common histopathological fi nding was squamous cell carcinoma in 87.5% of the charts reviewed. 83% of patients had a signifi cant association with smoking and alcoholism, that’s why, we recommend the implementation of a joint program in health promotion and disease prevention among the Venezuelan Society of Gastroenterology and Cancer Society of Venezuela, to fight against the risk factors of this disease...

Screens using RNAi and cDNA expression as surrogates for genetics in mammalian tissue culture cells.

We have developed methods for the automation of transfection-grade DNA preparation, high-throughput retroviral preparation, and highly parallel phenotypic screens to establish approaches that will allow investigators to examine in an unbiased manner the roles of proteins in mammalian cells. These methods have been used to raise or lower the levels of individual kinases in individual micro-well cultures either by cDNA or short hairpin RNA expression and will allow investigators to treat mammalian cells in culture in manners that are analogous to genetic screens in yeast. Our proof-of-principle experiments have been performed in human cells using repositories that represent over 75% of the protein, nucleotide, carbohydrate, lipid, and amino acid kinases in the human genome. These initial experiments have demonstrated the feasibility of two general types of screens. We have performed phenotypic screens to identify proteins with specific roles in a chosen function and genetic interaction screens to establish epistatic relations between different proteins. The results suggest that any phenotype that can be scored by a robust assay in tissue culture is amenable to these types of screens and that interactions between mammalian proteins can be established. These results point to the near-term goal of establishing comprehensive, unbiased screens that will allow queries on the roles of all human proteins.

Lesiones gástricas premalignas en familiares en primer grado de pacientes con cáncer gástrico evaluados en la consulta de gastroenterología de dos hospitales universitarios de Caracas / Premalignant gastric lesions in first-degree relatives of gastric cancer patients evaluated in the gastroenterology at two university hospitals in Caracas

Las alteraciones morfológicas de la mucosa gástrica que predisponen al desarrollo de cáncer gástrico son atrofia, metaplasia y displasia. Diversos factores etiológicos han sido estudiados, destacándose el parentesco en primer grado con una probabilidad de presentar cáncer gástrico 2 veces más que la población general. Diagnosticar lesiones gástricas premalignas en familiares de pacientes con cáncer gástrico, evaluados en las consultas de Gastroenterología del Hospital Vargas de Caracas y Hospital Militar "Dr. Carlos Arvelo". Se realizó un estudio descriptivo, prospectivo, longitudinal, desde Octubre 2008 a Mayo 2009. Se incluyeron 75 individuos con edades comprendidas entre 18 y 80 años, familiares en primer grado de pacientes con cáncer gástrico, a los cuales se les practicó endoscopia digestiva superior con toma de biopsia. En 51 pacientes femeninos (68%) y 24 (32%) masculinos, se encontraron los siguientes hallazgos endoscópicos: endoscopia normal 52%, gastropatía crónica 33%, úlcera gástrica 5,33%, pólipos gástricos 3,33% y úlcera duodenal 1,33%. El estudio histológico determinó que la atrofia estuvo presente en 25 pacientes (33,3%), metaplasia intestinal en 17 pacientes (22,67%), displasia en 4 pacientes (5,33%), la mitad de ellas de alto grado y 1 (1,33%) indefinido para displasia. Conclusión y recomendación: En el grupo estudiado se encontró un 42,6% de lesiones gástricas premalignas, de las cuales el 5,33% correspondió a displasia, ninguno de estos con lesiones endoscópicas de malignidad; lo que nos hace recomendar de rutina en todos los servicios de gastroenterología del país un programa de pesquisa en familiares en primer grado de pacientes de cáncer gástrico...

Morphological alterations of the gastric mucosa that predispose to the development of gastric cancer are atrophy, metaplasia and dysplasia. Various etiologic factors have been studied, especially in first degree relatives,with a probability of gastric cancer double than that of the general population. To diagnose premalignant gastric lesions in relatives of patients with gastric cancer evaluated in the Gastroenterology consultation of "Hospital Vargas de Caracas", and "Hospital Militar Dr. Carlos Arvelo". A descriptive, prospective, longitudinal study was made, from October 2008 to May 2009. We included 75 individuals aged 18 to 80 years in first degree relatives of patients with gastric cancer, who underwent upper gastrointestinal endoscopy with biopsy guided. We found 51 (68%) female and 24 (32%) male, with these endoscopic findings: normal in 52% of the patients, with chronic gastropathy 37, 33%, gastric ulcer in 5,3%, gastric polyps in 3,33% and duodenal ulcer in 1,33%. The histological study found that atrophy was present in 25 (33.3%) patients, intestinal metaplasia in 17 (22, 67%) and dysplasia in 4(5, 33%) and 1(1,33%) indefinite to dysplasia. In this group of patients it was found a 42.6% of premalignant gastric lesions, of which 5.33% corresponded to dysplasia. Neither one had endoscopic malignant lesions. We recommend as a routine in all Gastroenterology Divisions in the country a program of screening in first degree families of patients of gastric cancer...

p13suc1 acts in the fission yeast cell division cycle as a component of the p34cdc2 protein kinase.

cdc2+ encodes a protein kinase that is required during both G1 and G2 phases of the cell division cycle in fission yeast. suc1+ is an essential gene that was originally identified as a plasmid-borne sequence that could rescue certain temperature-sensitive cdc2 mutants. To investigate the role of the suc1+ gene product in the cell cycle p13suc1 has been expressed in Escherichia coli and purified. An immunoaffinity purified anti-p13suc1 polyclonal serum has been prepared and used to identify p13suc1 in fission yeast. The abundance of this protein did not alter either during the cell cycle or during entry into stationary phase. p13suc1 was found in yeast lysates in a complex with the cdc2+ gene product. Approximately 5% of cellular p34cdc2 was associated with p13suc1, and this fraction of p34cdc2 was active as a protein kinase. The stability of the complex was disrupted in yeast strains carrying temperature-sensitive alleles of cdc2 that are suppressible by overexpression of suc1+. The level of association between p13suc1 and p34cdc2 was not affected by cell cycle arrest in adverse nutritional conditions. p13suc1 is not a substrate of the p34cdc2 protein kinase. We propose instead that it acts as a regulatory component of p34cdc2 that facilitates interaction with other proteins.

Activation of human CDC2 protein as a histone H1 kinase is associated with complex formation with the p62 subunit.

p34 kinase, the product of the CDC2 gene, is a cell-cycle regulated protein kinase that is most active during mitosis. In HeLa cells, p34 kinase has previously been shown to exist in both a low- and a high-molecular-mass form, the latter of which is only found in cells in the G2/M phase of the cell cycle and contains a 62-kDa subunit. Here we show that although each form of the kinase phosphorylates casein in vitro, only the high-molecular-mass form uses histone H1 as substrate. The high-molecular-mass form of p34 kinase from nocodazole-treated HeLa cells was purified 6700-fold. The apparent molecular mass of the mitotic CDC2-encoded protein kinase complex was 220 kDa. The purified enzyme phosphorylated not only its endogenous 62-kDa subunit but also phosphorylated histone H1 with a Km of 3 microM and used ATP 40 times more efficiently than GTP (Km 54 microM and 2 mM, respectively). The enzyme activity was unaffected by cAMP, calcium/calmodulin, or by the heat-stable inhibitor of cAMP-dependent protein kinase. These characteristics are typical of growth-associated histone H1 kinase from different organisms. These results suggest that CDC2 protein may be activated as an M-phase-specific protein kinase in part by its association with the p62 subunit.

A role for the p34cdc2 kinase and phosphatases in the regulation of phosphorylation and disassembly of lamin B2 during the cell cycle.

While the p34cdc2 kinase is considered to be a critical regulator of mitosis, its function has not yet been directly linked to one of the key events during the onset of mitosis: nuclear envelope breakdown. Here we show that a major structural protein of the nuclear envelope, lamin B2, is phosphorylated by p34cdc2. Results from two-dimensional phosphopeptide mapping experiments demonstrate that the p34cdc2-specific phosphopeptides represent both mitotic and interphase specific phosphorylations of lamin B2 and include the major interphase phosphorylation site. In mitotic cells we detected two distinct forms of lamin B2 which differ in electrophoretic mobility and in degree of phosphorylation. The phosphorylation pattern of lamin B2 generated in vitro by p34cdc2 was more closely related to the less phosphorylated mitotic lamin B2, suggesting that another kinase(s) in addition to p34cdc2 is involved in generating the mitotic phosphorylation pattern. In addition, we show that treatment of interphase cells with okadaic acid, a potent phosphatase inhibitor, leads to the acquisition of mitosis-specific phosphopeptides and can reversibly increase the detergent-solubility of lamin B2. However, the M-phase-like phosphorylation of lamin B2 in itself is not sufficient to induce its disassembly from the nuclear lamina suggesting that an additional event(s) besides phosphorylation is required.

Identification of p34 and p13, human homologs of the cell cycle regulators of fission yeast encoded by cdc2+ and suc1+.

cdc2+ and CDC28 play central roles in the cell division cycles of the widely divergent yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. The genes encode protein kinases that show 62% protein sequence identity and are capable of cross-complementation. Monoclonal antibodies were raised against p34cdc2, and a subset recognize p36cdc28. The cross-reacting antibodies detected a 34 kd homolog of the p34cdc2/p36CDC28, protein in HeLa cells. Human p34 was also recognized by an affinity-purified polyclonal anti-p34cdc2 serum. Peptide mapping of p34cdc2, p36CDC28, and human p34 revealed complete conservation of four tryptophan residues in the three proteins. p34 thus appears to be closely related to the two yeast proteins. In addition, a p34 immune complex showed protein kinase activity in vitro, and HeLa cell p34 interacts with p13, the human homolog of the suc1+ gene product of S. pombe.