RESUMEN
We have examined the effects of topoisomerase inhibitors on the phosphorylation of histones in chromatin during the G2 and the M phases of the cell cycle. Throughout the G2 phase of BHK cells, addition of the topoisomerase II inhibitor VM-26 prevented histone H1 phosphorylation, accompanied by the inhibition of intracellular histone H1 kinase activity. However, VM-26 had no inhibitory effect on the activity of the kinase in vitro, suggesting an indirect influence on histone H1 kinase activity. Entry into mitosis was also prevented, as monitored by the absence of nuclear lamina depolymerization, chromosome condensation, and histone H3 phosphorylation. In contrast, the topoisomerase I inhibitor, camptothecin, inhibited histone H1 phosphorylation and entry into mitosis only when applied at early G2. In cells that were arrested in mitosis, VM-26 induced dephosphorylation of histones H1 and H3, DNA breaks, and partial chromosome decondensation. These changes in chromatin parameters probably reverse the process of chromosome condensation, unfolding condensed regions to permit the repair of strand breaks in the DNA that were induced by VM-26. The involvement of growth-associated histone H1 kinase in these processes raises the possibility that the cell detects breaks in the DNA through their effects on the state of DNA supercoiling in constrained domains or loops. It would appear that histone H1 kinase and topoisomerase II work coordinately in both chromosome condensation and decondensation, and that this process participates in the VM-26-induced G2 arrest of the cell.
Asunto(s)
Cromosomas/metabolismo , ADN-Topoisomerasas de Tipo II/fisiología , Histonas/metabolismo , Protamina Quinasa/metabolismo , Tenipósido/farmacología , Animales , Afidicolina , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Cromosomas/efectos de los fármacos , Daño del ADN , Demecolcina/farmacología , Diterpenos/farmacología , Fase G1 , Fase G2/fisiología , Metafase/fisiología , Mitosis/fisiología , Membrana Nuclear/metabolismo , Fosforilación , Protamina Quinasa/efectos de los fármacos , Inhibidores de Topoisomerasa I , Inhibidores de Topoisomerasa IIRESUMEN
Activation of the serine-threonine kinase p34cdc2 at an inappropriate time during the cell cycle leads to cell death that resembles apoptosis. Premature activation of p34cdc2 was shown to be required for apoptosis induced by a lymphocyte granule protease. The kinase was rapidly activated and tyrosine dephosphorylated at the initiation of apoptosis. DNA fragmentation and nuclear collapse could be prevented by blocking p34cdc2 activity with excess peptide substrate, or by inactivating p34cdc2 in a temperature-sensitive mutant. Premature p34cdc2 activation may be a general mechanism by which cells induced to undergo apoptosis initiate the disruption of the nucleus.
Asunto(s)
Apoptosis , Proteína Quinasa CDC2/metabolismo , Secuencia de Aminoácidos , Animales , Daño del ADN , Desoxirribonucleasas/farmacología , Activación Enzimática , Inducción Enzimática , Glicoproteínas de Membrana/farmacología , Ratones , Mitosis , Datos de Secuencia Molecular , Perforina , Fosforilación , Proteínas Citotóxicas Formadoras de Poros , Serina Endopeptidasas/farmacología , Células Tumorales CultivadasRESUMEN
A promising approach for cancer gene therapy is the combination of adenovirus vectors (AdV) with the suicide gene cytosine deaminase and uracil phosphoribosyl transferase (CDColon, two colonsUPRT). While such vectors have been tested in tumor cell lines and xenograft models, it is not clear how these therapeutic vectors would perform in primary human tumors. We, thus, examined the effect of the combination of a recombinant adenovirus expressing the CDColon, two colonsUPRT (AdCU) with 5-fluorocytosine (5-FC) on primary cancer cells isolated from the ascites or pleural fluids of patients with metastatic cancers. In such models, we have found a direct correlation between the patients' response to 5-FU and the response shown by the cancer cells in vitro, confirming the clinical relevance of this methodology. Our findings demonstrated that this combination was able to kill primary tumor cells, including those that had developed resistance to 5-FU. Furthermore, while proliferating cells were more susceptible to 5-FU, the combination was effective in both rapid and slow proliferating samples. Our study demonstrated that this gene therapy approach could provide an effective therapeutic option for cancers and is not affected by acquired 5-FU resistance. Also of importance is the effectiveness of this gene therapy approach on slower proliferating cells that is typical of the majority of cancers in vivo. This suggests a greater likelihood that it will be effective in a clinical setting.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Fluorouracilo/farmacología , Terapia Genética , Adenoviridae/genética , Apoptosis/genética , Resistencia a Antineoplásicos , Humanos , Transducción Genética , Células Tumorales CultivadasRESUMEN
Histone acetylation plays an important role in regulating chromatin structure and thus gene expression. Here we describe the functional characterization of HDAC4, a human histone deacetylase whose C-terminal part displays significant sequence similarity to the deacetylase domain of yeast HDA1. HDAC4 is expressed in various adult human tissues, and its gene is located at chromosome band 2q37. HDAC4 possesses histone deacetylase activity intrinsic to its C-terminal domain. When tethered to a promoter, HDAC4 represses transcription through two independent repression domains, with repression domain 1 consisting of the N-terminal 208 residues and repression domain 2 containing the deacetylase domain. Through a small region located at its N-terminal domain, HDAC4 interacts with the MADS-box transcription factor MEF2C. Furthermore, HDAC4 and MEF2C individually upregulate but together downmodulate c-jun promoter activity. These results suggest that HDAC4 interacts with transcription factors such as MEF2C to negatively regulate gene expression.
Asunto(s)
Histona Desacetilasas/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Mapeo Cromosómico , Cromosomas Humanos Par 2 , Clonación Molecular , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Histona Desacetilasas/genética , Humanos , Hibridación Fluorescente in Situ , Proteínas de Dominio MADS , Factores de Transcripción MEF2 , Datos de Secuencia Molecular , Familia de Multigenes , Factores Reguladores Miogénicos , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Represoras/genética , Homología de Secuencia de Aminoácido , Distribución Tisular , Factores de Transcripción/metabolismoRESUMEN
At any point during the progression of many tumor types, cells can develop a hyperploid DNA content. Hyperploid tumors are significant more aggressive, with a higher growth rate and a poor patient prognosis. Yeast genetics have implicated three important genes involved in DNA ploidy changes: cdc2, cyclin b, and a specific inhibitor of the p34(cdc2)/cyclin B kinase, rum1. Mutations in these genes uncoupled the dependence mitosis on DNA replication in the fission yeast, Saccharomyces pombe. It was proposed that the inactivation of the mitotic kinase complex, p34(cdc2)/cyclin B, induces a G(1), state wherein the cells re-replicate their DNA without an intervening mitosis. We show in this report that treatment of only M phase-arrested mouse cells, with the protein kinase inhibitor staurosporine, induced polyploidy. Nocodazole-arrested metaphase FT210 cells were pulsed with 100 ng/ml of staurosporine for 1 h. This 1-h treatment results in the inhibition of the mitotic p34(cdc2) kinase. The inhibition of the mitotic kinases leads to a reduction in the histone H1 and H3 mitotic-associated phosphorylations, chromosome decondensation and nuclear membrane reformation. When released into normal growth medium, these cells are reset to a G(1)state, re-replicate their DNA without completing mitosis, and become octaploid.
Asunto(s)
Alcaloides/farmacología , Inhibidores Enzimáticos/farmacología , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Mitosis/efectos de los fármacos , Poliploidía , Animales , Antineoplásicos/farmacología , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteína Quinasa CDC2/metabolismo , ADN de Neoplasias/genética , Fase G1/efectos de los fármacos , Histonas/metabolismo , Metafase/efectos de los fármacos , Ratones , Nocodazol/farmacología , Fosforilación , Estaurosporina , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
We have used the method of combined bromodeoxyuridine density label and radioactive label to measure the size of the repair patches appearing in the DNA of KB cells following treatment with UV-light or MMS. The repair patch size distribution was found to be the same for both agents, corresponding to the insertion of 34-40 nucleotides. These results, confirm recent results obtained by the bromodeoxyuridine-photolysis technique, that simple alkylating agents induce 'long patch' repair in human cells.
Asunto(s)
Reparación del ADN , Metilmetanosulfonato/farmacología , Rayos Ultravioleta , Bromodesoxiuridina , Línea Celular , Humanos , Peso MolecularRESUMEN
During excision repair of UV light or dimethyl sulphate (DMS)-induced damage to DNA the patch size for actively replicating KB or T98G cells is around 20 nucleotides. When confluent T98G cells or 'quiescent' KB cells are used the patch size is around 10 nucleotides. This value can be increased to around 20 nucleotides in T98G cells if a large excess of BrdUrd is included in the repair incubation medium. With 'quiescent' KB cells the patch size is not increased by excess BrdUrd. For all of these experimental conditions, when excision repair of UV or DMS damage takes place in the presence of aphidicolin, the patch size is found to be several times that found in its absence. Given the inhibitory specificity of aphidicolin for DNA polymerase alpha these results provide additional evidence that DNA polymerase alpha plays a role in the excision repair of DNA damaged by UV light or DMS. It is postulated that aphidicolin interrupts the processivity of the DNA polymerase alpha holoenzyme and allows an exonuclease to enlarge the repair site.
Asunto(s)
ADN Polimerasa II/antagonistas & inhibidores , Reparación del ADN/efectos de los fármacos , Diterpenos/farmacología , Afidicolina , Bromodesoxiuridina/metabolismo , Línea Celular , Replicación del ADN/efectos de los fármacos , HumanosRESUMEN
Recent research in cell biology as well as oncology research has focused on apoptosis or programmed cell death as a means of quantifying the induced effects of treatment. A hallmark of late-stage apoptosis is nuclear fragmentation in which DNA is degraded to release nucleosomes with their associated histones. In this work, a method was developed for detecting and measuring nucleosome concentration in vitro with magnetic resonance imaging (MRI). The indirect procedure used a commercially available secondary antibody-superparamagnetic iron oxide (SPIO) particle complex as a contrast agent that bound to primary antibodies against nucleosomal histones H4, H2A and H2B. Using a multiple-echo spin-echo sequence on a 1.5 T clinical MRI scanner, significant T2 relaxation enhancement as a function of in vitro nucleosomal concentration was measured. In addition, clustering or aggregation of the contrast agent was demonstrated with its associated enhancement in T2 effects. The T2 clustering enhancement showed a complex dependence on relative concentrations of nucleosomes, primary antibody and secondary antibody + SPIO. The technique supports the feasibility of using MRI measurements of nucleosome concentration in blood as a diagnostic, prognostic and predictive tool in the management of cancer.
Asunto(s)
Anticuerpos/inmunología , Apoptosis , Compuestos Férricos/química , Compuestos Férricos/metabolismo , Imagen por Resonancia Magnética/métodos , Imanes/química , Nucleosomas/metabolismo , Circulación Sanguínea , Estudios de Factibilidad , Células HL-60 , Humanos , Ligandos , Microesferas , Pronóstico , Reproducibilidad de los ResultadosRESUMEN
Since the first description of apoptosis, genetic and biochemical studies have led to a greater understanding of the multiple pathways that eukaryotic cells can take to terminate their existence. These findings have also proven useful in understanding the development of various diseases such as AIDS, Alzheimer's, and Parkinson's and have provided potential targets for possible therapies. Despite all these studies, the mechanism of chromatin condensation, a morphological hallmark of apoptosis, remains elusive. This review describes the work to date on the post-translational modifications of histones during apoptosis and discusses the models that have been presented to explain the apoptotic condensation of chromatin.
Asunto(s)
Apoptosis , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Acetilación , Animales , Cromatina/genética , Cromatina/metabolismo , Daño del ADN , Replicación del ADN/genética , Histonas/genética , Humanos , Fosforilación , Poli Adenosina Difosfato Ribosa , ProteínasRESUMEN
A modified method for measuring DNA repair patch sizes by the bromodeoxyuridine buoyant density shift method is presented. This method involves the digestion of total chromatin by Staphylococcal nuclease to produce small DNA fragments with a very homogeneous size distribution. Using this method to measure the size of repair patches following treatment with u.v.-light or dimethyl sulfate, a calculated incorporation of 40 nucleotides was obtained. This value is identical to previously published patch size estimations obtained by the buoyant density shift method with sonically produced DNA fragments. It is also in close agreement with values recently obtained by the bromodeoxyuridine photolysis method. Taken together, these independent results obtained with a variety of DNA damaging agents suggest that excision repair patch size is independent of the damaging agent.
Asunto(s)
Reparación del ADN , Nucleosomas/metabolismo , Bromodesoxiuridina/farmacología , Línea Celular , Reparación del ADN/efectos de la radiación , Replicación del ADN/efectos de los fármacos , Replicación del ADN/efectos de la radiación , Glioblastoma , Humanos , Cinética , Nucleasa Microcócica , Nucleosomas/efectos de los fármacos , Nucleosomas/efectos de la radiación , Rayos UltravioletaRESUMEN
Several groups of investigators have reported that the extent of repair replication following treatment of mammalian cells with dimethyl sulfate is increased by 3-aminobenzamide (3AB) over that occurring in its absence. Two plausable explanations for this phenomenon were tested. The first is that the number of nucleotides inserted per repair site, that is the repair patch size, is increased and the second is that the rate of repair is increased. Human T98G cells were treated with dimethyl sulfate and allowed to repair their DNA in the presence or absence of 3AB. It was found that the presence of 3AB did not increase the repair patch size nor the rate of removal of methylation products from the DNA, which is assumed to equal the rate of repair. The results of further experiments suggested that the 3AB effect can be explained by changes in nucleotide precursor pools.
Asunto(s)
Benzamidas/farmacología , Reparación del ADN/efectos de los fármacos , Células Cultivadas , Humanos , Hidroxiurea/farmacología , Metilación , Ésteres del Ácido Sulfúrico/farmacologíaRESUMEN
The considerable length of DNA in eukaryotic genomes requires packaging into chromatin to fit inside the small dimensions of the cell nucleus. Histone H1 functions in the compaction of chromatin into higher order structures derived from the repeating 'beads on a string' nucleosome polymer. Modulation of H1 binding activity is thought to be an important step in the potentiation/depotentiation of chromatin structure for transcription. It is generally accepted that H1 binds less tightly than other histones to DNA in chromatin and can readily exchange in living cells. Fusion proteins of Histone H1 and green fluorescent protein (GFP) have been shown to associate with chromatin in an apparently identical fashion to native histone H1. This provides a means by which to study histone H1-chromatin interactions in living cells. Here we have used human cells with a stably integrated H1.1-GFP fusion protein to monitor histone H1 movement directly by fluorescence recovery after photobleaching in living cells. We find that exchange is rapid in both condensed and decondensed chromatin, occurs throughout the cell cycle, and does not require fibre-fibre interactions. Treatment with drugs that alter protein phosphorylation significantly reduces exchange rates. Our results show that histone H1 exchange in vivo is rapid, occurs through a soluble intermediate, and is modulated by the phosphorylation of a protein or proteins as yet to be determined.
Asunto(s)
Cromatina/metabolismo , Histonas/metabolismo , ADN/metabolismo , Diclororribofuranosil Benzoimidazol/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Fosforilación , Unión Proteica , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Estaurosporina/farmacología , Células Tumorales CultivadasRESUMEN
Small proline-rich proteins, believed to be precursor proteins for the crosslinked envelope formation in cells undergoing squamous differentiation, are encoded by the SPRR genes. To further investigate the role of these proteins, the time course of increased synthesis of SPRR1 mRNA in nasal epithelia of rats exposed to cigarette smoke was determined, and the deduced amino acid sequence of the rat SPRR1 was compared with those of other species. Using the pig homologue (20K) antisense cRNA probe, high levels of SPRR1 transcript were detected by in situ hybridization in squamous epithelia that line the nasal vestibule and hard palate of the rat. Basal cells of both the vestibule and palate contained low levels of the transcript, and increasing amounts were detected in the squamous layers. In rats exposed to 250 mg/m3 (total particulate matter) cigarette smoke 6 h/day for 5 days, the number of small mucous cells increased in the respiratory epithelium of the nasal septum in the early stages of squamous differentiation, but were gradually replaced by squamous metaplastic cells. During this transition, hybridization of the 20K antisense cRNA probe increased in the epithelial and mesenchymal cells, indicating that SPRR1 protein could have roles in cellular differentiation other than as a building block of the crosslinked envelope. Similarly, high levels of SPRR1 transcript were detected in the nasal transitional epithelium lining internal walls and maxilloturbinates that had undergone squamous metaplasia after cigarette smoke exposure. At 5 days after the withdrawal of cigarette smoke exposure, the morphology of the midseptal epithelium returned to that of a pseudostratified mucociliary epithelium and the epithelia lining the maxilloturbinates to that of a transitional epithelium. Accompanying this change in morphology of the tissues, the levels of SPRR1 transcripts significantly decreased in the epithelia. However, in the mesenchyme no significant decrease was observed during this recovery. RNA prepared from the external nose surrounding the nasal vestibule contained a transcript of about 0.9 kb that hybridized to the 20K cDNA probe on Northern blot analysis. DNA sequence analysis of the transcript confirmed the identity as that of the SPRR mRNA with its characteristic repeat encoding the oligopeptide with the general consensus -EPC*PKVP-. However, the rat homologue rSPRR1 contained more repeats of the oligopeptide compared with those of higher mammals such as the rabbit, pig, and human, suggesting a possible inverse relation between number of repeats and evolution development. This finding suggests that the number of repeats in the protein may be redundant; however, the conserved sequence of the peptide indicates that this region is essential for the function of this protein.
Asunto(s)
Regulación de la Expresión Génica , Mucosa Nasal/metabolismo , Mucosa Nasal/patología , Biosíntesis de Proteínas , Proteínas , Humo/efectos adversos , Fumar , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular , Proteínas Ricas en Prolina del Estrato Córneo , Sondas de ADN , Epitelio/metabolismo , Epitelio/patología , Humanos , Hibridación in Situ , Masculino , Proteínas de la Membrana , Metaplasia , Datos de Secuencia Molecular , Plantas Tóxicas , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Conejos , Ratas , Ratas Endogámicas F344 , Homología de Secuencia de Aminoácido , Porcinos , NicotianaRESUMEN
In mammalian cells, hyperphosphorylation of histone H1 and phosphorylation of histone H3 correlate well with the G2 phase to metaphase condensation of chromosomes, and these phosphorylations most probably have a role in initiating and controlling the entry into mitosis. The protein kinase inhibitor staurosporine has been used to examine the role of H1 and H3 phosphorylations in controlling chromosome condensation in the mouse FM3A cell line. We present evidence that (i) staurosporine inhibits the protein kinases that phosphorylate histone H1 during mitosis, (ii) staurosporine also inhibits the histone H3-specific kinase, (iii) the inhibition of these kinase activities prevent cells from entering mitosis, and (iv) addition of staurosporine to cells already arrested at metaphase by nocodazole causes a rapid dephosphorylation of histones H1 and H3 and the decondensation of the metaphase chromosomes. The results show that the hyperphosphorylation of histone H1 and phosphorylation of histone H3 are required to maintain metaphase chromosomes in their condensed state.
Asunto(s)
Alcaloides/farmacología , Cromosomas/efectos de los fármacos , Histonas/metabolismo , Protamina Quinasa/antagonistas & inhibidores , Animales , Ciclo Celular/efectos de los fármacos , Línea Celular , Cromosomas/ultraestructura , Electroforesis en Gel de Poliacrilamida , Femenino , Fase G2/efectos de los fármacos , Histonas/aislamiento & purificación , Neoplasias Mamarias Experimentales , Metafase/efectos de los fármacos , Ratones , Mitosis/efectos de los fármacos , Fosfoproteínas/aislamiento & purificación , Fosfoproteínas/metabolismo , Fosforilación , Estaurosporina , Células Tumorales CultivadasRESUMEN
In mammalian cells, up to six serines and threonines in histone H1 are phosphorylated in vivo in a cell cycle dependent manner that has long been linked with chromatin condensation. Growth-associated H1 kinases, now known as cyclin-dependent kinases (CDKs), are thought to be the enzymes responsible for this process. This paper describes the phosphorylation of histone H1 by four different purified CDKs. The four CDKs phosphorylate only the cell cycle specific phosphorylation sites of H1, indicating that they belong to the kinase class responsible for growth-related H1 phosphorylation in vivo. All four CDKs phosphorylate all of the interphase and mitotic-specific H1 sites. In addition to the (S/T)PXK consensus phosphorylation sites, these four CDKs also phosphorylate a mitotic-specific in vivo H1 phosphorylation site that lacks this sequence. There is no site selectivity among the growth-related phosphorylation sites by any of the four CDKs because all four CDKs phosphorylate all relevant sites. The results imply that the cell cycle dependent H1 phosphorylations observed in vivo must involve differential accessibility of H1 sites at different stages of the cell cycle.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Histonas/metabolismo , Animales , Sitios de Unión , Células CHO , División Celular , Línea Celular , Cricetinae , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Histonas/aislamiento & purificación , Cinética , Ratones , Mapeo Peptídico , FosforilaciónRESUMEN
We have identified two intrinsically bent regions of DNA which flank the transcription start site of the rRNA gene from Physarum polycephalum. DNA fragments from both regions were analyzed by circular permutation polyacrylamide gel electrophoresis assay and computer modeling. Both types of analysis indicate that one fragment contains a relatively simple bend centered about 160 base pairs (bp) upstream of the transcription start site while the other fragment contains multiple bends, the most prominent of which is centered about 150 bp downstream of the start site. According to both gel mobilities and computer modeling we estimate that the net bending in each is about 45 degrees. These fragments were studied in detail by varying parameters of electrophoresis that are known to affect bending. Previous work indicates that anomalous mobility should decrease when temperature or ethidium bromide concentration is increased, whereas anomalous mobility should increase when polyacrylamide gel percentage is increased. The anomalous mobility of both fragments decreases as temperature is raised from 4 to 65 degrees C, although the bent structure centered at -160 bp is more temperature labile than the bend at +150 bp. Strikingly different behavior was observed for the two fragments as the polyacrylamide concentrations was varied. As polyacrylamide concentrations are increased from 6 to 10%, the anomalous mobility of the bend centered at -160 bp increases while that of the bend centered at +150 bp decreases. The bend centered at +150 bp is "straightened" at all ethidium concentrations tested. In sharp contrast and unexpectedly, the anomalous migration of the bend centered at -160 bp increases dramatically in 0.1 micrograms/ml ethidium bromide. Many of the mobility differences we observe suggest that the two regions studied represent structurally distinct forms of bent DNA. The location of these strongly bent regions on either side of a RNA polymerase I transcription start site suggests important roles for such structures in chromatin structure and transcription initiation.
Asunto(s)
ADN de Hongos/metabolismo , ADN Ribosómico/metabolismo , Genes Fúngicos , Physarum polycephalum/genética , ARN Polimerasa I/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , ADN de Hongos/química , ADN de Hongos/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Physarum polycephalum/enzimología , Plásmidos , ARN Ribosómico/genética , Mapeo Restrictivo , TermodinámicaRESUMEN
The form of inhibitor protein of the cAMP-dependent protein kinase (PKI) that has been most thoroughly studied is a protein purified from rabbit skeletal muscle. Beale et al. previously isolated a species of PKI from rat testis that appeared from its amino acid composition to be quite distinct from the rabbit skeletal muscle protein [Beale, E. G., Dedman, J. R. & Means, A. R. (1977) J. Biol. Chem. 252, 6322-6327]. The amino acid sequence of a form of rat testis PKI has now been determined both by sequencing overlapping peptide fragments for 95% of the protein and by the isolation of a cDNA clone containing the coding region for the 70-amino acid protein. The sequence of the 70-amino acid testis PKI displays a maximum of only 41% sequence identity with the previously sequenced 75-amino acid rabbit skeletal muscle PKI. However, the two forms have identical potency as inhibitors and the key amino acids of the pseudosubstrate site, shown to be critical for maximal inhibition with the rabbit skeletal muscle PKI, have been conserved in the testis protein. The rabbit skeletal muscle and rat testis PKIs most likely represent distinct isoforms. The nucleotide sequence of the rat testis PKI cDNA suggests that a second form of testis PKI, longer by 8 additional amino-terminal amino acids, might also be produced.
Asunto(s)
Proteínas Portadoras/genética , Péptidos y Proteínas de Señalización Intracelular , Inhibidores de Proteínas Quinasas , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Secuencia de Bases , Cromatografía DEAE-Celulosa , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Cinética , Masculino , Datos de Secuencia Molecular , Conejos , RatasRESUMEN
The mouse cell FT210 was isolated as a G2 phase mutant with a possible defect in the histone H1 kinase. We determined that a temperature-sensitive lesion in this cell line lies in the CDC2 gene. DNA sequence analysis revealed two point mutations in highly conserved regions of the gene: an isoleucine to valine change in the PSTAIR region, and a proline to serine change at the C-terminal region of the protein p34. These mutations cause the p34 protein kinase to become inactivated and degraded in FT210 cells at the restrictive temperature, 39 degrees C. The consequence of this temperature-induced inactivation of the CDC2 gene product is cell cycle arrest at the mid to late G2 phase, and this arrest can be alleviated by the introduction of the human CDC2 homolog.
Asunto(s)
Proteína Quinasa CDC2/genética , Fase G2 , Mutación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteína Quinasa CDC2/metabolismo , Línea Celular , Estabilidad de Enzimas , Cinética , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos , Homología de Secuencia de Ácido Nucleico , Temperatura , Transcripción Genética , TransfecciónRESUMEN
Chromosome condensation at mitosis correlates with the activation of p34cdc2 kinase, the hyperphosphorylation of histone H1 and the phosphorylation of histone H3. Chromosome condensation can also be induced by treating interphase cells with the protein phosphatase 1 and 2A inhibitors okadaic acid and fostriecin. Mouse mammary tumour FT210 cells grow normally at 32 degrees C, but at 39 degrees C they lose p34cdc2 kinase activity and arrest in G2 because of a temperature-sensitive lesion in the cdc2 gene. The treatment of these G2-arrested FT210 cells with fostriecin or okadaic acid resulted in full chromosome condensation in the absence of p34cdc2 kinase activity or histone H1 hyperphosphorylation. However, phosphorylation of histones H2A and H3 was strongly stimulated, partly through inhibition of histone H2A and H3 phosphatases, and cyclins A and B were degraded. The cells were unable to complete mitosis and divide. In the presence of the protein kinase inhibitor starosporine, the addition of fostriecin did not induce histone phosphorylation and chromosome condensation. The results show that chromosome condensation can take place without either the histone H1 hyperphosphorylation or the p34cdc2 kinase activity normally associated with mitosis, although it requires a staurosporine-sensitive protein kinase activity. The results further suggest that protein phosphatases 1 and 2A may be important in regulating chromosome condensation by restricting the level of histone phosphorylation during interphase, thereby preventing premature chromosome condensation.