RESUMEN
OBJECTIVES: Medication reconciliation is widely promoted by international health authorities. Its expansion requires human resources, which are limited and unequally distributed among health care facilities. Recent international studies support the involvement of pharmacy technician in the medication reconciliation process but his role remains unstructured in France. We aimed to assess pharmacy technicians' opinions and willingness to be involved in the medication reconciliation process expansion and to identify the levers and barriers of the project. METHODS: A field study was conducted among health facilities of our territory hospital group. Semi-structured interviews were carried out with different pharmacy technicians. Data were analyzed using a qualitative thematic analysis approach. RESULTS: Overall, 12 pharmacy technicians from 5 hospitals were interviewed and almost all assumed their rightful place in the medication reconciliation process (n=11), with a view to revaluating tasks. For all pharmacy technicians, the main barriers to participate in medication reconciliation were the lack of time and training. The spread of a "patient culture", the supervision by pharmacists, the desire to be part of the care team in the ward and additional training requests were major levers of change. CONCLUSIONS: Pharmacy technicians' role in expanding medication reconciliation process is legitimate and must be standardized in France. The deployment of the project requires to be formalized within a territory and should consider and develop local organisations.
Asunto(s)
Conciliación de Medicamentos/métodos , Servicio de Farmacia en Hospital/organización & administración , Técnicos de Farmacia , Actitud del Personal de Salud , Francia , Humanos , Organización y Administración , Farmacéuticos , Técnicos de Farmacia/educaciónRESUMEN
Intense visible nano-emitters are key objects for many technologies such as single photon source, bio-labels or energy convertors. Chalcogenide nanocrystals have ruled this domain for several decades. However, there is a demand for cheaper and less toxic materials. In this scheme, ZnO nanoparticles have appeared as potential candidates. At the nanoscale, they exhibit crystalline defects which can generate intense visible emission. However, even though photoluminescence quantum yields as high as 60% have been reported, it still remains to get quantum yield of that order of magnitude which remains stable over a long period. In this purpose, we present hybrid ZnO/polyacrylic acid (PAAH) nanocomposites, obtained from the hydrolysis of diethylzinc in presence of PAAH, exhibiting quantum yield systematically larger than 20%. By optimizing the nature and properties of the polymeric acid, the quantum yield is increased up to 70% and remains stable over months. This enhancement is explained by a model based on the hybrid type II heterostructure formed by ZnO/PAAH. The addition of PAAX (X = H or Na) during the hydrolysis of ZnEt2 represents a cost effective method to synthesize scalable amounts of highly luminescent ZnO/PAAX nanocomposites.
RESUMEN
Electropermeabilization is a physical method to deliver molecules into cells and tissues. Clinical applications have been successfully developed for antitumoral drug delivery and clinical trials for gene electrotransfer are currently underway. However, little is known about the mechanisms involved in this transfer. The main difficulties stem from the lack of single cell models which reliably replicate the complex in vivo environment. In order to increase our understanding of the DNA electrotransfer process, we exploited multicellular tumor spheroids as an ex vivo model of tumor. We used confocal microscopy to visualize the repartition of permeabilized cells in spheroids subjected to electric pulses. Our results reveal that even if cells can be efficiently permeabilized with electric fields, including those cells present inside the spheroids, gene expression is by contrast limited to the external layers of cells. Taken together, these results, in agreement with the ones obtained in tumors, indicate that the spheroid model is more relevant to an in vivo situation than cells cultured as monolayers. They validate the spheroid model as a way to study electro-mediated gene delivery processes.
Asunto(s)
Electroporación/métodos , Modelos Moleculares , Conformación Molecular , Esferoides Celulares/metabolismo , Animales , Técnicas de Transferencia de Gen , Células HCT116 , Humanos , Ratones , Ratones Endogámicos C57BL , PermeabilidadRESUMEN
Multicellular tumor spheroids, an in vitro 3-D model that simulates malignant-cell contacts within a tumor, can be used to evaluate tumor response to therapeutic agents. We found that MELN (derived from MCF-7 cells) cells grown in 3-D as spheroids, remain highly sensitive to estradiol in terms of growth, down-regulation of ERalpha expression and ERalpha-induced transcriptional activity. Estradiol induces cyclin D1 and CDK1 proteins in Ki-67 positive proliferating cells, whereas survivin is up-regulated in both Ki-67 positive proliferative outer layer of cells and around the necrotic zone in non-proliferating cells. OH-Tam inhibits both estradiol-induced transcriptional activity and estradiol-dependent growth of MELN spheroids. Consistent with its antiproliferative effect, we observed that OH-Tam induces an important decrease in the proportion of proliferating cells, positive for Ki-67, cyclin D1 and CDK1. But, in contrast to what was expected, OH-Tam treatment resulted in a decrease in the proportion of p21 positive cells. Furthermore, despite its ability to down-regulate survivin in MELN spheroids, OH-Tam did not trigger apoptosis. Taken together, these results indicate that this model, is more relevant to an in vivo situation than monolayer cultures. It could be useful to identify new markers of the response to endocrine treatment and to investigate the effects of drugs combination.
Asunto(s)
Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Estradiol/metabolismo , Moduladores de los Receptores de Estrógeno/farmacología , Tamoxifeno/análogos & derivados , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/inmunología , Proteína Quinasa CDC2/metabolismo , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Ciclina D , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Ciclinas/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Proteínas Inhibidoras de la Apoptosis , Antígeno Ki-67/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Esferoides Celulares , Survivin , Tamoxifeno/farmacología , Factores de TiempoRESUMEN
BRCA1 germline mutations predispose women to early onset, familial breast and ovarian cancer. BRCA1 has been recently implicated in the cellular response to agents that disrupt the mitotic spindle. In this report, we studied BRCA1 contribution to paclitaxel response in MCF-7 breast cancer cells. We show that MCF-7 cells transfected with BRCA1 siRNA display a significant increase in resistance to paclitaxel compared with the control cells. We next demonstrate that downregulation of BRCA1 reduces the mitotic index and triggers premature cyclin B1 degradation and decrease in Cdk1 activity following paclitaxel treatment, suggesting that BRCA1 downregulation results in precocious inactivation of the spindle checkpoint. These findings were confirmed by showing that BRCA1 downregulation induces premature sister-chromatids separation in MCF-7 cells following spindle damage. Furthermore, we show that BRCA1 up-regulates the expression of the protein kinase BubR1, essential component of the functional spindle checkpoint, whose downregulation is known to result in paclitaxel resistance in MCF-7 cells. Altogether, our findings support the notion that downregulation of BRCA1 expression mediates paclitaxel resistance through premature inactivation of spindle checkpoint in MCF-7 breast cancer cells. They link BRCA1 to the mitotic checkpoint that plays an essential role in the maintenance of chromosomal stability.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Proteína BRCA1/genética , Neoplasias de la Mama/metabolismo , Paclitaxel/farmacología , Huso Acromático/efectos de los fármacos , Proteína BRCA1/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Ciclo Celular/fisiología , Línea Celular Tumoral , Inestabilidad Cromosómica , Regulación hacia Abajo , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , ARN Interferente Pequeño/metabolismo , Huso Acromático/ultraestructura , Regulación hacia ArribaRESUMEN
Several studies have suggested that Bcl-2 phosphorylation, which occurs during mitotic arrest induced by paclitaxel, inhibits its antiapoptotic function. In the present study, we demonstrated that the level of phosphorylated Bcl-2 was threefold higher in mitochondria than in the nuclear membrane or endoplasmic reticulum. Our results show, in isolated mitochondria, that phosphorylation of Bcl-2 in mitosis does not modify either its integration into the mitochondrial membrane or the ability to release cytochrome c in response to Bid, a cytochrome c releasing agent. In HeLa cells, in which paclitaxel induces apoptosis, the nonphosphorylated form of Bcl-2 is degraded by a proteasome-dependent degradation pathway, whereas the phosphorylated forms of mitochondrial Bcl-2 appear to be resistant to proteasome-induced degradation. We found that low concentrations of recombinant Bid triggered a greater release of cytochrome c from mitochondria isolated from paclitaxel-treated HeLa cells than from mitochondria isolated from control HeLa cells. Taken together, these results show that Bcl-2 phosphorylation does not inhibit its function. On the contrary, Bcl-2 phosphorylation indirectly regulated its antiapoptotic action via protection against degradation. Indeed, in response to paclitaxel treatment, the level of Bcl-2 expression in mitochondria rather than its phosphorylation state could regulate the sensitivity of mitochondria to cytochrome c releasing agents in vitro.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Proteínas Portadoras/farmacología , Mitocondrias/efectos de los fármacos , Paclitaxel/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Apoptosis/fisiología , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Proteínas Portadoras/genética , Cisteína Endopeptidasas/metabolismo , Grupo Citocromo c/metabolismo , Retículo Endoplásmico/metabolismo , Células HeLa/efectos de los fármacos , Humanos , Membranas Intracelulares/fisiología , Mitocondrias/metabolismo , Complejos Multienzimáticos/metabolismo , Membrana Nuclear/metabolismo , Fosforilación , Complejo de la Endopetidasa Proteasomal , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Células Tumorales CultivadasRESUMEN
In this paper, we report, for the first time, the localization of the phosphorylation site of the fetoacinar pancreatic protein (FAPP), which is an oncofetal variant of the pancreatic bile salt-dependent lipase. Using Chinese hamster ovary (CHO) cells transfected with the cDNA encoding FAPP, we radiolabeled the enzyme with (32)P, and then the protein was purified by affinity chromatography on cholate-immobilized Sepharose column and submitted to a CNBr hydrolysis. Analysis of peptides by high pressure liquid chromatography, associated with the radioactivity profile, revealed that the phosphorylation site is located at threonine 340. Site-specific mutagenesis experiments, in which the threonine was replaced by an alanine residue, were used to invalidate the phosphorylation of FAPP and to study the influence of the modification on the activity and secretion of the enzyme. These studies showed that CHO cells, transfected with the mutated cDNA of FAPP, kept all of their ability to synthesize the protein, but the loss of the phosphorylation motif prevented the release of the protein in the extracellular compartment. However, the mutated enzyme, which was sequestrated in the transfected CHO cells, remains active on bile salt-dependent lipase substrates.
Asunto(s)
Proteínas Portadoras/metabolismo , Glicoproteínas/metabolismo , Lipasa , Esterol Esterasa/metabolismo , Animales , Secuencia de Bases , Células CHO , Proteínas Portadoras/química , Proteínas Portadoras/genética , Cricetinae , Cartilla de ADN , ADN Complementario , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Mutagénesis Sitio-Dirigida , Fosforilación , Esterol Esterasa/química , Treonina/metabolismo , TransfecciónRESUMEN
Microtubule damages induced by paclitaxel inhibit proteasome-dependent degradation of cyclin B, resulting in a sustained activation of cyclin B/cdc2 kinase and a cell cycle arrest in mitosis. It has been previously shown that this kinase activity is also required for paclitaxel-induced apoptosis. We found here that paclitaxel increased cdc2 mRNA and protein levels and led to an accumulation of cdc2 in the active dephosphorylated form in NIH-OVCAR-3 cells. The addition of cycloheximide inhibited the paclitaxel-induced increase in cdc2 protein level, further indicating that paclitaxel stimulates cdc2 synthesis. This increase in cdc2 synthesis is a consequence of paclitaxel-induced arrest in mitosis. Indeed, dual analysis of DNA and cdc2 protein contents indicated that cdc2 up-regulation occurred in cells arrested with a G2/M DNA content. Furthermore, no up-regulation of cdc2 protein was observed when paclitaxel-treated cells were prevented from entering mitosis by treatment with purvalanol A, a cyclin-dependent kinase (CDK) inhibitor, or stimulated to exit mitosis with 2-AP, a non-specific kinase inhibitor. In addition, when paclitaxel-induced apoptosis was inhibited by Bcl-2 over-expression, cdc2 up-regulation did not occur, leading to a lower level of activation of the cyclin B/cdc2 complex. Taken together, these results indicated that paclitaxel-induced cdc2 protein synthesis participates in a positive feedback loop designed to increase the activity of cyclin B/cdc2 kinase and thus may play a role in paclitaxel-induced apoptosis.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis/fisiología , Proteína Quinasa CDC2/metabolismo , Paclitaxel/farmacología , 2-Aminopurina/farmacología , Antimetabolitos/farmacología , Cicloheximida/farmacología , ADN/efectos de los fármacos , ADN/metabolismo , Activación Enzimática , Fase G2/efectos de los fármacos , Fase G2/genética , Humanos , Fosforilación , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/metabolismo , Células Tumorales Cultivadas/efectos de los fármacos , Regulación hacia ArribaRESUMEN
To elucidate the mechanisms of the mammalian cell defense against cross-linking agents, we studied previously cellular responses to mitomycin C (MMC) treatment in two MMC-hypersensitive hamster cell mutants' V-H4 and V-C8, as well as their parental cell line V79. In the present report, we investigated whether alterations in cell cycle checkpoints and induction of apoptosis could be responsible for the MMC hypersensitivity of the V-H4 and V-C8 mutant cell lines. First, we found that parental and mutant cells exhibited similar cell cycle responses to MMC concentrations of equivalent cytotoxicity, arguing against a defective cell cycle checkpoint in hypersensitive cell lines. In contrast, we showed that mutant cells underwent greater levels of apoptosis following MMC treatment than parental cells. These findings indicate that increased induction of apoptosis contributes to the hypersensitivity of V-H4 and V-C8 cells to the growth inhibitory effect of MMC. This differential apoptotic response was observed with both equimolar and equitoxic MMC doses and was specific to the cross-linking agent MMC, suggesting that control of the apoptotic process is altered in both MMC-hypersensitive mutants. The defective genes in V-H4 and V-C8 cells would then function in the regulation of an apoptotic pathway triggered by MMC-induced damage and independent of p53-mediated transcription.
Asunto(s)
Apoptosis , Mitomicina/farmacología , Animales , Antibióticos Antineoplásicos/farmacología , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Cricetinae , Cricetulus , MutaciónRESUMEN
This work describes the characterization of an immunoreactive form of bile salt-dependent lipase (BSDL) expressed by the human pancreatic tumoral Mia PaCa-2 cell line. This BSDL-related protein, which has an M(r) of 70 kDa, is enzymatically active and poorly secreted. Furthermore, a protein with the same electrophoretic migration can also be immunoprecipitated with polyclonal antibodies specific for the human pancreatic BSDL after in vitro translation of RNA isolated from Mia PaCa-2 cells. These data indicated that this BSDL-related protein might be poorly, or not, glycosylated. Reverse transcription and amplification of RNA extracted from Mia PaCa-2 cells using primers able to specifically amplify the full-length mRNA of the human BSDL resulted in a detectable 1.8-kb cDNA product, shorter than that of BSDL (2.2 kb). The sequence of this transcript corresponds to the mRNA sequence that codes for the mature human pancreatic BSDL. However, a deletion of 330 bp is located within the 3'-domain of this cDNA. Therefore data allowed us to conclude that the 70-kDa BSDL-related protein is a 612 amino acid length protein and represents a truncated form of BSDL. The deletion of 110 amino acids occurs in the C-terminal region of the protein, which encompasses 6 tandemly repeated sequences instead of the 16 normally present in the sequence of BSDL. Because feto-acinar pancreatic protein (FAPP), which is the oncofetal counterpart of BSDL, is a C-terminally truncated isoform of BSDL, it is suggested that the 70-kDa BSDL-related protein expressed in MiaPaCa-2 cells could be representative of the protein moiety of FAPP.
Asunto(s)
Lipasa , Neoplasias Pancreáticas/enzimología , Esterol Esterasa/biosíntesis , Esterol Esterasa/inmunología , Empalme Alternativo , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/genética , Sistema Libre de Células/metabolismo , Glicoproteínas/genética , Glicosilación , Humanos , Inmunoadsorbentes , Isoenzimas/biosíntesis , Isoenzimas/genética , Isoenzimas/inmunología , Isoenzimas/aislamiento & purificación , Datos de Secuencia Molecular , Páncreas/metabolismo , Neoplasias Pancreáticas/patología , Biosíntesis de Proteínas , ARN Mensajero/biosíntesis , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Esterol Esterasa/genética , Esterol Esterasa/aislamiento & purificación , Secuencias Repetidas en Tándem/genética , Células Tumorales CultivadasRESUMEN
Growth of human breast adenocarcinoma MCF-7 cells as a tumor on nude mice is dependent on estrogen. It has been shown that estrogen withdrawal (EW) induces a partial regression of the tumor via an inhibition of cell proliferation and an induction of apoptosis. We investigated in this in vivo model the underlying molecular mechanisms of the hormone-dependent regulation of cell cycle machinery and apoptosis. We found that, 2 days after EW, the tumor protein levels of p21 rose, whereas those of Rb proteins decreased in parallel with the decrease in the proportion of tumor cells in S phase and the increase of the tumor apoptotic index. Between 3 and 7 days after EW, apoptosis was inhibited and tumor proliferation returned to the control value. There was a concomitant decline in p21 and an elevation of Rb tumor protein content. Slight variations of cyclin D protein level were observed in MCF-7 tumors over the time course following EW treatment. Bcl-2 overexpression not only inhibited apoptosis induced by EW but also modulated hormone-dependent cell cycle regulation. First, the analysis of phosphorylation status of Rb protein and the measurement of the proportion of tumor cells in S phase indicated that Bcl-2 overexpression results in a decrease of DNA synthesis induced by estradiol. Furthermore, after EW, Bcl-2-induced inhibition of hormone-dependent apoptosis was associated with an inhibition of Rb protein downregulation, a sustained level of p21 protein, and a prolonged inhibition of cell cycle progression. These results suggest that, in human hormone-dependent breast cancers, cross-talk exists between the signaling pathways which lead to regulation of cell cycle progression and apoptosis.
Asunto(s)
Apoptosis , Neoplasias de la Mama/patología , Ciclo Celular/fisiología , Estradiol/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/genética , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/análisis , Femenino , Genes de Retinoblastoma , Genes bcl-2 , Humanos , Etiquetado Corte-Fin in Situ , Ratones , Ratones Desnudos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Receptores de Estrógenos/fisiología , Proteínas Recombinantes/metabolismo , Proteína de Retinoblastoma/biosíntesis , Transfección , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Bile-salt-dependent lipase (BSDL, EC 3.1.1.-) is an enzyme expressed by the pancreatic acinar cells and secreted as a component of the pancreatic juice of all examined species. During its secretion route BSDL is associated with intracellular membranes. This association allows the complete glycosylation of the enzyme or participates in the inhibition of the enzyme activity, which can deleterious for the acinar pancreatic cell. Thereafter, the human BSDL is phosphorylated by a serine/threonine protein kinase and released from intracellular membranes. In the present study, we show that the rat pancreatic BSDL, expressed by AR4-2J cells used as a model, is phosphorylated by a protein kinase that is insensitive to inhibitors of protein kinases A, C or G and that the phosphorylation process is favoured by okadaic acid (an inhibitor of protein phosphatases 1 and 2A). However, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB), which is a specific inhibitor of casein kinase II, abolishes the phosphorylation in vitro of BSDL within micro- somes of AR4-2J pancreatic cells. We showed further that the alpha-subunit of casein kinase II co-locates with BSDL within the lumenal compartment of the Golgi. Genistein, which perturbs the trans-Golgi network, also inhibits the phosphorylation of BSDL, suggesting that this post-translational modification of BSDL probably occurred within this cell compartment. The inhibition of the phosphorylation of BSDL by DRB also decreases the rate at which the enzyme is secreted. Under the same conditions, the rate of alpha-amylase secretion was not modified. These data strongly suggest that phosphorylation is a post-translational event, which appears to be essential for the secretion of BSDL.
Asunto(s)
Páncreas/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Esterol Esterasa/metabolismo , Animales , Quinasa de la Caseína II , Compartimento Celular , Línea Celular , Diclororribofuranosil Benzoimidazol/farmacología , Inhibidores Enzimáticos/farmacología , Genisteína/farmacología , Humanos , Técnicas In Vitro , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Ratas , Esterol Esterasa/química , alfa-Amilasas/metabolismoRESUMEN
Treatment of NIH-OVCAR-3 cells with paclitaxel, a microtubule-stabilizing agent, induces mitotic arrest and apoptosis, but also Bcl-2 phosphorylation. We report here that Bcl-2 phosphorylation precedes Bcl-2 down-regulation and that both events are closely associated with mitotic arrest, but are not sufficient for paclitaxel to trigger apoptosis. Indeed, when paclitaxel-treated cells were induced to exit mitosis in the presence of 2-aminopurine, Bcl-2 phosphorylation and Bcl-2 down-regulation were both inhibited. In contrast, when apoptosis was inhibited by a caspase inhibitor or Bcl-2 over-expression, Bcl-2 phosphorylation and down-regulation still occurred. Furthermore, we show that Bcl-2 is degraded in mitosis by the proteasome-dependent pathway since Bcl-2 down-regulation is inhibited by proteasome inhibitors such as MG132, Lactacystin and LLnL. Taken together these results indicate that mitotic spindle damage results in post-translational modifications of Bcl-2 by phosphorylation and degradation.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Microtúbulos/fisiología , Complejos Multienzimáticos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Apoptosis , Femenino , Regulación Neoplásica de la Expresión Génica , Genes bcl-2 , Humanos , Leupeptinas/farmacología , Microtúbulos/efectos de los fármacos , Mitosis , Neoplasias Ováricas , Paclitaxel/toxicidad , Fosforilación , Complejo de la Endopetidasa Proteasomal , Proteínas Proto-Oncogénicas c-bcl-2/genética , Células Tumorales Cultivadas , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
In this article, we report the nucleotide sequence of the cDNA encoding an isoform of bile-salt-dependent lipase (BSDL) expressed by human hepatoma cells. The BSDL is a 100-kDa glycoprotein normally expressed by the human pancreas. Using a polyclonal antibody raised against an internal peptide located between Ile(327) and Glu(350) of the human pancreatic BSDL, we have immunodetected an isoform of human pancreatic BSDL, with an apparent molecular mass of about 62 kDa. This isoform of BSDL was mainly associated with the cytosol of a human hepatoma cell line (HepG2), the remaining protein being found in the microsome fraction. In addition, esterolytic activity on p-nitrophenyl hexanoate measured in microsomes and cytosol appeared very low and was weakly stimulated by bile salts, such as taurocholate. In contrast to human pancreatic BSDL, which is secreted as a component of pancreatic juice, this isoform appeared to be retained in the HepG2 cells. Reverse transcription, followed by PCR and amplification, performed on RNA extracted from HepG2 cells using specific primers hybridizing to the sequence coding for the entire normal human pancreatic BSDL, allowed us to amplify a 1. 7-kb transcript that appeared to be 0.5 kb shorter than the transcript of the pancreatic enzyme (2.2 kb). From the sequence of the transcript thus obtained, a protein with a molecular mass of 62 kDa might be predicted, which is in good agreement with the size of the isoform of BSDL immunodetected in HepG2 cells. The N-terminal amino-acid sequence, deduced from the 1.7-kb transcript sequence, matched that of the pancreatic BSDL. However, the C-terminal domain appeared truncated, bearing only a single mucin-like sequence compared with sixteen for the human pancreatic BSDL. The actual intracellular function of this human BSDL hepatoma isoform remains to be elucidated.
Asunto(s)
Microsomas Hepáticos/enzimología , Esterol Esterasa/genética , Esterol Esterasa/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Ácidos y Sales Biliares/farmacología , Western Blotting , Clonación Molecular , Reacciones Cruzadas , Citosol/efectos de los fármacos , Citosol/enzimología , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/inmunología , Isoenzimas/metabolismo , Neoplasias Hepáticas/enzimología , Microsomas Hepáticos/efectos de los fármacos , Datos de Secuencia Molecular , Peso Molecular , Páncreas/enzimología , Biosíntesis de Proteínas , ARN Mensajero/análisis , ARN Mensajero/genética , Esterol Esterasa/química , Esterol Esterasa/inmunología , Células Tumorales CultivadasRESUMEN
Activation of protein kinase C (PKC) inhibits cell cycle progression at the G1/S and G2/M transitions. We found that phorbol 12-myristate 13-acetate (PMA) induced upregulation of p21, not only in MCF-7 cells arrested in the G1 phase as previously shown, but also in cells delayed in the G2 phase. This increase in p21 in cells accumulated in the G1 and G2/M phases of the cell cycle after PMA treatment was inhibited by the PKC inhibitor GF109203X. This indicates that PKC activity is required for PMA-induced p21 upregulation and cell cycle arrest in the G1 and G2/M phases of the cell cycle. To further assess the role of p21 in the PKC-induced G2/M cell cycle arrest independently of its G1 arrest, we used aphidicolin-synchronised MCF-7 cells. Our results show that, in parallel with the inhibition of cdc2 activity, PMA addition enhanced the associations between p21 and either cyclin B or cdc2. Furthermore, we found that after PMA treatment p21 was able to associate with the active Tyr-15 dephosphorylated form of cdc2, but this complex was devoid of kinase activity indicating that p21 may play a role in inhibition of cdc2 induced by PMA. Taken together, these observations provide evidence that p21 is involved in integrating the PKC signaling pathway to the cell cycle machinery at the G2/M cell cycle checkpoint.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Ciclinas/metabolismo , Fase G2/fisiología , Mitosis/fisiología , Proteína Quinasa C/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Afidicolina/farmacología , Proteína Quinasa CDC2/antagonistas & inhibidores , Ciclina B/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Citometría de Flujo , Fase G2/efectos de los fármacos , Humanos , Indoles/farmacología , Maleimidas/farmacología , Mitosis/efectos de los fármacos , Fosforilación , Pruebas de Precipitina , Proteína Quinasa C/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Acetato de Tetradecanoilforbol/antagonistas & inhibidores , Células Tumorales Cultivadas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
We have demonstrated over-expression of the cyclin-dependent kinase inhibitor p21 in various ovarian-cancer cell lines as well as in ovarian-tumor biopsies. This increase in p21 expression relative to that observed in normal ovarian epithelial cells is unrelated to proliferation index. In the present study, we found that p21 is functional, since the protein extracted from IGROVI cells is still able to inhibit cdk2-kinase activity. We then investigated how IGROVI cells overcome the growth-inhibitory function of p21. Immunofluorescence assays and subcellular fractionation showed that p21 is located in cytoplasm and nucleus both in normal and in tumoral cells. Compared with normal ovarian epithelial cells in culture, the increase in level of p21 in IGROVI cells was found to be associated with increased expression of cdk2, cyclin-A and PCNA proteins. In IGROVI cells, p21 is associated with inactive cdk2/cyclin-A complex, indicating that it acts as an inhibitory factor rather than an assembly factor. Over-expression of cdk2 and of cyclin A observed in IGROVI cells allows them to escape to p21-inhibitory activity. The fact that cells from ovarian-tumor biopsies exhibited a concomitant increase in p21 and in its partners cdk2 and PCNA suggest that ovarian-tumor cells can tolerate high levels of functional p21 via over-expression of other cell-cycle-regulatory proteins.
Asunto(s)
Quinasas CDC2-CDC28 , Ciclina A/biosíntesis , Quinasas Ciclina-Dependientes/biosíntesis , Ciclinas/biosíntesis , Neoplasias Ováricas/metabolismo , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Proteínas Serina-Treonina Quinasas/biosíntesis , Animales , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Femenino , Humanos , Conejos , Células Tumorales CultivadasRESUMEN
1. Pancreatic bile-salt-dependent lipase has been detected in human plasma where it has the capability to modify normal low- and high-density lipoprotein composition and structure and to reduce the atherogenicity of oxidized low-density lipoprotein (Shamir R, Johnson WJ, Morlock-Fitzpatrick K, Zolfaghari R, Li L, Mas E, Lombardo D, Morel DW, Fisher EA. Pancreatic carboxyl ester lipase: a circulating enzyme that modifies normal and oxidized lipoproteins in vitro. J Clin Invest 1996; 97: 1696-704). 2. In the present study, we investigated the effect of glycation and particularly that of human serum albumin on the activity of bile-salt-dependent lipase. In vitro, bile-salt-dependent lipase activity decreased in the presence of human serum albumin; however, this was less pronounced in the presence of glycated human serum albumin. In vivo, bile-salt-dependent lipase specific activity was about 2-fold higher in the sera of diabetic patients than in the sera of normal subjects. 3. A significant increase in the specific activity of bile-salt-dependent lipase related to the serum level of glycation was observed. The increase in bile-salt-dependent lipase specific activity was not related to the glucose concentration in serum suggesting that glycation of bile-salt-dependent lipase could not be involved in the observed effects. Although the stability of serum bile-salt-dependent lipase was important enough to allow a systemic action of the enzyme on lipoproteins, it could not explain the higher activity of the enzyme in diabetic serum. 4. We concluded that bile-salt-dependent lipase could be helpful against the premature development of atherosclerosis in diabetes.
Asunto(s)
Diabetes Mellitus Tipo 1/enzimología , Lipasa/metabolismo , Albúmina Sérica/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Ácidos y Sales Biliares/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Ácidos Grasos/metabolismo , Femenino , Fructosamina/sangre , Glicosilación , Humanos , Lipasa/sangre , Masculino , Persona de Mediana Edad , Pruebas de PrecipitinaRESUMEN
Bile salt-dependent lipase (BSDL, E.C. 3.1.1.-) is a digestive enzyme secreted by the pancreatic acinar cell. Once in the duodenum, the enzyme, upon activation by primary bile salts, hydrolyzes dietary lipid esters such as cholesteryl esters and lipid-soluble vitamin esters. This enzyme is partially transferred from the duodenum or pancreas to the circulation where it has been postulated to exert a systemic action on atheroma-generating oxidized-low density lipoprotein (LDL). In the present study, sera from 40 healthy normolipidemic volunteers were used to investigate the possible linkage between circulating BSDL, lipids, and lipoproteins. We showed, firstly, that pancreatic-like BSDL activity can be detected in these serums. Secondly, BSDL activity increased significantly with the level of LDL-cholesterol and was also positively linked to the serum concentration of Apo B100 and Apo A-I. Thirdly, we also established that BSDL was associated with LDL, in part by a specific interaction with Apo B100, while no interaction was found with Apo A-I. No linkage with other recorded parameters (triglycerides, phospholipids, and high density lipoprotein-cholesterol) was detected. Because an increase in LDL-cholesterol represents an important risk factor for atheroma, the concomitant increase in BSDL, which can metabolize atherogenic LDL, suggests for the first time that this circulating enzyme may exert a positive effect against atherosclerosis.
Asunto(s)
Ácidos y Sales Biliares/farmacología , Lípidos/sangre , Páncreas/enzimología , Esterol Esterasa/sangre , Adulto , Anciano , Anciano de 80 o más Años , Apolipoproteína A-I/metabolismo , Apolipoproteína B-100 , Apolipoproteínas B/sangre , LDL-Colesterol/sangre , Duodeno/enzimología , Femenino , Humanos , Lipoproteínas LDL/sangre , Masculino , Persona de Mediana EdadRESUMEN
All-trans retinoic acid (ATRA) has been previously shown to inhibit the proliferation of some human ovarian carcinoma cell lines, and this inhibition was accompanied by cellular changes that were indicative of differentiation (Caliaro et al, 1994). In this work, a pretreatment of these adenocarcinoma cells with ATRA, for their respective doubling time, enhanced cisplatin (CDDP) cytotoxicity in the cell ines that were sensitive to its antiproliferative effect, but not in the ATRA-resistant ones. Results were assessed using median effect analysis in two ATRA-sensitive cell lines (OVCCR1 and NIHOVCAR3 cells) and in one ATRA-insensitive cell line (IGROV1 cells). Synergy between these two agents was observed only in cells sensitive to ATRA, regardless of their relative sensitivity to CDDP. Potential mechanisms for this synergy were investigated. ATRA did not increase the cellular platinum content, did not decrease the cellular glutathione and had no influence on the metallothionein IIA mRNA levels in NIHOVCAR3 cells. Moreover, the protein kinase C (PKC) activity was modulated by this differentiating agent in all cell lines tested, indicating that this activity was not directly involved in this potentiation. However, an ATRA inhibition of glutathione-S-transferase activity associated with an increase in the total DNA adducts formation could explain the potentiation of the CDDP cytotoxicity observed in NIHOVCAR3 cells. Finally, the ATRA modulation of the epidermal growth factor (EGF) receptor mRNA level could also be implicated in this synergy.
Asunto(s)
Antineoplásicos/toxicidad , Cisplatino/toxicidad , Receptores ErbB/biosíntesis , Metalotioneína/biosíntesis , Tretinoina/toxicidad , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacocinética , Aductos de ADN/metabolismo , Cartilla de ADN , Sinergismo Farmacológico , Femenino , Glutatión/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Cinética , Neoplasias Ováricas , Platino (Metal)/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas , Ensayo de Tumor de Célula MadreRESUMEN
It has been shown recently that expression of p21 is enhanced by paclitaxel. This cytotoxic compound induces mitotic spindle damage resulting in blockade of the mitotic cell cycle associated or not with apoptotic cell death. In the present study, we showed that, in MCF-7 cells, paclitaxel induced accumulation of p21 in cells with a G2/M DNA content, corresponding to cells either in abnormal mitosis or in an interphase-like state (decondensed chromatin) with multiple nuclei. In MCF-7 cells, the increase in p21 was subsequent to the mitotic arrest and was associated with the exit from abnormal mitosis leading to formation of cells with micronuclei. In this cell line, we noted a relationship between the elevation of p21 expression and the inhibition of p34cdc2 activity. High levels of p21 protein were also found to be associated with inactive p34cdc2/cyclin B protein complex after treatment with paclitaxel. Treatment with p21 antisense oligonucleotide partially blocked induction of p21 expression by paclitaxel and significantly reduced survival of MCF-7 cells exposed to this agent. In NIH-OVCAR-3 cells, which are deficient in basal and paclitaxel-induced p21 expression, paclitaxel led to a prolonged activation of p34cdc2 and a delayed mitotic exit associated with apoptotic cell death. These observations suggest that p21 is not required for the mitotic arrest in response to paclitaxel, but argue in favor of a role for this inhibitor in facilitating the exit from abnormal mitosis. This effectively enhances cell survival after paclitaxel-induced spindle damage.