RÉSUMÉ
Zac1, a new zinc-finger protein that regulates both apoptosis and cell cycle arrest, is abundantly expressed in many proliferative/differentiation areas during brain development. In the present work, we studied Zac1 gene expression and protein in experimental seizure models following i.p. injection of pentylenetetrazole (PTZ) or kainic acid (KA). Following KA treatment, an early and intense up-regulation of Zac1 is detected in the limbic areas, such as the hippocampus, cortex and amygdaloid and hypothalamic nuclei. Pre-treatment with MK-801, an antagonist of the NMDA receptors, fully blocks the effect of KA in the hippocampus, whereas it only attenuates KA-induced Zac1 up-regulation in the other areas of the limbic system. A reduced induction is obtained with PTZ-treated animals, specifically in the entorhinal and piriform cortices as well as in amygdaloid and hypothalamic nuclei. Thus, Zac1 is highly induced in the seizure models that generate strong neuronal stimulation and/or extensive cell damage (cell death), reinforcing its putative role in the control of the cell cycle and/or apoptosis. Moreover, strong induction is observed in the granular cells of the dentate gyrus (which are resistant to neurodegeneration) and in some glial cells of the dentate gyrus and subventricular zone, suggesting that Zac1 may be implicated in the mechanisms of neural plasticity following injury.
Sujet(s)
Protéines du cycle cellulaire/métabolisme , Système limbique/métabolisme , Neurones/métabolisme , Crises épileptiques/physiopathologie , Facteurs de transcription/métabolisme , Animaux , Apoptose , Protéines du cycle cellulaire/génétique , Convulsivants/pharmacologie , Maléate de dizocilpine/pharmacologie , Synergie des médicaments , Antagonistes des acides aminés excitateurs/pharmacologie , Expression des gènes/effets des médicaments et des substances chimiques , Gènes suppresseurs de tumeur , Protéine gliofibrillaire acide/métabolisme , Immunohistochimie , Acide kaïnique/pharmacologie , Système limbique/anatomopathologie , Mâle , Souris , Souris de lignée ICR , Pentétrazol/pharmacologie , Protéines proto-oncogènes c-fos/métabolisme , ARN messager/métabolisme , Crises épileptiques/anatomopathologie , Distribution tissulaire , Facteurs de transcription/génétique , Doigts de zincRÉSUMÉ
Bid plays an essential role in Fas-mediated apoptosis of the so-called type II cells. In these cells, following cleavage by caspase 8, the C-terminal fragment of Bid translocates to mitochondria and triggers the release of apoptogenic factors, thereby inducing cell death. Here we report that Bid is phosphorylated by casein kinase I (CKI) and casein kinase II (CKII). Inhibition of CKI and CKII accelerated Fas-mediated apoptosis and Bid cleavage, whereas hyperactivity of the kinases delayed apoptosis. When phosphorylated, Bid was insensitive to caspase 8 cleavage in vitro. Moreover, a mutant of Bid that cannot be phosphorylated was found to be more toxic than wild-type Bid. Together, these data indicate that phosphorylation of Bid represents a new mechanism whereby cells control apoptosis.
Sujet(s)
Apoptose/physiologie , Protéines de transport/métabolisme , Caspases/métabolisme , Protein kinases/métabolisme , Protein-Serine-Threonine Kinases/métabolisme , Séquence d'acides aminés , Animaux , Protéine Bid , Protéines de transport/génétique , Casein Kinase II , Casein Kinases , Caspase 8 , Caspase-9 , Fractionnement cellulaire , Lignée cellulaire , Protéines de liaison à l'ADN/métabolisme , Granzymes , Humains , Immunotransfert , Souris , Données de séquences moléculaires , Phosphorylation , Inhibiteurs de protéines kinases , Protein kinases/isolement et purification , Protein-Serine-Threonine Kinases/antagonistes et inhibiteurs , Structure tertiaire des protéines , Protéines proto-oncogènes c-bcl-2/métabolisme , Protéines recombinantes/génétique , Protéines recombinantes/métabolisme , Serine endopeptidases/métabolisme , Antigènes CD95/métabolismeRÉSUMÉ
The circadian clock located in the suprachiasmatic nucleus (SCN) organizes autonomic and behavioral rhythms into a near 24 hr time that is adjusted daily to the solar cycle via a direct projection from the retina, the retinohypothalamic tract (RHT). This neuronal pathway costores the neurotransmitters PACAP and glutamate, which seem to be important for light-induced resetting of the clock. At the molecular level the clock genes mPer1 and mPer2 are believed to be target for the light signaling to the clock. In this study, we investigated the possible role of PACAP-type 1 receptor signaling in light-induced resetting of the behavioral rhythm and light-induced clock gene expression in the SCN. Light stimulation at early night resulted in larger phase delays in PACAP-type 1 receptor-deficient mice (PAC1(-)/-) compared with wild-type mice accompanied by a marked reduction in light-induced mPer1, mPer2, and c-fos gene expression. Light stimulation at late night induced mPer1 and c-fos gene expression in the SCN to the same levels in both wild type and PAC1(-)/- mice. However, in contrast to the phase advance seen in wild-type mice, PAC1(-)/- mice responded with phase delays after photic stimulation. These data indicate that PAC1 receptor signaling participates in the gating control of photic sensitivity of the clock and suggest that mPer1, mPer2, and c-fos are of less importance for light-induced phase shifts at night.
Sujet(s)
Rythme circadien/physiologie , Régulation de l'expression des gènes/physiologie , Protéines nucléaires/métabolisme , Récepteur hormone hypophysaire/déficit , Cycles d'activité/physiologie , Cycles d'activité/effets des radiations , Animaux , Protéines du cycle cellulaire , Rythme circadien/effets des radiations , Croisements génétiques , Obscurité , Régulation de l'expression des gènes/effets des radiations , Immunohistochimie , Lumière , Mâle , Souris , Lignées consanguines de souris , Souris knockout , Activité motrice/génétique , Activité motrice/effets des radiations , Neuropeptides/métabolisme , Protéines nucléaires/génétique , Protéines circadiennes Period , Stimulation lumineuse , Polypeptide activateur de l'adénylcyclase hypophysaire , Protéines proto-oncogènes c-fos/métabolisme , ARN messager/biosynthèse , Récepteurs au polypeptide activateur de l'adénylcyclase hypophysaire , Récepteurs au polypeptide activateur de l'adénylcyclase hypophysaire , Récepteur hormone hypophysaire/génétique , Transduction du signal/physiologie , Transduction du signal/effets des radiations , Noyau suprachiasmatique/cytologie , Noyau suprachiasmatique/métabolisme , Facteurs de transcriptionRÉSUMÉ
ZAC encodes a zinc finger protein with antiproliferative activity, is maternally imprinted and is a candidate for the tumor suppressor gene on 6q24. ZAC expression is frequently lost in breast and ovary tumor-derived cell lines and down-regulated in breast primary tumors. In this report, we describe ZACDelta2, an alternatively spliced variant of ZAC lacking the sequence encoding the two N-terminal zinc fingers. Messenger RNAs encoding ZAC or ZACDelta2 were equally abundant and both proteins were nuclear. ZACDelta2 displayed an improved transactivation activity and an enhanced affinity for a ZAC binding site, suggesting that the two N-terminal zinc fingers negatively regulated ZAC binding to its target DNA sequences. Both proteins were equally efficient in preventing colony formation, indicating similar overall antiproliferative activities. However, these activities resulted from a differential regulation of apoptosis vs cell cycle progression since ZACDelta2 was more efficient at induction of cell cycle arrest than ZAC, whereas it was the reverse for apoptosis induction. Hence, these data further underline that ZAC gene is critically controlled, both at the transcriptional level through imprinting and at the functional level through alternative splicing.
Sujet(s)
Épissage alternatif , Protéines du cycle cellulaire/métabolisme , Gènes suppresseurs de tumeur/physiologie , Transactivateurs/métabolisme , Doigts de zinc/physiologie , Épissage alternatif/physiologie , Apoptose/effets des médicaments et des substances chimiques , Séquence nucléotidique , Technique de Western , Tumeurs du sein/métabolisme , Protéines du cycle cellulaire/génétique , Chloramphenicol O-acetyltransferase/métabolisme , Amorces ADN/composition chimique , Protéines de liaison à l'ADN/génétique , Protéines de liaison à l'ADN/métabolisme , Protéines de liaison à l'ADN/physiologie , Électrophorèse sur gel d'agar , Cellules épithéliales/métabolisme , Femelle , Cytométrie en flux , Séquence riche en GC/génétique , Délétion de gène , Humains , Techniques immunoenzymatiques , Rein/métabolisme , Données de séquences moléculaires , Réaction de polymérisation en chaîne , ARN messager/métabolisme , Transactivateurs/génétique , Facteurs de transcription/génétique , Facteurs de transcription/métabolisme , Facteurs de transcription/physiologie , Protéines suppresseurs de tumeursRÉSUMÉ
ZAC is a recently isolated zinc finger protein that induces apoptosis and cell cycle arrest. The corresponding gene is imprinted maternally through an unknown mechanism and maps to 6q24-q25, within the minimal interval harboring the gene responsible for transient neonatal diabetes mellitus (TNDM) and a tumor suppressor gene involved in breast cancer. Because of its functional properties, imprinting status, and expression pattern in mammary cell lines and tumors, ZAC is the best candidate so far for both disease conditions. In the present work, we delineated ZAC genomic organization and mapped its transcriptional start site. It is noteworthy that the ZAC promoter localized to the CpG island harboring the methylation imprint associated with TNDM and methylation of this promoter silenced its activity. These data indicate that the methylation mark may have a direct effect on the silencing of the ZAC imprinted allele. Our findings further strengthen the hypothesis that ZAC is the gene responsible for TNDM and suggest a novel mechanism for ZAC inactivation in breast tumors.
Sujet(s)
Protéines du cycle cellulaire/génétique , Méthylation de l'ADN , Diabète/génétique , Gènes suppresseurs de tumeur , Régions promotrices (génétique) , Transactivateurs/génétique , Facteurs de transcription , Allèles , Division cellulaire , Cartographie chromosomique , Chromosomes humains de la paire 6 , Clonage moléculaire , Ilots CpG , Exons , Extinction de l'expression des gènes , Empreinte génomique , Humains , Nouveau-né , Introns , Modèles génétiques , RT-PCR , Distribution tissulaire , Transcription génétique , Transfection , Cellules cancéreuses en culture , Protéines suppresseurs de tumeursRÉSUMÉ
In situ hybridization (ISH) is widely used to detect DNA and RNA sequences within the cell and tissue sections. The important step in performing this technique is tissue fixation. We investigated the influence of the pH of the fixative on the outcome of ISH. Our studies indicate that alkaline formaldehyde dramatically increases the ISH signal with RNA probes. The increase in signal was observed for detection of low as well as for high abundance messages. The sensitivity of the method was increased 5- to 6-fold.
Sujet(s)
Fixateurs , Formaldéhyde , Gènes suppresseurs de tumeur , Hybridation in situ/méthodes , Facteurs de transcription , Protéines du cycle cellulaire/génétique , Protéines du cycle cellulaire/métabolisme , Humains , Concentration en ions d'hydrogène , Sondes d'ARN , ARN messager/métabolisme , Transactivateurs/génétique , Transactivateurs/métabolisme , Protéines suppresseurs de tumeursRÉSUMÉ
The ZAC gene encodes a new zinc-finger protein that concomitantly induces apoptosis and cell cycle arrest and localizes to chromosome 6q24-q25, a well-known hot spot related to cancer. ZAC is highly expressed in the anterior pituitary gland, and its ablation by antisense targeting promotes pituitary cell proliferation. Here we investigate ZAC status in pituitary tumors to evaluate its role in pituitary tumorigenesis. Interest ingly, a strong reduction or absence of ZAC mRNA and protein expres sion was detected in nonfunctioning pituitary adenomas, whereas in clin ically active pituitary neoplasias, the decrease in ZAC expression was variable. Loss of expression was not associated with a mutation of the ZAC gene. Our observations suggest that alternative mechanisms of gene inactivation and/or altered regulation of the ZAC gene occur in nonfunctioning pituitary adenomas.
Sujet(s)
Adénomes/métabolisme , Protéines du cycle cellulaire/biosynthèse , Protéines du cycle cellulaire/génétique , Gènes suppresseurs de tumeur , Tumeurs de l'hypophyse/métabolisme , Transactivateurs/biosynthèse , Transactivateurs/génétique , Facteurs de transcription , Technique de Western , Division cellulaire , ADN complémentaire/métabolisme , Récepteurs ErbB/biosynthèse , Humains , Immunohistochimie , Hybridation in situ , Mutation , Oligonucléotides antisens/métabolisme , Hypophyse/métabolisme , ARN messager/métabolisme , RT-PCR , Protéines suppresseurs de tumeurs , Doigts de zincRÉSUMÉ
Loss of chromosome 6q21-qter is the second most frequent loss of chromosomal material in sporadic breast neoplasms suggesting the presence of at least one tumor suppressor gene on 6q. We recently isolated a cDNA encoding a new zinc finger protein which we named ZAC according to its functional properties, namely induction of apoptosis and control of cell cycle progression. ZAC is expressed in normal mammary gland and maps to 6q24-q25, a recognized breast cancer hot spot on 6q. In the present report, we investigated the possible inactivation of ZAC in breast cancer cell lines and primary tumors. We detected no mutation in ZAC coding region in a panel of 45 breast tumors with allelic imbalance of 6q24-q25. However, a survey of eight breast cancer cell lines showed a deeply reduced (three cell lines) or complete loss of (five cell lines) ZAC expression. Treatment of three of these cell lines with the methylation-interfering agent 5-azacytidine induced ZAC re-expression. In addition, Northern blot and RNase protection assay analysis of ZAC expression in 23 unselected primary breast tumors showed a reduced expression in several samples. Together with its functional properties and chromosomal localization, these findings substantiate ZAC as a good candidate for the tumor suppressor gene on 6q24-q25.
Sujet(s)
Tumeurs du sein/génétique , Protéines du cycle cellulaire/génétique , Délétion de gène , Régulation de l'expression des gènes , Gènes suppresseurs de tumeur , Transactivateurs/génétique , Facteurs de transcription , Région mammaire/métabolisme , Protéines du cycle cellulaire/biosynthèse , Protéines du cycle cellulaire/métabolisme , Méthylation de l'ADN , Régulation négative/génétique , Cellules épithéliales/métabolisme , Humains , Perte d'hétérozygotie , Mutation , Transactivateurs/biosynthèse , Transactivateurs/métabolisme , Cellules cancéreuses en culture , Protéines suppresseurs de tumeurs , Doigts de zinc/génétiqueRÉSUMÉ
Pituitary adenylate cyclase-activating polypeptides and PAC1-R are expressed during early embryogenesis and PACAP's neurotrophic action supports a role in neuronal development. In the adult brain PACAP functions as a neuroprotective factor that attenuates the neuronal damage resulting from various insults. The tumor suppressor gene p53 and the new zinc finger protein Zac regulate apoptosis and cell cycle arrest through unrelated pathways and both genes are up-regulated under cerebral ischemia. We report here that p53 and Zac induce expression of the PAC1-R gene. By this mechanism p53 and Zac could fine-tune the balance between death promoting and protective signals and may thus fulfil a dual role in ischemia.
Sujet(s)
Gènes p53 , Protéines de tissu nerveux/génétique , Récepteur hormone hypophysaire/génétique , Doigts de zinc , Animaux , Apoptose/physiologie , Encéphalopathie ischémique/métabolisme , Cycle cellulaire/physiologie , Différenciation cellulaire/physiologie , Humains , Récepteurs au polypeptide activateur de l'adénylcyclase hypophysaire , Récepteurs au polypeptide activateur de l'adénylcyclase hypophysaireRÉSUMÉ
Zac1 is a new zinc finger protein that concomitantly controls apoptosis and cell cycle arrest through separate pathways. The mouse Zac1 gene is mainly expressed in the pituitary gland and in different brain areas. In this study regional and cellular expression of Zac1 in the pituitary gland was determined by in situ hybridization. Zac1 messenger RNA was abundantly expressed in the anterior pituitary lobe compared with that in the intermediate and posterior lobes. Zac1 transcripts were found in all hormone-secreting cell types, with the highest levels in GH- and PRL-producing cells. To investigate the impact of Zac1 in pituitary cell proliferation, we ablated the endogenous Zac1 gene by antisense treatment in two murine cell types, AtT-20 and TtT/GF, that are representative of granular and agranular cell lineages, respectively. The decline in Zac1 protein levels under antisense treatment was accompanied by increased DNA synthesis in clonal corticotroph and folliculo-stellate cells, as demonstrated by enhanced [3H]thymidine incorporation (36% and 50%, respectively). Antisense oligonucleotides against Zac1 controlled cell proliferation in a dose-dependent way, and mutagenized antisense oligonucleotides were inert. Conclusively, our data provide the first evidence of a role for Zac1 in pituitary growth control.
Sujet(s)
Protéines du cycle cellulaire/antagonistes et inhibiteurs , Protéines du cycle cellulaire/génétique , Expression des gènes/physiologie , Gènes suppresseurs de tumeur , Adénohypophyse/physiologie , Hypophyse/cytologie , Transactivateurs/antagonistes et inhibiteurs , Transactivateurs/génétique , Facteurs de transcription , Animaux , Division cellulaire/physiologie , Lignée cellulaire , Cellules cultivées , Immunohistochimie , Hybridation in situ , Mâle , Souris , Lignées consanguines de souris , Oligonucléotides antisens/pharmacologie , Hypophyse/métabolisme , ARN messager/métabolisme , Distribution tissulaire , Transfection/physiologieRÉSUMÉ
We previously reported the identification of mZac, a novel mouse zinc finger protein that shared with p53 the ability to regulate concomitantly apoptosis and cell cycle progression. We describe here the isolation, chromosomal localization, and functional in vitro characterization of its human homolog. hZAC is a widely expressed zinc finger protein that reveals transactivation and DNA-binding activity. hZAC inhibits tumor cell growth through induction of apoptotic cell death and G1 arrest. Thus hZAC, like its mouse counterpart, displays antiproliferative properties through pathways known to be central to the activity of p53. We mapped hZAC on chromosome 6q24-q25, a region frequently deleted in many solid tumors. Indeed, allelic loss at 6q24-q25 has been shown in breast and ovary cancers, melanomas, astrocytomas, and renal cell carcinomas. Furthermore, Abdollahi et al. [Abdollahi, A., Godwin, A. K., Miller, P. D., Getts, L. A., Schultz, D. C., Tagushi, T., Testa, J. R. & Hamilton, T. C. (1997) Cancer Res. 57, 2029-2034] recently isolated ZAC through its loss of expression in a surface epithelial ovary tumor model and accordingly named it Lot for "lost on transformation." In view of these observations, the functional properties we report here provide further arguments to consider hZAC as a tumor suppressor gene candidate.
Sujet(s)
Protéines du cycle cellulaire/génétique , Chromosomes humains de la paire 6 , Gènes suppresseurs de tumeur , Tumeurs/génétique , Transactivateurs/génétique , Facteurs de transcription , Doigts de zinc/génétique , Séquence d'acides aminés , Animaux , Apoptose/génétique , Technique de Southern , Protéines du cycle cellulaire/composition chimique , Protéines du cycle cellulaire/métabolisme , Cartographie chromosomique , Protéines de liaison à l'ADN/génétique , Protéines de liaison à l'ADN/métabolisme , Humains , Souris , Données de séquences moléculaires , Tumeurs/anatomopathologie , Similitude de séquences d'acides aminés , Transactivateurs/composition chimique , Transactivateurs/métabolisme , Activation de la transcription , Cellules cancéreuses en culture , Protéines suppresseurs de tumeursRÉSUMÉ
We reported recently the cloning of the type I PACAP receptor by a functional expression cloning technique. Unexpectedly, we observed additional PACAP-positive pools that turned out to encode the wild-type form of the tumor suppressor gene p53 and the novel zinc finger protein Zac1, which regulates apoptosis and cell cycle arrest. Both Zac1 and p53 caused, under transient or stably regulated expression, induction of the type I PACAP receptor by transcriptional mechanisms. Transactivation of the type I PACAP receptor gene by Zac1 and p53 points to a subtle balance between death promoting and protective mechanisms. The control of these processes is central to various physiological conditions ranging from development to senescence, whereas dysregulation may lead to overt pathological outcomes, notably cancer, immune deficiency syndromes, and neurodegenerative disorders.
Sujet(s)
Protéines du cycle cellulaire/génétique , Régulation de l'expression des gènes , Gènes suppresseurs de tumeur , Récepteur hormone hypophysaire/génétique , Transactivateurs/génétique , Facteurs de transcription , Protéine p53 suppresseur de tumeur/métabolisme , Animaux , Apoptose , Cycle cellulaire , Clonage moléculaire , Humains , Récepteurs au polypeptide activateur de l'adénylcyclase hypophysaire , Récepteurs au polypeptide activateur de l'adénylcyclase hypophysaire , Récepteur hormone hypophysaire/biosynthèse , Récepteur hormone hypophysaire/physiologie , Protéines recombinantes/biosynthèse , Transcription génétique , Activation de la transcription , Protéines suppresseurs de tumeurs , Doigts de zincRÉSUMÉ
Pituitary adenylate cyclase-activating polypeptides (PACAP-27 and -38) are neuropeptides of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon family. PACAP receptors are expressed in different brain regions including the cerebellum. We used primary culture of rat cerebellar granule neurons to study the effect of PACAP-38 on apoptosis induced by potassium deprivation. We demonstrated that serum and potassium withdrawal induces a mixture of apoptosis and necrosis rather than apoptosis only. We showed that PACAP-38 increased survival of cerebellar neurons in a dose-dependent manner by specifically decreasing the extent of apoptosis estimated by DNA fragmentation. PACAP-38 induced activation of the extracellular signal-regulated kinase (ERK)-type of MAP kinase through a cAMP-dependent pathway. PD98059, an inhibitor of MEK (MAP kinase kinase), completely abolished the anti-apoptotic effect of PACAP-38, suggesting that MAP kinase pathway activation is necessary for PACAP-38 effect.
Sujet(s)
Apoptose/physiologie , Cervelet/cytologie , Cervelet/physiologie , Neurones/effets des médicaments et des substances chimiques , Neuropeptides/pharmacologie , Neuroprotecteurs/pharmacologie , Animaux , Apoptose/effets des médicaments et des substances chimiques , Calcium-Calmodulin-Dependent Protein Kinases/métabolisme , Cellules cultivées , Fragmentation de l'ADN , Antienzymes/pharmacologie , Flavonoïdes/pharmacologie , Mitogen-Activated Protein Kinase Kinases , Nécrose , Neurones/cytologie , Neurones/physiologie , Neuropeptides/physiologie , Polypeptide activateur de l'adénylcyclase hypophysaire , Potassium/pharmacologie , Protein kinases/métabolisme , Rats , Récepteurs au polypeptide activateur de l'adénylcyclase hypophysaire , Récepteur hormone hypophysaire/génétique , Récepteur hormone hypophysaire/physiologieRÉSUMÉ
The proliferation rate of a cell population reflects a balance between cell division, cell cycle arrest, differentiation and apoptosis. The regulation of these processes is central to development and tissue homeostasis, whereas dysregulation may lead to overt pathological outcomes, notably cancer and neurodegenerative disorders. We report here the cloning of a novel zinc finger protein which regulates apoptosis and cell cycle arrest and was accordingly named Zac1. In vitro Zac1 inhibited proliferation of tumor cells, as evidenced by measuring colony formation, growth rate and cloning in soft agar. In vivo Zac1 abrogated tumor formation in nude mice. The antiproliferative activity of Zac1 was due to induction of extensive apoptosis and of G1 arrest, which proceeded independently of retinoblastoma protein and of regulation of p21(WAF1/Cip1), p27Kip1, p57Kip2 and p16INK4a expression. Zac1-mediated apoptosis was unrelated to cell cycle phase and G1 arrest was independent of apoptosis, indicating separate control of apoptosis and cell cycle arrest. Zac1 is thus the first gene besides p53 which concurrently induces apoptosis and cell cycle arrest.
Sujet(s)
Apoptose/génétique , Encéphale/physiologie , Protéines du cycle cellulaire/génétique , Phase G1/génétique , Hypophyse/physiologie , Facteurs de transcription , Doigts de zinc/génétique , Séquence d'acides aminés , Animaux , Protéines du cycle cellulaire/isolement et purification , Protéines du cycle cellulaire/métabolisme , Clonage moléculaire , Régulation de l'expression des gènes tumoraux , Gènes suppresseurs de tumeur , Mâle , Souris , Souris de lignée BALB C , Souris nude , Données de séquences moléculaires , Protéines recombinantes/biosynthèse , Analyse de séquence d'ADN , Distribution tissulaire , Protéine p53 suppresseur de tumeur/métabolismeRÉSUMÉ
The tetracycline-dependent expression system has gained increasing popularity for the expression of any gene of interest. Careful choice of the expression vector has been suggested to exploit the full potential of this system. A novel tetracycline-sensitive expression vector based on a modified mouse mammary tumor virus promoter achieved considerably improved regulatory properties in a series of cell lines tested under transient and stable conditions. Therefore, the applicability of the tetracycline-dependent expression system can be largely enhanced by careful adaptation of the expression vector to the host cell line.
Sujet(s)
Régulation de l'expression des gènes , Vecteurs génétiques , Virus de la tumeur mammaire de la souris/génétique , Tétracycline/pharmacologie , Animaux , Lignée cellulaire , Régulation de l'expression des gènes/effets des médicaments et des substances chimiques , Cellules HeLa , Humains , Cellules LLC-PK1 , Mammifères , Souris , Régions promotrices (génétique) , Suidae , TransfectionRÉSUMÉ
Serum and potassium withdrawal-induced cell death of cerebellar granule cells is one of the most popular models used for studying neuronal apoptosis in vitro, and it is generally assumed that compounds preventing cell death in this model prevent apoptosis. In the present study we demonstrate that serum and potassium withdrawal induces a mixture of apoptosis and necrosis rather than apoptosis only. Each of these death mechanisms could be blocked by activation of different pathways, such as raising cAMP production or stimulation of the IGF-1 receptor. Studies on serum and potassium withdrawal-induced cell death of cerebellar granule cells should therefore carefully assess whether a given compound is preventing apoptosis or necrosis using appropriate techniques.
Sujet(s)
Apoptose/physiologie , Mort cellulaire/physiologie , Cervelet/cytologie , Neurones/cytologie , Potassium/pharmacologie , Androstadiènes/pharmacologie , Animaux , Apoptose/effets des médicaments et des substances chimiques , Mort cellulaire/effets des médicaments et des substances chimiques , Survie cellulaire , Cellules cultivées , Cervelet/effets des médicaments et des substances chimiques , Cervelet/anatomopathologie , 4H-1-Benzopyran-4-ones/pharmacologie , Colforsine/pharmacologie , Milieux de culture , Milieux de culture sans sérum , ADN/analyse , Fragmentation de l'ADN , Antienzymes/pharmacologie , Facteur de croissance IGF-I/pharmacologie , Morpholines/pharmacologie , Nécrose , Neurones/effets des médicaments et des substances chimiques , Neurones/anatomopathologie , Neuropeptides/pharmacologie , Neuroprotecteurs/pharmacologie , Phosphatidylinositol 3-kinases , Phosphotransferases (Alcohol Group Acceptor)/antagonistes et inhibiteurs , Polypeptide activateur de l'adénylcyclase hypophysaire , Rats , Rat Wistar , WortmannineRÉSUMÉ
Pituitary adenylate cyclase-activating polypeptides (PACAP-27 and PACAP-38) are neuropeptides of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon family. PACAP receptors are expressed in different brain regions, including cerebellum. We used primary culture of rat cerebellar granule neurons to study the effect of PACAP-38 on apoptosis induced by potassium deprivation. We demonstrated that PACAP-38 increased survival of cerebellar neurons in a dose-dependent manner by decreasing the extent of apoptosis estimated by DNA fragmentation. PACAP-38 induced activation of the extracellular signal-regulated kinase (ERK)-type of mitogen-activated protein (MAP) kinase through a cAMP-dependent pathway. PD98059, an inhibitor of MEK (MAP kinase kinase), completely abolished the antiapoptotic effect of PACAP-38, suggesting that MAP kinase pathway activation is necessary for PACAP-38 action.
Sujet(s)
Apoptose/effets des médicaments et des substances chimiques , Calcium-Calmodulin-Dependent Protein Kinases/métabolisme , Cervelet/effets des médicaments et des substances chimiques , Neurones/effets des médicaments et des substances chimiques , Neuropeptides/pharmacologie , Neuroprotecteurs/pharmacologie , Agents neuromédiateurs/pharmacologie , Animaux , Cellules cultivées , Cervelet/cytologie , Polypeptide activateur de l'adénylcyclase hypophysaire , Rats , Rat Wistar , Récepteurs au polypeptide activateur de l'adénylcyclase hypophysaire , Récepteur hormone hypophysaire/métabolisme , Récepteur peptide intestinal vasoactif/métabolismeRÉSUMÉ
Rat anterior pituitary tissue expresses mRNA for PVR1 and PVR3, as well as a low level of PVR2. The PVR1 appears to be highly expressed in gonadotroph-like cells, while somatotroph-like cells apparently express the PVR3. We have recently demonstrated the expression of mRNA for both PVR2 and PVR3 in corticotroph-like AtT20 cells (FIG.3). If normal corticotrophs express the same mRNA as AtT20 cells, this may partly explain the low levels of PVR2 seen in normal pituitary tissue. Significant levels of at least two PVR1 splice variants mRNAs (PVR1s and PVR1hop) were expressed in clonal gonadotroph-like alpha T3-1 cells and normal rat anterior pituitary tissue. However, these splice variants are reported to have almost identical pharmacological characteristics in terms of binding, and the activation of AC and PLC. Further experiments are necessary to determine the functional consequences of differential splice variant expression in such cells. Interestingly, all three pituitary-cell lines studied expressed mRNA for the PVR3 (FIG.3), whereas earlier binding studies demonstrate a predominance of PACAP-preferring binding sites on normal anterior pituitary-cell membranes. In addition, it is clear that the different PVR subtypes can couple to different intracellular messenger systems. Thus it will be important to determine the expression of the different PVR subtypes in normal anterior pituitary-cell types if we are to begin to understand the regulation of pituitary-cell regulation by PACAP. Such questions form the basis of some of the ongoing studies in our laboratory.