Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 35
Filter
Add more filters










Publication year range
1.
Oncogene ; 36(33): 4682-4691, 2017 08 17.
Article in English | MEDLINE | ID: mdl-28394338

ABSTRACT

High-risk and MYCN-amplified neuroblastomas are among the most aggressive pediatric tumors. Despite intense multimodality therapies, about 50% of these patients succumb to their disease, making the search for effective therapies an absolute priority. Due to the important functions of poly (ADP-ribose) polymerases, PARP inhibitors have entered the clinical settings for cancer treatment and are being exploited in a variety of preclinical studies and clinical trials. PARP inhibitors based combination schemes have also been tested in neuroblastoma preclinical models with encouraging results. However, the expression of PARP enzymes in human neuroblastoma and the biological consequences of their inhibition remained largely unexplored. Here, we show that high PARP1 and PARP2 expression is significantly associated with high-risk neuroblastoma cases and poor survival, highlighting its previously unrecognized prognostic value for human neuroblastoma. In vitro, PARP1 and 2 are abundant in MYCN amplified and MYCN-overexpressing cells. In this context, PARP inhibitors with high 'PARP trapping' potency, such as olaparib or talazoparib, yield DNA damage and cell death preceded by intense signs of replication stress. Notwithstanding the activation of a CHK1-CDC25A replication stress response, PARP-inhibited MYCN amplified and overexpressing cells fail to sustain a prolonged checkpoint and progress through mitosis in the presence of damaged DNA, eventually undergoing mitotic catastrophe. CHK1-targeted inhibition of the replication stress checkpoint exacerbated this phenotype. These data highlight a novel route for cell death induction by PARP inhibitors and support their introduction, together with CHK1 inhibitors, in therapeutic approaches for neuroblastomas with high MYC(N) activity.


Subject(s)
DNA Replication/drug effects , Mitosis/drug effects , N-Myc Proto-Oncogene Protein/metabolism , Neuroblastoma/drug therapy , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Poly(ADP-ribose) Polymerases/metabolism , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Checkpoint Kinase 1/metabolism , Child , Humans , Kaplan-Meier Estimate , N-Myc Proto-Oncogene Protein/genetics , Poly (ADP-Ribose) Polymerase-1/genetics , Poly(ADP-ribose) Polymerase Inhibitors/administration & dosage , Poly(ADP-ribose) Polymerases/genetics
2.
Oncogene ; 32(38): 4572-8, 2013 Sep 19.
Article in English | MEDLINE | ID: mdl-23108393

ABSTRACT

The serum- and glucocorticoid-regulated kinase (Sgk1) is essential for hormonal regulation of epithelial sodium channel-mediated sodium transport and is involved in the transduction of growth factor-dependent cell survival and proliferation signals. Growing evidence now points to Sgk1 as a key element in the development and/or progression of human cancer. To gain insight into the mechanisms through which Sgk1 regulates cell proliferation, we adopted a proteomic approach to identify up- or downregulated proteins after Sgk1-specific RNA silencing. Among several proteins, the abundance of which was found to be up- or downregulated upon Sgk1 silencing, we focused our attention of RAN-binding protein 1 (RANBP1), a major effector of the GTPase RAN. We report that Sgk1-dependent regulation of RANBP1 has functional consequences on both mitotic microtubule activity and taxol sensitivity of cancer cells.


Subject(s)
Carcinoma/genetics , Carcinoma/metabolism , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Drug Resistance, Neoplasm/genetics , Immediate-Early Proteins/metabolism , Nuclear Proteins/genetics , Protein Serine-Threonine Kinases/metabolism , Transcription, Genetic , Apoptosis/drug effects , Apoptosis/genetics , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Humans , Paclitaxel/pharmacology , Phosphorylation , Proteomics , RNA Interference , Sp1 Transcription Factor/metabolism
3.
Oncogene ; 28(15): 1748-58, 2009 Apr 16.
Article in English | MEDLINE | ID: mdl-19270727

ABSTRACT

Mitotic microtubule (MT)-targeting drugs are widely used to treat cancer. The GTPase Ran regulates multiple processes, including mitotic spindle assembly, spindle pole formation and MT dynamics; Ran activity is therefore essential to formation of a functional mitotic apparatus. The RanBP1 protein, which binds Ran and regulates its interaction with effectors, is overexpressed in many cancer types. Several observations indicate that RanBP1 contributes to regulate the function of the mitotic apparatus: RanBP1 inactivation yields hyperstable MTs and induces apoptosis during mitosis, reminiscent of the effects of the MT-stabilizing drug taxol. Here we have investigated the influence of RanBP1 on spontaneous and taxol-induced apoptosis in transformed cells. We report that RanBP1 downregulation by RNA interference activates apoptosis in several transformed cell lines regardless of their p53 status, but not in the caspase-3-defective MCF-7 breast cancer cell line. Furthermore, RanBP1-interfered cells show an increased apoptotic response to taxol compared to their counterpart with normal or high RanBP1 levels, and this response is caspase-3 dependent. These results indicate that RanBP1 can modulate the outcome of MT-targeting therapeutic protocols.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Caspase 3/physiology , Nuclear Proteins/physiology , Paclitaxel/pharmacology , Apoptosis , Cell Line, Tumor , Down-Regulation , HeLa Cells , Humans , Microtubules/drug effects , Nuclear Proteins/antagonists & inhibitors , Tumor Suppressor Protein p53/physiology
4.
Oncogene ; 27(51): 6539-49, 2008 Nov 20.
Article in English | MEDLINE | ID: mdl-18663358

ABSTRACT

Mitotic spindle assembly is a highly regulated process, crucial to ensure the correct segregation of duplicated chromosomes in daughter cells and to avoid aneuploidy, a common feature of tumors. Among the most important spindle regulators is Aurora-A, a mitotic centrosomal kinase frequently overexpressed in tumors. Here, we investigated the role of Aurora-A in spindle pole organization in human cells. We show that RNA interference-mediated Aurora-A inactivation causes pericentriolar material fragmentation in prometaphase, yielding the formation of spindles with supernumerary poles. This fragmentation does not necessarily involve centrioles and requires microtubules (MTs). Aurora-A-depleted prometaphases mislocalize the MT-stabilizing protein colonic hepatic tumor-overexpressed gene (ch-TOG), which abnormally accumulates at spindle poles, as well as the mitotic centromere-associated kinesin (MCAK), the major functional antagonist of ch-TOG, which delocalizes from poles. ch-TOG is required for extrapole formation in prometaphases lacking Aurora-A, because co-depletion of Aurora-A and ch-TOG mitigates the fragmented pole phenotype. These results indicate a novel function of Aurora-A, the regulation of ch-TOG and MCAK localization, and highlight a common pathway involving the three factors in control of spindle pole integrity.


Subject(s)
Microtubule-Associated Proteins/physiology , Protein Serine-Threonine Kinases/physiology , Spindle Apparatus/metabolism , Aurora Kinases , Humans , Kinesins/metabolism , Metaphase/drug effects , Microtubule-Associated Proteins/antagonists & inhibitors , Mitosis/drug effects , Models, Biological , Protein Binding , Protein Serine-Threonine Kinases/antagonists & inhibitors , RNA, Small Interfering/pharmacology , Signal Transduction/drug effects , Signal Transduction/physiology , Spindle Apparatus/drug effects , Spindle Apparatus/physiology , Tissue Distribution , Tumor Cells, Cultured
5.
Cell Mol Life Sci ; 64(15): 1891-914, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17483873

ABSTRACT

Mitosis is the most potentially dangerous event in the life of a cell, during which the cell genetic identity is transmitted to daughters; errors at this stage may yield aneuploid cells that can initiate a genetically unstable clone. The small GTPase Ran is the central element of a conserved signaling network that has a prominent role in mitotic regulation. Pioneering studies with amphibian oocytes indicated that Ran, in the GTP-bound form, activates factors that regulate spindle assembly and dynamics. An increasing body of data indicate higher specificity and complexity in mitotic control operated by Ran in somatic cells. Newly identified target factors of Ran operate with different specificity, and it is emerging that mitotic progression requires the precise positioning of Ran network components and effectors at specific sites of the mitotic apparatus according to a highly regulated schedule in space and time. In this review we summarize our current understanding of Ran control of mitosis and highlight the specificity of mechanisms operating in mammalian somatic cells.


Subject(s)
Mitosis/physiology , ran GTP-Binding Protein/physiology , Animals , Biological Transport, Active , Cell Cycle/physiology , Centrosome/physiology , Humans , Interphase/physiology , Microtubules/physiology , Models, Biological , Spindle Apparatus/physiology
6.
Mol Biol Cell ; 15(8): 3751-7, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15181149

ABSTRACT

We recently demonstrated that the p53 oncosuppressor associates to centrosomes in mitosis and this association is disrupted by treatments with microtubule-depolymerizing agents. Here, we show that ATM, an upstream activator of p53 after DNA damage, is essential for p53 centrosomal localization and is required for the activation of the postmitotic checkpoint after spindle disruption. In mitosis, p53 failed to associate with centrosomes in two ATM-deficient, ataxiatelangiectasia-derived cell lines. Wild-type ATM gene transfer reestablished the centrosomal localization of p53 in these cells. Furthermore, wild-type p53 protein, but not the p53-S15A mutant, not phosphorylatable by ATM, localized at centrosomes when expressed in p53-null K562 cells. Finally, Ser15 phosphorylation of endogenous p53 was detected at centrosomes upon treatment with phosphatase inhibitors, suggesting that a p53 dephosphorylation step at centrosome contributes to sustain the cell cycle program in cells with normal mitotic spindles. When dissociated from centrosomes by treatments with spindle inhibitors, p53 remained phosphorylated at Ser15. AT cells, which are unable to phosphorylate p53, did not undergo postmitotic proliferation arrest after nocodazole block and release. These data demonstrate that ATM is required for p53 localization at centrosome and support the existence of a surveillance mechanism for inhibiting DNA reduplication downstream of the spindle assembly checkpoint


Subject(s)
Centrosome/chemistry , Mitosis , Protein Serine-Threonine Kinases/metabolism , Serine/metabolism , Tumor Suppressor Protein p53/analysis , Tumor Suppressor Protein p53/metabolism , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins , Cell Line , Centrosome/metabolism , DNA-Binding Proteins , Humans , Mutation/genetics , Nocodazole/pharmacology , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/genetics , Serine/genetics , Spindle Apparatus/drug effects , Spindle Apparatus/metabolism , Tubulin/analysis , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Proteins
7.
J Biol Chem ; 276(22): 19205-13, 2001 Jun 01.
Article in English | MEDLINE | ID: mdl-11376010

ABSTRACT

Growing evidence indicates a central role for p53 in mediating cell cycle arrest in response to mitotic spindle defects so as to prevent rereplication in cells in which the mitotic division has failed. Here we report that a transient inhibition of spindle assembly induced by nocodazole, a tubulin-depolymerizing drug, triggers a stable activation of p53, which can transduce a cell cycle inhibitory signal even when the spindle-damaging agent is removed and the spindle is allowed to reassemble. Cells transiently exposed to nocodazole continue to express high levels of p53 and p21 in the cell cycle that follows the transient exposure to nocodazole and become arrested in G(1), regardless of whether they carry a diploid or polyploid genome after mitotic exit. We also show that p53 normally associates with centrosomes in mitotic cells, whereas nocodazole disrupts this association. Together these results suggest that the induction of spindle damage, albeit transient, interferes with the subcellular localization of p53 at specific mitotic locations, which in turn dictates cell cycle arrest in the offspring of such defective mitoses.


Subject(s)
Centrosome/metabolism , Tumor Suppressor Protein p53/biosynthesis , Tumor Suppressor Protein p53/metabolism , Anaphase , Antineoplastic Agents/pharmacology , Blotting, Western , Cell Cycle , Cell Line , Cell Separation , Flow Cytometry , Fluorescent Antibody Technique, Indirect , G1 Phase , Humans , K562 Cells , Metaphase , Microscopy, Fluorescence , Mitosis , Nocodazole/pharmacology , Ploidies , Proto-Oncogene Proteins p21(ras)/metabolism , Time Factors , Transfection , Tubulin/metabolism , Up-Regulation
8.
FEBS Lett ; 487(3): 397-403, 2001 Jan 05.
Article in English | MEDLINE | ID: mdl-11163365

ABSTRACT

Human lymphoblastoid cells of normal origin and from genetic instability syndromes, i.e. Fanconi anemia (FA) group C and ataxia telangectasia, were continuously exposed to extremely low frequency magnetic field (ELF-MF). We report that ELF-MF, though not perturbing cell cycle progression, increases the rate of cell death in normal cell lines. In contrast, cell death is not affected in cells from genetic instability syndromes; this reflects a specific failure of the apoptotic response. Reintroduction of complementation group C in FA cells re-established the apoptotic response to ELF-MF. Thus, genes implicated in genetic instability syndromes are relevant in modulating the response of cells to ELF-MF.


Subject(s)
Cell Death , Magnetics/adverse effects , Apoptosis , Ataxia Telangiectasia/genetics , Ataxia Telangiectasia/pathology , Cell Cycle , Cell Line , Fanconi Anemia/genetics , Fanconi Anemia/pathology , Humans , Lymphocytes/cytology , Microscopy, Electron , Mutation , Transfection
9.
Mech Dev ; 97(1-2): 211-5, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11025228

ABSTRACT

We have examined the murine genes encoding transcription factors E2F1, -3, -5 and -6 in gametes and early embryos. All genes are expressed as maternal transcripts and all are efficiently transcribed after the blastocyst stage. Between those two stages, each E2F mRNA is transcribed with a distinctive and unique pattern. E2F proteins are also differentially expressed and compartmentalized in pre-implantation embryos.


Subject(s)
Carrier Proteins , Cell Cycle Proteins , DNA-Binding Proteins , Germ Cells/metabolism , Transcription Factors/genetics , 3T3 Cells , Animals , E2F Transcription Factors , E2F1 Transcription Factor , E2F3 Transcription Factor , E2F5 Transcription Factor , E2F6 Transcription Factor , Embryonic and Fetal Development , Female , Gene Expression Regulation, Developmental , Male , Mice , Mice, Inbred C57BL , Retinoblastoma-Binding Protein 1 , Transcription Factor DP1
10.
Genomics ; 68(3): 253-63, 2000 Sep 15.
Article in English | MEDLINE | ID: mdl-10995567

ABSTRACT

We identified in the EST database murine and human sequences similar, but not identical, to the members of the PC3/BTG/TOB family of cell cycle inhibitors. A conserved domain (aa 50-68) of the PC3 protein, the prototype member of the family, was used as a query. That domain has been shown by us to be necessary for the antiproliferative activity of PC3. A murine EST clone and a highly homologous human EST clone, containing the entire ORF, were chosen for sequencing. Comparison to databases and a phylogenetic tree analysis indicated that these EST clones are the mouse and human homologues of a gene that represents a novel member of the PC3/BTG/TOB family. This gene, named PC3B, is endowed with marked antiproliferative activity, being able to induce G(1) arrest, and is highly expressed in testis, in oocyte, and in preimplantation embryos. Analysis of its expression during murine development indicated a specific localization in the olfactory epithelium at midgestation, suggesting that PC3B might be involved in the differentiation of this neuronal structure. Human PC3B mapped to chromosome 11q23, as indicated by radiation hybrid analysis.


Subject(s)
Cell Cycle Proteins/genetics , Olfactory Mucosa/metabolism , Amino Acid Sequence , Animals , Aspartic Acid Endopeptidases/chemistry , Aspartic Acid Endopeptidases/genetics , Base Sequence , Cell Cycle/genetics , Cell Cycle Proteins/chemistry , Cloning, Molecular , Conserved Sequence , Evolution, Molecular , Expressed Sequence Tags , Humans , Mice , Molecular Sequence Data , Multigene Family , Olfactory Mucosa/cytology , Open Reading Frames , Phylogeny , Proprotein Convertases , Sequence Alignment , Sequence Homology, Amino Acid
11.
Cell Growth Differ ; 11(8): 455-65, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10965850

ABSTRACT

Ran-binding protein (RanBP) 1 is a major regulator of the Ran GTPase and is encoded by a regulatory target gene of E2F factors. The Ran GTPase network controls several cellular processes, including nucleocytoplasmic transport and cell cycle progression, and has recently also been shown to regulate microtubule nucleation and spindle assembly in Xenopus oocyte extracts. Here we report that RanBP1 protein levels are cell cycle regulated in mammalian cells, increase from S phase to M phase, peak in metaphase, and abruptly decline in late telophase. Overexpression of RanBP1 throughout the cell cycle yields abnormal mitoses characterized by severe defects in spindle polarization. In addition, microinjection of anti-RanBP1 antibody in mitotic cells induces mitotic delay and abnormal nuclear division, reflecting an abnormal stabilization of the mitotic spindle. Thus, regulated RanBP1 activity is required for proper execution of mitosis in somatic cells.


Subject(s)
Nuclear Proteins/physiology , Spindle Apparatus/ultrastructure , ran GTP-Binding Protein/physiology , 3T3 Cells , Animals , Antibodies/immunology , Cell Cycle , Cell Nucleus/ultrastructure , Chromatin/ultrastructure , Fluorescent Antibody Technique, Indirect , Mammals/physiology , Mice , Microinjections , Mitosis , Nuclear Proteins/immunology , Nuclear Proteins/metabolism , Spindle Apparatus/physiology , ran GTP-Binding Protein/immunology , ran GTP-Binding Protein/metabolism
12.
Gene ; 253(2): 161-70, 2000 Aug 08.
Article in English | MEDLINE | ID: mdl-10940553

ABSTRACT

Per genes encode components of the circadian clocks controlling metabolic and behavioural rhythms. The human Per1 cDNA, RIGUI, was previously isolated and mapped on chromosome 17p12 (Sun, Z.S., Albrecht, U., Zhuchenko, O., Bailey, J., Eichele, G., Lee, C.C., 1997. RIGUI, a putative mammalian orthologue of the Drosophila period gene. Cell 90, 1003-1011). We have now isolated the entire genomic locus containing the human Per1 gene, in a search for genes associated with CpG-rich sequences. The hPer1 gene spans 15kb of human genomic DNA and is composed of 23 exons, flanked by 5' and 3' regulatory regions. Comparison of the hPer1 genomic clone with the dbEST database revealed homologies with putative alternative transcripts. Functional mapping within the 5' CpG-rich regulatory region enabled us to locate the hPer1 promoter core in a 510bp-long sequence centred around a TATA box, which supports high levels of hPer1 transcription. A second regulatory region was formally identified in intron 1, which appears to exert a negative role in transcriptional control of hPer1. These regions may be differentially involved in tissue-specificity, and/or circadian regulation, of the human hPer1 gene transcription.


Subject(s)
Genes/genetics , Nuclear Proteins/genetics , Promoter Regions, Genetic/genetics , 3T3 Cells , Animals , Base Sequence , Cell Cycle Proteins , Chloramphenicol O-Acetyltransferase/genetics , Chloramphenicol O-Acetyltransferase/metabolism , Chromosome Mapping , Chromosomes, Human, Pair 17/genetics , Cloning, Molecular , DNA/chemistry , DNA/genetics , DNA/isolation & purification , Drosophila Proteins , Exons , Humans , In Situ Hybridization, Fluorescence , Introns , Mice , Molecular Sequence Data , Period Circadian Proteins , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Analysis, DNA , Transcription, Genetic
13.
J Cell Sci ; 112 ( Pt 20): 3537-48, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10504302

ABSTRACT

We have characterized a nuclease hypersensitive chromatin fraction from murine spermatozoa. Endogenous nuclease activity can be induced in mouse epididymal spermatozoa by appropriate stimuli and cause the localized degradation of chromosomal DNA. Based on these observations, we have isolated nuclease hypersensitive chromatin regions released from spermatozoa in the supernatant of pelleted sperm cells, and have cloned and characterized the DNA. Gel electrophoresis of end-labelled released DNA fragments showed a typical nucleosomal distribution. Peripherally distributed nucleohistones were visualized by immunofluorescence in sperm nuclei, and histones were identified by western blot in sperm chromatin. Moreover, the released DNA is enriched in retroposon DNA from a variety of families. FISH and immunofluorescence analysis showed that retroposon DNA and nucleohistone chromatin co-localize and are both peripherically distributed in nuclei of spermatozoa. In contrast, a major satellite DNA probe, used for control, co-localizes with highly condensed chromatin in the central region of sperm nuclei. The nuclear Ran and RCC1 proteins were also visualized in the dorsal margin of sperm nuclei, and were abundantly released with the hypersensitive chromatin fraction. Together, these results indicate that nucleohistone chromatin fraction(s) with typical features of 'active' chromatin are present in murine spermatozoa, are hypersensitive to nuclease cleavage, enriched in retroposon DNA and organized in nucleosomal domains. These observations suggest that nucleohistone domains identify a fraction of the sperm genome which may be functional during early embryogenesis.


Subject(s)
Chromatin/ultrastructure , Nucleosomes/ultrastructure , Retroelements , Spermatozoa/ultrastructure , Animals , Cell Nucleus/physiology , Cell Nucleus/ultrastructure , Epididymis/physiology , Genomic Library , Humans , In Situ Hybridization, Fluorescence , Male , Mice , Nucleosomes/genetics , Sperm Motility , Spermatozoa/physiology
14.
Exp Cell Res ; 250(2): 339-50, 1999 Aug 01.
Article in English | MEDLINE | ID: mdl-10413588

ABSTRACT

We have studied the response of human transformed cells to mitotic spindle inhibition. Two paired cell lines, K562 and its parvovirus-resistant KS derivative clone, respectively nonexpressing and expressing p53, were continuously exposed to nocodazole. Apoptotic cells were observed in both lines, indicating that mitotic spindle impairment induced p53-independent apoptosis. After a transient mitotic delay, both cell lines exited mitosis, as revealed by flow-cytometric determination of MPM2 antigen and cyclin B1 expression, coupled to cytogenetic analysis of sister centromere separation. Both cell lines exited mitosis without chromatid segregation. K562 p53-deficient cells further resumed DNA synthesis, giving rise to cells with a DNA content above 4C, and reentered a polyploid cycle. In contrast, KS cells underwent a subsequent G1 arrest in the tetraploid state. Thus, G1 arrest in tetraploid cells requires p53 function in the rereplication checkpoint which prevents the G1/S transition following aberrant mitosis; in contrast, p53 expression is dispensable for triggering the apoptotic response in the absence of mitotic spindle.


Subject(s)
Apoptosis , Cell Cycle Proteins , DNA Replication/genetics , Genes, p53 , Spindle Apparatus/physiology , Apoptosis/drug effects , Cell Cycle/drug effects , Centromere/drug effects , Centromere/metabolism , Chromosome Segregation/drug effects , Cyclin B/analysis , Cyclin B1 , DNA/biosynthesis , DNA Fragmentation/drug effects , DNA Replication/drug effects , Dose-Response Relationship, Drug , Flow Cytometry , Humans , Kinesins , Mitotic Index/drug effects , Nocodazole/pharmacology , Phosphoproteins/analysis , Polyploidy , Spindle Apparatus/drug effects , Tumor Cells, Cultured , Tumor Suppressor Protein p53/physiology
15.
Nucleic Acids Res ; 27(14): 2852-9, 1999 Jul 15.
Article in English | MEDLINE | ID: mdl-10390525

ABSTRACT

The CpG-rich promoter of the retinoblastoma tumor suppressor gene (Rb-1) is normally unmethylated. However, aberrant methylation of CpG dinucleotides within the Rb-1 promoter has been depicted in certain tumors, which determines transcriptional inactivity of the gene and absence of the pRb retinoblastoma protein. Here we have concentrated on an E2F-binding site in the Rb-1 promoter. We show that the E2F site is required for cell-cycle regulated Rb-1 transcription in non-transformed cells. The function of the E2F site is associated with its ability to interact with several activating factors of the E2F family. In contrast, in vitro methylation of two tandemly arranged CpGs in the E2F recognition site prevents binding by E2F factors, and determines instead the recruitment of the general repressor methylcytosine-binding protein 2 (MeCP2). These results suggest that the interaction of MeCP2 with the methylated version of the E2F site may represent a step towards Rb-1 promoter inactivity in tumor cells.


Subject(s)
Carrier Proteins , Cell Cycle Proteins , Chromosomal Proteins, Non-Histone , Cytosine/metabolism , DNA Methylation , DNA-Binding Proteins/metabolism , Promoter Regions, Genetic/genetics , Retinoblastoma Protein/genetics , Transcription Factors/metabolism , 3T3 Cells , Animals , Base Sequence , Binding Sites , CpG Islands/genetics , DNA/genetics , DNA/metabolism , E2F Transcription Factors , Methyl-CpG-Binding Protein 2 , Mice , Models, Genetic , Mutation , Repressor Proteins/metabolism , Response Elements/genetics , Retinoblastoma-Binding Protein 1 , S Phase , Transcription Factor DP1 , Transfection , Up-Regulation
16.
Bioessays ; 21(3): 221-30, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10333731

ABSTRACT

In this review, we will focus on the role played by transcription factors of the E2F/DP family in controlling the expression of genes that carry out important cell-cycle control functions, thereby ensuring ordered progression through the mammalian cell division cycle. The emerging picture is that cell-cycle progression depends on the execution of a regulatory cascade of gene expression, driven by E2F/DP transcription factors, which are in turn regulated by the products of some of these genes. That E2F factors are potent regulators of cell-cycle checkpoints in mammalian cells is supported by experiments demonstrating that ectopic expression of individual E2F family members is sufficient to modulate cell proliferation and apoptosis. It is also clear that deregulation of E2F activity will result in the loss of particular checkpoint controls, thereby predisposing cells to malignant conversion.


Subject(s)
Carrier Proteins , Cell Cycle Proteins , Cell Cycle/physiology , DNA-Binding Proteins , Gene Expression Regulation , Signal Transduction , Transcription Factors/metabolism , ran GTP-Binding Protein , Animals , Apoptosis , E2F Transcription Factors , GTP-Binding Proteins/genetics , Humans , Nuclear Proteins/genetics , Retinoblastoma-Binding Protein 1 , S Phase , Transcription Factor DP1 , Transcription Factors/genetics , Transcription, Genetic
17.
J Biol Chem ; 274(15): 10339-48, 1999 Apr 09.
Article in English | MEDLINE | ID: mdl-10187822

ABSTRACT

The gene encoding Ran-binding protein 1 (RanBP1) is transcribed in a cell cycle-dependent manner. The RanBP1 promoter contains two binding sites for E2F factors, named E2F-c, located proximal to the transcription start, and E2F-b, falling in a more distal promoter region. We have now induced site-directed mutagenesis in both sites. We have found that the distal E2F-b site, together with a neighboring Sp1 element, actively controls up-regulation of transcription in S phase. The proximal E2F-c site plays no apparent role in cycling cells yet is required for transcriptional repression upon growth arrest. Protein binding studies suggest that each E2F site mediates specific interactions with individual E2F family members. In addition, transient expression assays with mutagenized promoter constructs indicate that the functional role of each site is also dependent on its position relative to other regulatory elements in the promoter context. Thus, the two E2F sites play opposite genetic functions and control RanBP1 transcription through distinct molecular mechanisms.


Subject(s)
Carrier Proteins , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Fungal Proteins/genetics , GTP-Binding Proteins/genetics , Leucine Zippers , Nuclear Proteins/genetics , Transcription Factors/metabolism , Transcription, Genetic , ran GTP-Binding Protein , 3T3 Cells , Animals , E2F Transcription Factors , Fungal Proteins/physiology , G1 Phase , GTP-Binding Proteins/physiology , Mice , Mutagenesis, Site-Directed , Nuclear Proteins/physiology , Promoter Regions, Genetic , Retinoblastoma-Binding Protein 1 , S Phase , Structure-Activity Relationship , Transcription Factor DP1
18.
J Biol Chem ; 273(1): 495-505, 1998 Jan 02.
Article in English | MEDLINE | ID: mdl-9417108

ABSTRACT

The murine Htf9-a/RanBP1 and Htf9-c genes are divergently transcribed from a shared TATA-less promoter. Transcription of both genes is initiated on complementary DNA strands and is controlled by cell cycle-dependent mechanisms. The bidirectional promoter harbors a genomic footprint flanking the major transcription start site of both genes. Transient promoter assays showed that the footprinted element is important for transcription of both genes. Protein-binding experiments and antibody assays indicated that members of the retinoid X receptor family interact with the double-stranded site. In addition, distinct factors interact with single DNA strands of the element. Double-stranded binding factors were highly expressed in liver cells, in which neither gene is transcribed, while single-stranded binding proteins were abundant in cycling cells, in which transcription of both genes is efficient. In vivo S1 analysis of the promoter depicted an S1-sensitive organization in cells in which transcription of both genes is active; S1 sensitivity was not detected in conditions of transcriptional repression. Thus, the same element is a target for either retinoid X receptor factors, or for single-stranded binding proteins, and form distinct complexes in different cellular conditions depending on the DNA conformation in the binding site.


Subject(s)
DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , GTP-Binding Proteins/genetics , Nuclear Proteins/genetics , Promoter Regions, Genetic , Proteins/genetics , Transcriptional Activation , ran GTP-Binding Protein , 3T3 Cells , Animals , Base Sequence , Binding Sites , Cell Extracts , DNA Footprinting , DNA-Binding Proteins/genetics , Fibroblasts/metabolism , Liver/metabolism , Mice , Molecular Sequence Data , Mutation , RNA-Binding Proteins , Receptors, Retinoic Acid/genetics , Receptors, Retinoic Acid/metabolism , Retinoid X Receptors , Transcription Factors/genetics , Transcription Factors/metabolism
19.
J Cell Sci ; 110 ( Pt 19): 2345-57, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9410874

ABSTRACT

RanBP1 is a molecular partner of the Ran GTPase, which is implicated in the control of several processes, including DNA replication, mitotic entry and exit, cell cycle progression, nuclear structure, protein import and RNA export. While most genes encoding Ran-interacting partners are constitutively active, transcription of the RanBP1 mRNA is repressed in non proliferating cells, is activated at the G1/S transition in cycling cells and peaks during S phase. We report here that forced expression of the RanBP1 gene disrupts the orderly execution of the cell division cycle at several stages, causing inhibition of DNA replication, defective mitotic exit and failure of chromatin decondensation during the telophase-to-interphase transition in cells that achieve nuclear duplication and chromosome segregation. These results suggest that deregulated RanBP1 activity interferes with the Ran GTPase cycle and prevents the functioning of the Ran signalling system during the cell cycle.


Subject(s)
Cell Cycle/genetics , GTP-Binding Proteins/genetics , Gene Expression Regulation , Nuclear Proteins/genetics , ran GTP-Binding Protein , 3T3 Cells , Animals , Cell Cycle/drug effects , Cell Nucleus/drug effects , Cell Nucleus/genetics , Culture Media, Serum-Free/pharmacology , GTP-Binding Proteins/biosynthesis , Gene Expression Regulation/drug effects , Mice , Mitosis/genetics , Nuclear Proteins/biosynthesis , S Phase/genetics
20.
Biochem J ; 325 ( Pt 1): 277-86, 1997 Jul 01.
Article in English | MEDLINE | ID: mdl-9224656

ABSTRACT

The murine Htf9-a/RanBP1 and Htf9-c genes are divergently transcribed from a bidirectional promoter. The Htf9-a gene encodes the RanBP1 protein, a major partner of the Ran GTPase. The divergently transcribed Htf9-c gene encodes a protein sharing similarity with yeast and bacterial nucleic acid-modifying enzymes. We report here that both mRNA species produced by the Htf9-associated genes are regulated during the cell cycle progression, peak in S phase and decrease during mitosis. Transient expression experiments with reporter constructs showed that cell cycle expression is controlled at the transcriptional level, because the bidirectional Htf9 promoter is down-regulated in growth-arrested cells, is activated at the G1/S transition and reaches maximal activity in S phase, though with a different efficiency for each orientation. We have delimited specific promoter regions controlling S phase activity in one or both orientations: identified elements contain recognition sites for members belonging to both the E2F and Sp1 families of transcription factors. Together, the results suggest that the sharing of the regulatory region supports co-regulation of the Htf9-a/RanBP1 and Htf9-c genes in a common window of the cell cycle.


Subject(s)
Cell Cycle/physiology , GTP-Binding Proteins/biosynthesis , GTP-Binding Proteins/genetics , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , Promoter Regions, Genetic , Protein Biosynthesis , Proteins/genetics , Transcription, Genetic , ran GTP-Binding Protein , 3T3 Cells , Amino Acid Sequence , Animals , Casein Kinase II , Cell Cycle/drug effects , GTP-Binding Proteins/chemistry , Gene Expression Regulation , Hydroxyurea/pharmacology , Leucine Zippers , Mice , Molecular Sequence Data , Nocodazole/pharmacology , Nuclear Proteins/chemistry , Phosphorylation , Protein Kinase C/metabolism , Protein Serine-Threonine Kinases/metabolism , Proteins/chemistry , RNA-Binding Proteins , Sequence Alignment , Sequence Homology, Amino Acid , Transcription, Genetic/drug effects
SELECTION OF CITATIONS
SEARCH DETAIL
...