Biomedical subjects

P Lavia

Publications and source records attributed to P Lavia.

At least 19 recordsLinked to original sources

PubMed2004

p53 localization at centrosomes during mitosis and postmitotic checkpoint are ATM-dependent and require serine 15 phosphorylation.

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

Ataxia Telangiectasia Mutated Proteins↗

PubMed2001

p53 displacement from centrosomes and p53-mediated G1 arrest following transient inhibition of the mitotic spindle.

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.

Anaphase↗

PubMed2001

Normal and cancer-prone human cells respond differently to extremely low frequency magnetic fields.

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.

Apoptosis↗

PubMed2000

Cloning of PC3B, a novel member of the PC3/BTG/TOB family of growth inhibitory genes, highly expressed in the olfactory epithelium.

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.

Amino Acid Sequence↗

PubMed2000

The human per1 gene: genomic organization and promoter analysis of the first human orthologue of the Drosophila period gene.

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.

3T3 Cells↗

PubMed2000

E2F transcription factors are differentially expressed in murine gametes and early embryos.

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.

3T3 Cells↗

PubMed2000

Regulated Ran-binding protein 1 activity is required for organization and function of the mitotic spindle in mammalian cells in vivo.

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.

3T3 Cells↗

PubMed1999

p53-independent apoptosis and p53-dependent block of DNA rereplication following mitotic spindle inhibition in human cells.

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.

Apoptosis↗

PubMed1999

Cytosine methylation transforms an E2F site in the retinoblastoma gene promoter into a binding site for the general repressor methylcytosine-binding protein 2 (MeCP2).

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.

3T3 Cells↗

PubMed1999

Two E2F sites control growth-regulated and cell cycle-regulated transcription of the Htf9-a/RanBP1 gene through functionally distinct mechanisms.

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.

3T3 Cells↗

PubMed1999

E2F target genes and cell-cycle checkpoint control.

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.

Animals↗

PubMed1999

A fraction of mouse sperm chromatin is organized in nucleosomal hypersensitive domains enriched in retroposon DNA.

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.

Animals↗

PubMed1998

Interactions with single-stranded and double-stranded DNA-binding factors and alternative promoter conformation upon transcriptional activation of the Htf9-a/RanBP1 and Htf9-c genes.

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.

3T3 Cells↗

PubMed1997

Expression of the murine RanBP1 and Htf9-c genes is regulated from a shared bidirectional promoter during cell cycle progression.

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.

3T3 Cells↗

PubMed1997

Deregulated expression of the RanBP1 gene alters cell cycle progression in murine fibroblasts.

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.

3T3 Cells↗

PubMed1996

Different effects of histone H1 on de novo DNA methylation in vitro depend on both the DNA base composition and the DNA methyltransferase.

We have characterized the inhibition exerted by histone H1 on the activity of human placenta DNA (cytosine-5-)-methyltransferase. Our experiments demonstrate that the extent of inhibition depends on the DNA base composition, AT-rich substrates being more severely affected than GC-rich substrates and CpG-rich islands. With bacterial SssI methylase, the effect is completely reversed since its activity on AT-rich substrates undergoes a 4-5-fold stimulation upon the addition of H1. Poly(L-lysine) mimicks H1 effects, suggesting an essential role of lysine residues in both the inhibitory and stimulatory effects of H1. By comparison of the different behaviors of the two enzymes, the inhibitory effect over the eukaryotic enzyme might be accounted for by hypothesizing a competition between minor groove-binding motifs (SPKK-like) present in placenta methylase as well as in histone H1.

Base Composition↗

PubMed1995

Transcriptional control of the Htf9-A/RanBP-1 gene during the cell cycle.

The mouse Htf9-a gene encodes a small hydrophilic protein, named Ran-binding protein 1 (RanBP-1), for its interaction with the Ras-related Ran protein; RanBP-1 binds the biologically active form of Ran. Ran has been implicated in a variety of nuclear events including DNA replication, RNA export and protein import, and monitoring completion of DNA synthesis before the onset of mitosis; it biological activity is thought to be regulated in S phase. We have investigated whether expression of the Htf9-a/RanBP-1 gene is linked to cell proliferation. Expression of the Htf9-a/RanBP-1 transcript appeared to be dependent on the proliferating state of the cells and peaked in S phase. We have identified a promoter region required for Htf9-a cell cycle-dependent expression and for responsiveness to cell cycle regulators. An E2F-binding site was identified within the cell cycle-regulated promoter region. Expression of the E1A oncoprotein prevented Htf9-a down-regulation in quiescent cells; in addition, both pRb and its relative p107 inhibited transcription from the Htf9-a promoter. These results link the control of Htf9-a/RanBP-1 expression to the cell cycle progression.

3T3 Cells↗

PubMed1993

Cell type-specific interactions of transcription factors with a housekeeping promoter in vivo.

Mammalian housekeeping promoters represent a class of regulatory elements different from those of tissues-specific genes, lacking a TATA box and associated with CG-rich DNA. We have compared the organization of the housekeeping Htf9 promoter in different cell types by genomic footprinting. The sites of in vivo occupancy clearly reflected local combinations of tissue-specific and ubiquitous binding factors. The flexibility of the Htf9 promoter in acting as the target of cell-specific combinations of factors may ensure ubiquitous expression of the Htf9-associated genes.

3T3 Cells↗