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Biomedical subjects

Giuseppe Biamonti

Publications and source records attributed to Giuseppe Biamonti.

15 recordsLinked to original sources
PubMed2006

Cellular response to etoposide treatment.

Etoposide is a potent anti-tumor drug that belongs to the class of topoisomerase poisons. Although its molecular target, i.e. DNA topoisomerase II, has been identified more than 20 years ago, the cellular response to etoposide is still poorly understood. The cytotoxicity of the drug stems from its ability to stabilize a covalent complex between DNA topoisomerase II and DNA that results in a high level of DNA damage. Here, we review the present knowledge about the strategy used by the cells to deal with the etoposide-induced DNA damage. New and unanticipated effects of topoisomerase II poisoning on cell metabolism are recently emerging, among which the ability to activate cell cycle checkpoint pathways and to affect gene expression at different levels, including chromatin remodeling and alternative splicing of gene transcripts. The elucidation of the effects of etoposide on cell metabolism will increase our ability to exploit this drug in cancer therapy and will expand our comprehension of the cancerous cell.

Animals↗
PubMed2006

The dispersal of replication proteins after Etoposide treatment requires the cooperation of Nbs1 with the ataxia telangiectasia Rad3-related/Chk1 pathway.

In mammalian cells, DNA replication takes place in functional subnuclear compartments, called replication factories, where replicative factors accumulate. The distribution pattern of replication factories is diagnostic of the different moments (early, mid, and late) of the S phase. This dynamic organization is affected by different agents that induce cell cycle checkpoint activation via DNA damage or stalling of replication forks. Here, we explore the cell response to etoposide, an anticancer drug belonging to the topoisomerase II poisons. Etoposide does not induce an immediate block of DNA synthesis and progressively affects the distribution of replication proteins in S phase. First, it triggers the formation of large nuclear foci that contain the single-strand DNA binding protein replication protein A (RPA), suggesting that lesions produced by the drug are processed into extended single-stranded regions. These RPA foci colocalize with DNA replicated at the beginning of the treatment. Etoposide also triggers the dispersal of replicative proteins, proliferating cell nuclear antigen and DNA ligase I, from replication factories. This event requires the activity of the ataxia telangiectasia Rad3-related (ATR) checkpoint kinase. By comparing the effect of the drug in cell lines defective in different DNA repair and checkpoint pathways, we show that, along with the downstream kinase Chk1, the Nbs1 protein, mutated in the Nijmegen breakage syndrome, is also relevant for this response and for ATR-dependent phosphorylation. Finally, our analysis evidences a critical role of Nbs1 in the etoposide-induced inhibition of DNA replication in early S phase.

Antineoplastic Agents, Phytogenic↗
PubMed2005

Cell motility is controlled by SF2/ASF through alternative splicing of the Ron protooncogene.

Ron, the tyrosine kinase receptor for the Macrophage-stimulating protein, is involved in cell dissociation, motility, and matrix invasion. DeltaRon, a constitutively active isoform that confers increased motility to expressing cells, is generated through the skipping of exon 11. We show that abnormal accumulation of DeltaRon mRNA occurs in breast and colon tumors. Skipping of exon 11 is controlled by a silencer and an enhancer of splicing located in the constitutive exon 12. The strength of the enhancer parallels the relative abundance of DeltaRon mRNA and depends on a sequence directly bound by splicing factor SF2/ASF. Overexpression and RNAi experiments demonstrate that SF2/ASF, by controlling the production of DeltaRon, activates epithelial to mesenchymal transition leading to cell locomotion. The effect of SF2/ASF overexpression is reverted by specific knockdown of DeltaRon mRNA. This demonstrates a direct link between SF2/ASF-regulated splicing and cell motility, an activity important for embryogenesis, tissue formation, and tumor metastasis.

Alternative Splicing↗
PubMed2005

Structural and functional characterization of noncoding repetitive RNAs transcribed in stressed human cells.

Thermal and chemical stresses induce the formation in human cells of novel and transient nuclear structures called nuclear stress bodies (nSBs). These contain heat shock factor 1 (HSF-1) and a specific subset of pre-mRNA processing factors. Nuclear stress bodies are assembled on specific pericentromeric heterochromatic domains containing satellite III (SatIII) DNA. In response to stress, these domains change their epigenetic status from heterochromatin to euchromatin and are transcribed in poly-adenylated RNAs that remain associated with nSBs. In this article, we describe the cloning, sequencing, and functional characterization of these transcripts. They are composed of SatIII repeats and originate from the transcription of multiple sites within the SatIII arrays. Interestingly, the level of SatIII RNAs can be down-regulated both by antisense oligonucleotides and small interfering RNAs (siRNA). Knockdown of SatIII RNA by siRNAs requires the activity of Argonaute 2, a component of the RNA-induced silencing complex. Down-regulation of satellite III RNAs significantly affects the recruitment of RNA processing factors to nSBs without altering the association of HSF-1 with these structures nor the presence of acetylated histones within nSBs. Thus, satellite III RNAs have a major role in the formation of nSBs.

Animals↗
PubMed2004

Subnuclear distribution of the largest subunit of the human origin recognition complex during the cell cycle.

In eukaryotes, initiation of DNA replication requires the activity of the origin recognition complex (ORC). The largest subunit of this complex, Orc1p, has a critical role in this activity. Here we have studied the subnuclear distribution of the overexpressed human Orc1p during the cell cycle. Orc1p is progressively degraded during S-phase according to a spatio-temporal program and it never colocalizes with replication factories. Orc1p is resynthesized in G1. In early G1, the protein is distributed throughout the cell nucleus, but successively it preferentially associates with heterochromatin. This association requires a functional ATP binding site and a protein region partially overlapping the bromo-adjacent homology domain at the N-terminus of Orc1p. The same N-terminal region mediates the in vitro interaction with heterochromatin protein 1 (HP1). Fluorescence resonance energy transfer (FRET) experiments demonstrate the interaction of human Orc1p and HP1 in vivo. Our data suggest a role of HP1 in the recruitment but not in the stable association of Orc1p with heterochromatin. Indeed, the subnuclear distribution of Orc1p is not affected by treatments that trigger the dispersal of HP1.

Adenosine Triphosphate↗
PubMed2004

RNA recognition motif 2 directs the recruitment of SF2/ASF to nuclear stress bodies.

Heat shock induces the transcriptional activation of large heterochromatic regions of the human genome composed of arrays of satellite III DNA repeats. A number of RNA-processing factors, among them splicing factor SF2/ASF, associate with these transcription factors giving rise to nuclear stress bodies (nSBs). Here, we show that the recruitment of SF2/ASF to these structures is mediated by its second RNA recognition motif. Amino acid substitutions in the first alpha-helix of this domain, but not in the beta-strand regions, abrogate the association with nSBs. The same mutations drastically affect the in vivo activity of SF2/ASF in the alternative splicing of adenoviral E1A transcripts. Sequence analysis identifies four putative high-affinity binding sites for SF2/ASF in the transcribed strand of the satellite III DNA. We have verified by gel mobility shift assays that the second RNA-binding domain of SF2/ASF binds at least one of these sites. Our analysis suggests that the recruitment of SF2/ASF to nSBs is mediated by a direct interaction with satellite III transcripts and points to the second RNA-binding domain of the protein as the major determinant of this interaction.

Alternative Splicing↗
PubMed2004

Modular structure of the human lamin B2 replicator.

The cis-acting elements necessary for the activity of DNA replication origins in metazoan cells are still poorly understood. Here we report a thorough characterization of the DNA sequence requirements of the origin associated with the human lamin B2 gene. A 1.2-kb DNA segment, comprising the start site of DNA replication and located within a large protein-bound region, as well as a CpG island, displays origin activity when moved to different ectopic positions. Genomic footprinting analysis of both the endogenous and the ectopic origins indicates that the large protein complex is assembled in both cases around the replication start site. Replacement of this footprinted region with an unrelated sequence, maintaining the CpG island intact, abolishes origin activity and the interaction with hORC2, a subunit of the origin recognition complex. Conversely, the replacement of 17 bp within the protected region reduces the extension of the protection without affecting the interaction with hORC2. This substitution does not abolish the origin activity but makes it more sensitive to the integration site. Finally, the nearby CpG island positively affects the efficiency of initiation. This analysis reveals the modular structure of the lamin B2 origin and supports the idea that sequence elements close to the replication start site play an important role in origin activation.

Base Sequence↗
PubMed2004

A single polypyrimidine tract binding protein (PTB) binding site mediates splicing inhibition at mouse IgM exons M1 and M2.

Splicing of mouse immunoglobulin (IgM) exons M1 and M2 is directed by two juxtaposed regulatory elements, an enhancer and an inhibitor, located within the M2 exon. A primary function of the enhancer is to counteract the inhibitor, allowing splicing to occur. Here we show that the inhibitor contains two binding sites for polypyrimidine tract binding protein (PTB). Mutational analysis indicates that only one of these sites is necessary and sufficient to direct splicing inhibition both in vitro and in vivo. We demonstrate that the difference in activity of the two sites is explained by proximity to the intron. We further show that the presence of the enhancer results in the disruption of the PTB-inhibitor interaction, enabling splicing to occur. In the absence of the enhancer, splicing can be artificially activated by immuno-inhibition of PTB. Collectively, our results indicate that a single PTB binding site can function as an inhibitor that regulates alternative splicing both in vitro and in vivo.

Alternative Splicing↗
PubMed2003

Transcriptional activation of a constitutive heterochromatic domain of the human genome in response to heat shock.

Heat shock triggers the assembly of nuclear stress bodies that contain heat shock factor 1 and a subset of RNA processing factors. These structures are formed on the pericentromeric heterochromatic regions of specific human chromosomes, among which chromosome 9. In this article we show that these heterochromatic domains are characterized by an epigenetic status typical of euchromatic regions. Similarly to transcriptionally competent portions of the genome, stress bodies are, in fact, enriched in acetylated histone H4. Acetylation peaks at 6 h of recovery from heat shock. Moreover, heterochromatin markers, such as HP1 and histone H3 methylated on lysine 9, are excluded from these nuclear districts. In addition, heat shock triggers the transient accumulation of RNA molecules, heterogeneous in size, containing the subclass of satellite III sequences found in the pericentromeric heterochromatin of chromosome 9. This is the first report of a transcriptional activation of a constitutive heterochromatic portion of the genome in response to stress stimuli.

Acetylation↗
PubMed2003

Early mitotic degradation of the homeoprotein HOXC10 is potentially linked to cell cycle progression.

Hox proteins are transcription factors involved in controlling axial patterning, leukaemias and hereditary malformations. Here, we show that HOXC10 oscillates in abundance during the cell cycle, being targeted for degradation early in mitosis by the ubiquitin-dependent proteasome pathway. Among abdominal-B subfamily members, the mitotic proteolysis of HOXC10 appears unique, since the levels of the paralogous HOXD10 and the related homeoprotein HOXC13 are constant throughout the cell cycle. When two destruction box motifs (D-box) are mutated, HOXC10 is stabilized and cells accumulate in metaphase. HOXC10 appears to be a new prometaphase target of the anaphase-promoting complex (APC), since its degradation coincides with cyclin A destruction and is suppressed by expression of a dominant-negative form of UbcH10, an APC-associated ubiquitin-conjugating enzyme. Moreover, HOXC10 co-immunoprecipitates the APC subunit CDC27, and its in vitro degradation is reduced in APC-depleted extracts or by competition with the APC substrate cyclin A. These data imply that HOXC10 is a homeoprotein with the potential to influence mitotic progression, and might provide a link between developmental regulation and cell cycle control.

Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome↗
PubMed2003

Cell cycle-dependent phosphorylation of human DNA ligase I at the cyclin-dependent kinase sites.

We have described previously that, during S-phase, human DNA ligase I is phosphorylated on Ser66, a casein kinase II site. Here we investigate the phosphorylation status of DNA ligase I during the cell cycle by gel shift analysis and electrospray mass spectrometry. We show that three residues (Ser51, Ser76, and Ser91), which are part of cyclin-dependent kinase sites, are phosphorylated in a cell cycle-dependent manner. Phosphorylation of Ser91 occurs at G1/S transition and depends on a cyclin binding site in the C-terminal part of the protein. This modification is required for the ensuing phosphorylation of Ser76 detectable in G2/M extracts. The substitution of serines at positions 51, 66, 76, and 91 with aspartic acid to mimic the phosphorylated enzyme hampers the association of DNA ligase I with the replication foci. We suggest that the phosphorylation of DNA ligase I and possibly other replicative enzymes is part of the mechanism that directs the disassembly of the replication machinery at the completion of S-phase.

Amino Acid Motifs↗
PubMed2003

Is DNA sequence sufficient to specify DNA replication origins in metazoan cells?

DNA replication occupies a central position in the cell cycle and, therefore, in the development and life of multicellular organisms. During the last 10 years, our comprehension of this important process has considerably improved. Although the mechanisms that coordinate DNA replication with the other moments of the cell cycle are not yet fully understood, it is known that they mainly operate through DNA replication origins and the protein complexes bound to them. In eukaryotes, the packaging status of chromatin seems to be part of the mechanism that controls whether or not and when during the S-phase a particular origin will be activated. Intriguingly, the protein complexes bound to DNA replication origins appear to be directly involved in controlling chromatin packaging. In this manner they can also affect gene expression. In this review we focus on DNA replication origins in metazoan cells and on the relationship between these elements and the structural and functional organization of the genome.

Animals↗
PubMed2002

Cell cycle-dependent dynamic association of cyclin/Cdk complexes with human DNA replication proteins.

We have previously described the isolation of a replication competent (RC) complex from calf thymus, containing DNA polymerase alpha, DNA polymerase delta and replication factor C. Here, we describe the isolation of the RC complex from nuclear extracts of synchronized HeLa cells, which contains DNA replication proteins associated with cell-cycle regulation factors like cyclin A, cyclin B1, Cdk2 and Cdk1. In addition, it contains a kinase activity and DNA polymerase activities able to switch from a distributive to a processive mode of DNA synthesis, which is dependent on proliferating cell nuclear antigen. In vivo cross-linking of proteins to DNA in synchronized HeLa cells demonstrates the association of this complex to chromatin. We show a dynamic association of cyclins/Cdks with the RC complex during the cell cycle. Indeed, cyclin A and Cdk2 associated with the complex in S phase, and cyclin B1 and Cdk1 were present exclusively in G(2)/M phase, suggesting that the activity, as well the localization, of the RC complex might be regulated by specific cyclin/Cdk complexes.

Animals↗
PubMed2002

Human chromosomes 9, 12, and 15 contain the nucleation sites of stress-induced nuclear bodies.

We previously reported the identification of a novel nuclear compartment detectable in heat-shocked HeLa cells that we termed stress-induced Src-activated during mitosis nuclear body (SNB). This structure is the recruitment center for heat shock factor 1 and for a number of RNA processing factors, among a subset of Serine-Arginine splicing factors. In this article, we show that stress-induced SNBs are detectable in human but not in hamster cells. By means of hamster>human cell hybrids, we have identified three human chromosomes (9, 12, and 15) that are individually able to direct the formation of stress bodies in hamster cells. Similarly to stress-induced SNB, these bodies are sites of accumulation of hnRNP A1-interacting protein and heat shock factor 1, are usually associated to nucleoli, and consist of clusters of perichromatin granules. We show that the p13-q13 region of human chromosome 9 is sufficient to direct the formation of stress bodies in hamster>human cell hybrids. Fluorescence in situ hybridization experiments demonstrate that the pericentromeric heterochromatic q12 band of chromosome 9 and the centromeric regions of chromosomes 12 and 15 colocalize with stress-induced SNBs in human cells. Our data indicate that human chromosomes 9, 12, and 15 contain the nucleation sites of stress bodies in heat-shocked HeLa cells.

3T3 Cells↗