Biomedical subjects

Olivier Brison

Publications and source records attributed to Olivier Brison.

10 recordsLinked to original sources

PubMed2005

High frequency trans-splicing in a cell line producing spliced and polyadenylated RNA polymerase I transcripts from an rDNA-myc chimeric gene.

The 2G1MycP2Tu1 cell line was obtained following transfection of human colon carcinoma cells from the SW613-S cell line with a plasmid carrying a genomic copy of the human MYC gene. 2G1MycP2Tu1 cells produce MYC mRNAs and proteins of abnormal size. In order to analyze the structure of these abnormal products, a cDNA library constructed using RNA isolated from these cells was screened with a MYC probe. Fifty clones were studied by DNA sequencing. The results indicated that a truncated copy of the MYC gene had integrated into an rDNA transcription unit in 2G1MycP2Tu1 cells. This was confirmed by northern blot analysis, PCR amplification on genomic DNA and fluorescent in situ hybridization (FISH) experiments on metaphase chromosomes. 2G1MycP2Tu1 cells produce hybrid rRNA-MYC RNA molecules that are polyadenylated and processed by splicing reactions involving natural and cryptic splice sites. These transcripts are synthesized by RNA polymerase I, as confirmed by actinomycin D sensitivity experiments, suggesting that 3' end processing and splicing are uncoupled from transcription in this case. 2G1MycP2Tu1 cells also produce another type of chimeric mRNAs consisting of correctly spliced exons 2 and 3 of the MYC gene fused to one or more extraneous 5' exons by proper splicing to the acceptor sites of MYC exon 2. These foreign exons belong to 33 different genes, which are located on 14 different chromosomes. These observations and the results of FISH and Southern blotting experiments lead us to conclude that trans-splicing events occur at high frequency in 2G1MycP2Tu1 cells.

Artificial Gene Fusion↗

PubMed2005

Use of adenoviral E1A protein to analyze K18 promoter deregulation in colon carcinoma cells discloses a role for CtBP1 and BRCA1.

BACKGROUND: The promoter of the keratin 18 (K18) gene is 5- to 10-fold more active in tumorigenic (T-type) cell clones derived from the SW613-S human colon carcinoma cell line than in non-tumorigenic (NT-type) clones. We have reported previously that the mechanism responsible for this differential activity is acting on the minimal K18 promoter (TATA box and initiation site). This mechanism does not require the binding of a factor to a specific site on the DNA but involves the acetylation of a non-histone substrate. To get further insight into this mechanism, we investigated the effect of the adenovirus E1A protein on the activity of the K18 promoter, both in T and NT cells. RESULTS: Wild type adenovirus E1A protein and C-terminal deletion mutants inhibit the K18 promoter, specifically in T-type cells. The domain responsible for this inhibitory effect is located in the 12-25 region of the viral protein. E1A mutants that have lost this region but retain the PLDLS motif (the C-terminal binding site for CtBP1) stimulate the K18 promoter, specifically in NT cells. The inhibitory or stimulatory effects of the different E1A mutants are not dependent on a particular sequence of the promoter. An E1A N-terminal deletion mutant carrying point mutations in the PLDLS motif cannot stimulate the K18 promoter. CtBP1 interacts with CtIP, which is a known partner of BRCA1, itself a component of the RNA polymerase II holoenzyme. The stimulatory effect of two BRCA1 mutants, specifically in NT cells, implicates a tripartite BRCA1-CtIP-CtBP1 complex in the regulation of the K18 promoter. CONCLUSION: Since we have shown previously that the K18 promoter is stimulated by deacetylase inhibitors, specifically in NT cells, we conclude that the activity of the promoter is repressed in NT cells by a mechanism involving the recruitment, by a BRCA1/CtIP complex, of CtBP1 and associated deacetylases to the preinitiation complex. We propose a model depicting the mechanism responsible for the differential activity of the K18 promoter between T and NT cells of the SW613-S cell line.

Adenovirus E1A Proteins↗

PubMed2004

Alternative pathways of MYCN gene copy number increase in primary neuroblastoma tumors.

Neuroblastomas, tumors of the sympathetic nervous system, account for 7-10% of the cancers of childhood. Genetic studies have shown, and this study has confirmed, that neuroblastomas are very heterogeneous; no single genetic change common to all neuroblastomas has yet been identified. One genetic aberration found frequently in this pediatric tumor is MYCN gene amplification. Recently we identified a new subset of tumors showing MYCN gain (small increases in gene number arising from unbalanced translocation). To investigate whether gain precedes amplification or is an independent event, we surveyed 200 primary tumors for MYCN copy number with fluorescence in situ hybridization; 152 of 200 (76%) were MYCN single-copy tumors, whereas 48 of 200 (24%) tumors harbored MYCN abnormalities: 36 of the 48 (75%) had MYCN amplification and 12 (25%) had MYCN gain. Among the 36 with MYCN amplified gene, we found four that also showed gain. In three tumors exhibiting simultaneous gain and amplification, these two events were detected in neighboring cells. In the fourth case we detected only MYCN gain in metastatic neuroblasts in the bone marrow, but both MYCN amplification and gain in the primary tumor. The detailed study of these four cases suggests that there may be several different mechanisms leading to increase in MYCN copy number. Further studies in other human malignancies are necessary to determine whether simultaneous gain and amplification are specific to neuroblastoma or constitute a general mechanism by which tumor cells can acquire selective growth advantage.

Child↗

PubMed2004

The 18S rRNA from Odontophrynus americanus 2n and 4n (Amphibia, Anura) reveals unusual extra sequences in the variable region V2.

The nucleotide sequence of the rDNA 18S region isolated from diploid and tetraploid species of the amphibian Odontophrynus americanus was determined and used to predict the secondary structure of the corresponding 18S rRNA molecules. Comparison of the primary and secondary structures for the 2n and 4n species confirmed that these species are very closely related. Only three nucleotide substitutions were observed, accounting for 99% identity between the 18S sequences, whereas several changes were detected by comparison with the Xenopus laevis 18S sequence (96% identity). Most changes were located in highly variable regions of the molecule. A noticeable feature of the Odontophrynus 18S rRNA was the presence of unusual extra sequences in the V2 region, between helices 9 and 11. These extra sequences do not fit the model for secondary structure predicted for vertebrate 18S rRNA.

Animals↗

PubMed2004

[Cytogenetics, cytogenomics and cancer: 2004 update].

Cytogenetics has clearly established the key role chromosomal rearrangements play in neoplastic initiation and progression. Investigation methods have evolved considerably from banding analysis of chromosome morphology to fluorescence in situ hybridization (FISH) and now to comparative genomic hybridization (CGH) on chromosomes or micro-array analysis of DNA. In addition to its contributions to the description, prognosis and understanding of oncogenesis and tumor progression, cytogenomics provides the information required for the rational use of new targeted therapies among which Imatimib is the most achieved example.

Chromosome Aberrations↗

PubMed2003

Multiple effects of paclitaxel are modulated by a high c-myc amplification level.

Paclitaxel affects microtubule stability by binding to beta-tubulin, thus leading to cell accumulation in the G(2)/M phase, polyploidization, and apoptosis. Because both cell proliferation and apoptosis could be somehow regulated by the protooncogene c-myc, in this work we have investigated whether the c-myc amplification level could modulate the multiple effects of paclitaxel. To this aim, paclitaxel was administered to SW613-12A1 and -B3 human colon carcinoma cell lines (which are characterized by a high and low c-myc endogenous amplification level, respectively), and to the B3mycC5 cell line, with an enforced exogenous expression of c-myc copies. In this experimental system, we previously demonstrated that a high endogenous/exogenous level of amplification of c-myc enhances serum deprivation- and DNA damage-induced apoptosis. Accordingly, the present results indicate that a high c-myc amplification level potentiates paclitaxel cytotoxicity, confers a multinucleated phenotype, and promotes apoptosis to a great extent, thus suggesting that c-myc expression level is relevant in modulating the cellular responses to paclitaxel. We have recently shown in HeLa cells that the phosphorylated form of c-Myc accumulates in the nucleus, as distinct nucleolar and extranucleolar spots; here, we demonstrated that, after the treatment with paclitaxel, phosphorylated c-Myc undergoes redistribution, becoming diffused in the nucleoplasm.

Antineoplastic Agents, Phytogenic↗

PubMed2003

Effect of apoptogenic stimuli on colon carcinoma cell lines with a different c-myc expression level.

We have recently demonstrated that a high c-myc endogenous amplification level confers an apoptosis-prone phenotype to serum-deprived colon carcinoma SW613-S cells. The aim of this study was to gain new insights into the features of c-myc-dependent apoptosis, by extending our analysis to different apoptogenic stimuli. The study was carried out on clones, derived from the human colon carcinoma SW613-S cell line, which harbor different levels of endogenous c-myc amplification, and on isogenic cell lines with an enforced c-myc overexpression. Our results indicate that cells with endogenous or transfected exogenous c-myc overexpression (SW613-12A1 and -2G1mycP2Tu1 cell lines, respectively), activate the apoptotic machinery in response to the treatment with etoposide, doxorubicin and vitamin D3, which induce apoptosis through the death receptor Fas. The low levels of c-myc expression present in SW613-B3 and -B3mycC5, seem to be unable to activate Fas-mediated apoptosis, thus suggesting that only a high c-myc expression can bypass the lack of Fas receptor. Apoptosis induction mediated by DNA damage and long-term culture was independent of c-myc expression. A pathway of apoptosis characterized by the activation of the enzyme L-DNase II, was observed in both 12A1 and B3 cell lines.

Antineoplastic Agents↗

PubMed2002

Megakaryocyte polyploidization is associated with a functional gene amplification.

It is believed that polyploidy induces an orchestrated increase in gene expression. To know whether all alleles remain functional during megakaryocyte polyploidization, we used a well-established fluorescence in situ hybridization technique which allows one to simultaneously detect pre-mRNAs and assess ploidy level in a single cell. All alleles of GPIIb, GPIIIa, VWF, beta-actin, hsp70, c-mpl, Fli-1, and FOG-1 genes are transcriptionally active in megakaryocytes from 4N to 32N. All X chromosomes in male cells are transcriptionally active but only half of them are transcriptionally active in female megakaryocytes, as revealed by the transcriptional activity of the GATA-1 gene. Nuclear untranslated XIST RNA accumulates on the inactivated X chromosomes, indicating that they are subjected to a normal inactivation process. Altogether, our results demonstrate that megakaryocyte polyploidization results in a functional gene amplification whose likely function is an increase in protein synthesis parallel with cell enlargement.

Alleles↗

PubMed2002

Transcriptional deregulation of the keratin 18 gene in human colon carcinoma cells results from an altered acetylation mechanism.

We are investigating the mechanism responsible for the overexpression of the keratin 18 (K18) gene in tumorigenic clones from the SW613-S human colon carcinoma cell line, as compared with non-tumorigenic clones. We have previously shown that this mechanism affects the minimal K18 promoter (TATA box and initiation site). We report here that treatment of the cells with histone deacetylase inhibitors stimulates the activity of the promoter in non-tumorigenic cells but has no effect in tumorigenic cells, resulting in a comparable activity of the promoter in both cell types. The adenovirus E1A protein inhibits the activity of the K18 promoter specifically in tumorigenic cells. This inhibition can be reversed by an excess of CBP protein. The conserved region 1 (CR1) of E1A, which is involved in the interaction with the CBP/p300 co-activators, is necessary to the inhibitory capacity of E1A. A 79 amino acid long N-terminal fragment of E1A, encompassing the two domains of E1A necessary and sufficient for binding to CBP (N-terminus and CR1), has the same differential inhibitory capacity on the K18 promoter as wild-type E1A. Forced recruitment of GAL4-CBP fusion proteins to the K18 promoter results in a greater stimulation of its activity in non-tumorigenic than in tumorigenic cells. The histone acetyltransferase activity of CBP is essential for this differential stimulation and the presence of the CBP2 domain greatly augments the activation capacity of the fusion protein. Chromatin immunoprecipitation experiments carried out with anti-acetylated histone antibodies showed no difference in the level of histone acetylation in the region of the K18 promoter between the two cell types. The structure of chromatin in the promoter region is similar in tumorigenic and non-tumorigenic cells, as determined by mapping of DNase I hypersensitive sites and probing the accessibility of the DNA to restriction endonucleases. From all these results we conclude that alteration of an acetylation mechanism involving the CBP (or p300) protein and acting on a non-histone substrate is responsible for the higher activity of the K18 promoter in tumorigenic cells of the SW613-S cell line.

Acetylation↗

PubMed2002

Molecular evolution of ribosomal intergenic spacers in Odontophrynus americanus 2n and 4n (Amphibia: Anura).

Ribosomal intergenic spacers (IGSs) of Odontophrynus americanus 2n and 4n were cloned, restriction mapped, and partially sequenced. Three distinct regions, namely alpha, beta, and delta, were identified in the IGSs. The alpha and beta regions flanked the 28S and 18S rRNA genes, respectively, conserving an identical restriction pattern at each ploidy level. The delta region, located between alpha and beta, was highly variable in size and restriction pattern, enclosing different BamHI subrepeats (B-SR), 87- to 530-bp-long. Sequence analysis showed that B-SRs were composed mainly of different arrangements of similar blocks of sequences. Another family of repetitive sequences was found in the delta region, clustered inside large BamHI fragments. These subrepeats are 189-bp-long and, although very similar in diploid and tetraploid IGSs, show a pattern of concerted evolution. A hypothetical functional role for the 189-bp repeats is discussed in view of their predicted secondary structure and presence of potential E2 binding sites inside diploid subrepeats. Although the same structural elements were present both in diploid and tetraploid IGSs, the higher level of repeatability of tetraploid IGSs suggests that common ancestor sequences have undergone several rounds of amplification after O. americanus polyploidy.

Animals↗