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Alain Puisieux

Publications and source records attributed to Alain Puisieux.

At least 19 recordsLinked to original sources
PubMed2006

Prognostic significance of urokinase plasminogen activator and plasminogen activator inhibitor-1 mRNA expression in lymph node- and hormone receptor-positive breast cancer.

BACKGROUND: One of the most thoroughly studied systems in relation to its prognostic relevance in patients with breast cancer, is the plasminogen activation system that comprises of, among others, the urokinase Plasminogen Activator (uPA) and its main inhibitor, the Plasminogen Activator Inhibitor-1 (PAI-1). In this study, we investigated the prognostic value of uPA and PAI-1 at the mRNA level in lymph node- and hormone receptor-positive breast cancer. METHODS: The study included a retrospective series of 87 patients with hormone-receptor positive and axillary lymph node-positive breast cancer. All patients received radiotherapy, adjuvant anthracycline-based chemotherapy and five years of tamoxifen treatment. The median patient age was 54 and the median follow-up time was 79 months. Distant relapse occurred in 30 patients and 22 patients died from breast cancer during follow-up. We investigated the prognostic value of uPA and PAI-1 at the mRNA level as measured by real-time quantitative RT-PCR. RESULTS: uPA and PAI-1 gene expression was not found to be correlated with any of the established clinical and pathological factors. Metastasis-free Survival (MFS) and Breast Cancer specific Survival (BCS) were significantly shorter in patients expressing high levels of PAI-1 mRNA (p < 0.0001; p < 0.0001; respectively). In Cox multivariate analysis, the level of PAI-1 mRNA appeared to be the strongest prognostic factor for MFS (Hazard Ratio (HR) = 10.12; p = 0.0002) and for BCS (HR = 13.17; p = 0.0003). Furthermore, uPA gene expression was not significantly associated neither with MFS (p = 0.41) nor with BCS (p = 0.19). In a Cox-multivariate regression analysis, uPA expression did not demonstrate significant independent prognostic value. CONCLUSION: These findings indicate that high PAI-1 mRNA expression represents a strong and independent unfavorable prognostic factor for the development of metastases and for breast cancer specific survival in a population of hormone receptor- and lymph node-positive breast cancer patients.

Breast Neoplasms↗
PubMed2006

UVB-induced G2 arrest of human melanocytes involves Cdc2 sequestration by Gadd45a in nuclear speckles.

Exposure to solar UVB radiation is involved in the development of cutaneous melanoma. We previously showed that human melanocytes and melanoma cells respond to UVB radiation via a p53-independent pathway involving GADD45A activation. Here, we determined that UVB-induction of Gadd45a is necessary for G(2) arrest and that Gadd45a and its partner p21(Waf1) colocalize in nuclear bodies called Nuclear Speckles. We further observed that UVB-induced G(2) arrest is associated with Cdc2 accumulation in these Nuclear Speckles. Knock-down of Gadd45a expression by RNA interference prevents both UVB-induced Cdc2 accumulation in Nuclear Speckles and G(2) arrest. Our results demonstrate that UVB-induced G(2) arrest of melanoma cells is Gadd45a-dependent. Furthermore, we show that Cdc2 sequestration by Gadd45a occurs in Nuclear Speckles, suggesting a new role for these nuclear bodies, so far only linked to RNA maturation.

CDC2 Protein Kinase↗
PubMed2006

A p21/WAF1 mutation favors the appearance of drug resistance to paclitaxel in human noncancerous epithelial mammary cells.

We investigated the mechanisms responsible for paclitaxel resistance in HME-1 cells (human mammary epithelial cells immortalized with hTERT). These cells were exposed to paclitaxel (10 pM for 7 days) and 20 cellular surviving populations (PSP) were obtained. PSP demonstrated high levels of resistance to paclitaxel cytotoxicity as compared with HME-1 cells. Activation of mdr-1 gene expression was observed in 2 PSP. Protein expression analysis using a C-terminal targeted antibody showed that 13 PSP were negative for p21/WAF1 expression after ionizing radiation (6 Gy) or doxorubicin (100 nM) treatment. Sequencing of the 3 exons of the CDKN1A gene revealed that 13 PSP contained a point mutation in exon 2. This mutation consisted in a T insertion at codon 104 leading to a premature STOP codon appearance. Mismatch amplification mutation assay and RFLP-PCR confirmed the presence of the mutation in 16 PSP. Western blot using an N-terminal targeted antibody demonstrated that the C-terminal-truncated p21/WAF1 protein (14 kDa) was indeed expressed in the 13 PSP. Our data suggest that p21/WAF1 inactivation may confer a strong resistance to paclitaxel in noncancerous breast epithelial cells harboring a p21/WAF1 mutant.

Amino Acid Sequence↗
PubMed2006

p53 as a target for anti-cancer drug development.

Loss of p53 function compromises genetic homeostasis in cells exhibiting deregulated DNA replication and/or DNA damage, and prevents normal cytotoxic responses to cancer therapies. Genetic and pharmacological approaches are being developed with the ultimate goal of restoring or controlling p53 functions in cancer patients. Progress has recently been made in the clinical use of replication-deficient virus carrying wt-TP53 (Ad5CMV-p53) and/or cancer-selective oncolytic adenoviruses (ONYX-015). These strategies demonstrated clinical activity as monotherapy and were synergistic with traditional chemotherapy agents in the treatment of some types of cancer. In addition, pharmacological methods are under development to either stimulate wild-type p53 protein function, or induce p53 mutant proteins to resume wild-type functions. These methods are based on small chemicals (CP-31388, PRIMA-1), peptides (CDB3) or single-chain Fv antibody fragments corresponding to defined p53 domains. Here, we discuss the mechanisms underlying these approaches and their perspectives for cancer therapy.

Adenoviridae↗
PubMed2006

[Cancer cells escape from failsafe programs in a simple Twist].

A major obstacle to the expansion of abnormal cells with significant proliferative potential is the induction of either cellular senescence or programmed cell death. Consequently, oncogene-driven hyperproliferation must be associated with apoptosis inhibition to allow malignant outgrowth. The oncogenic cooperation of N-Myc and Twist 1 in the development of neuroblastoma, the most common and deadly solid tumour of childhood, perfectly illustrates such a process. N-Myc promotes cell proliferation whereas Twist 1 counteracts its pro-apoptotic properties by knocking-down the ARF/p53 pathway. This observation provides a mechanistic explanation for the rarity of p53 mutations in neuroblastomas. It also highlights the involvement of two crucial regulators of embryogenesis in human cancer development. In this review, we discuss the possible role of Twist 1 in tumour progression, based on the numerous recent studies reporting its overexpression in a variety of human cancers.

Animals↗
PubMed2006

Systematic mRNA analysis for the effect of MLH1 and MSH2 missense and silent mutations on aberrant splicing.

A substantial proportion of MLH1 and MSH2 gene mutations in hereditary nonpolyposis colon cancer syndrome (HNPCC) families are characterized by nucleotide substitutions, either within the coding sequence (missense or silent mutations) or in introns. The question of whether these mutations affect the normal function of encoding mismatch DNA repair proteins and thus lead to the predisposition to cancer is determinant in genetic testing. Recent studies have suggested that some nucleotide substitutions can induce aberrant splicing by disrupting cis-transcription elements such as exonic enhancers (ESEs). ESE disruption has been proposed to be the mechanism that underlies the presumed pathological missense mutations identified in HNPCC families. To investigate the prevalence of aberrant splicing resulting from nucleotide substitutions, and its relevance to predicted ESEs, we conducted a systematic RNA screening of a series of 60 patients who carried unrelated exonic or intronic mutations in MLH1 or MSH2 genes. Aberrant splicing was found in 15 cases, five of which were associated with exonic mutations. We evaluated the link between those splicing mutations and predicted putative ESEs by using the computational tools ESEfinder and RESCUE-ESE. Our study shows that the algorithm-based ESE prediction cannot be definitely correlated to experimental observations from RNA screening. By using minigene constructs and in vitro transcription assay, we demonstrated that nucleotide substitutions are the direct cause of the splicing defect. This is the first systematic screening for the effect of missense and silent mutations on splicing in HNPCC patients. The pathogenic splicing mutations identified in this study will contribute to the assessment of "unclassified variants" in genetic counseling. Our results also suggest that one must use caution when determining the pathogenic effect of a missense or silent mutation using ESE prediction algorithms. Analysis at the RNA level is therefore necessary.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Mutational targets in colorectal cancer cells with microsatellite instability.

Cancers arise from the sequential acquisition of genetic alterations in specific genes. The high number of mutations in cancer cells led to the hypothesis that an early step in tumor progression is the generation of a genetic instability. The potent role of genetic instability in initiation and progression of colorectal cancers has been well defined in hereditary nonpolyposis colon cancer (HNPCC) syndrome. HNPCC is a common hereditary disorder caused by germline mutations of DNA mismatch repair (MMR) genes. Somatic loss of the normal allele of the predisposition gene leads to a strong "mutator phenotype", characterized by a high rate of mutations in repetitive sequences. Nevertheless, the observation of frequent alterations of key growth regulatory genes in MMR-deficient cells such as NF1, APC, p53, K-Ras, with no significant excess of frameshift mutations and changes at short coding repeats, suggest that even in the presence of an inherited tendency to genomic instability, tumor progression is mainly driven by a process of natural selection.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Metastasis: a question of life or death.

The metastatic process is highly inefficient--very few of the many cells that migrate from the primary tumour successfully colonize distant sites. One proposed mechanism to explain this inefficiency is provided by the cancer stem cell model, which hypothesizes that micrometastases can only be established by tumour stem cells, which are few in number. However, recent in vitro and in vivo observations indicate that apoptosis is an important process regulating metastasis. Here we stress that the inhibition of cell death, apart from its extensively described function in primary tumour development, is a crucial characteristic of metastatic cancer cells.

Animals↗
PubMed2006

[Immunotherapy of poor-prognosis neuroblastoma in children: from bench to bedside].

During the last two decades, improvements in the induction and consolidation treatment phases in patients with high-risk neuroblastoma have not translated into significant increases in survival rates. Efforts to improve outcome have used high-dose chemotherapy with stem cell rescue and more recently, differentiating (retinoids) and antiangiogenic agents. In parallel, immunotherapy has become an increasingly important part of the treatment of high-risk neuroblastoma. We review here the biological concepts underlying these new approaches and their clinical applications, with a particular emphasis on applications that manipulate the immune system, including monoclonal antibodies, gene-modified tumor cells (vaccines) or immune effectors.

Child↗
PubMed2005

Protein chip array profiling analysis of sera from neuroblastoma patients.

Neuroblastoma, the most common extracranial solid tumour in children, is characterised by highly heterogeneous clinical behaviour; patients are stratified into risk categories according to a combination of clinical and biological markers. However, identifying non-invasive prognostic markers predicting outcome independently from current risk-stratification features remains critical for better disease monitoring. Using the SELDI-TOF-MS technology (surface-enhanced laser desorption/ionization time-of-flight mass spectrometry), we found a serum biomarker that strongly correlates with prognosis in neuroblastoma patients. Subsequent peptide mapping identified this biomarker as SAA protein. In support of this observation, high SAA levels were detected by ELISA in the sera of patients with poor prognosis neuroblastoma. Based on this finding, promises and limitations of the approach are discussed.

Enzyme-Linked Immunosorbent Assay↗
PubMed2005

C. elegans homologue of the Caf1 gene, which encodes a subunit of the CCR4-NOT complex, is essential for embryonic and larval development and for meiotic progression.

The evolutionary conserved CCR4-NOT multi subunit complex is involved in different aspects of mRNA metabolism, including mRNA synthesis initiation and mRNA deadenylation (shortening of the poly(A) tail) in yeast and higher eukaryotes. Here we report the characterization of the gene encoding the Caf1 subunit of this complex in Caenorhabditis elegans, ccf-1, and the phenotypes associated with its inactivation. Use of staged populations and of mutants strains with altered germline showed that ccf-1 is predominantly expressed in embryos and adults. Loss of ccf-1 function, by both RNAi and a deletion allele, caused early embryonic and larval lethality. It also resulted in sterility in both males and hermaphrodites by blocking germ cell development at the pachytene stage of meiosis I. These results reveal that ccf-1 is an essential factor for both somatic and germline development in C. elegans. Functional analysis of ccf-1 may contribute to the understanding of the molecular role of the CCR4-NOT complex.

Animals↗
PubMed2005

The neurogene BTG2TIS21/PC3 is transactivated by DeltaNp73alpha via p53 specifically in neuroblastoma cells.

The p53 gene and its homologue p73 are rarely mutated in neuroblastoma. In recent studies, we showed that overexpression of DeltaNp73alpha, an isoform lacking the N-terminal transactivation (TA) domain, surprisingly induces p53 protein accumulation in the wild-type (wt) p53 human neuroblastoma line SH-SY5Y. As can be expected owing to its dominant-negative effect, DeltaNp73alpha inhibits Waf1/p21 gene expression, but equally importantly, it upregulates BTG2TIS21/PC3, another p53 target gene. This effect is not observed in neuroblastoma cells that express a mutated p53. To better understand the DeltaNp73-mediated transactivation of the BTG2TIS21/PC3 gene we performed luciferase assays with two reporter plasmids harboring long and short BTG2 promoter sequences in three human neuroblastoma cell lines and one breast cancer cell line. Our results demonstrate that BTG2TIS21/PC3 transactivation by DeltaNp73alpha depends on both p53 status (as it is not observed in a p53-/- neuroblastoma cell line) and cellular context (as it occurs in a p53+/+ neuroblastoma cell line but not in a p53+/+ breast tumor cell line). The fact that DeltaNp73alpha may either inhibit or stimulate wt-p53 transcriptional activity, depending on both the p53 target gene and the cellular context, was confirmed by real-time quantitative PCR. Moreover, transactivation of the BTG2TIS21/PC3 promoter requires a complete DeltaNp73alpha C-terminus sequence as it is not observed with DeltaNp73beta, which lacks most of the C-terminal domain. We have previously shown that DeltaNp73alpha is the only p73 isoform expressed in undifferentiated neuroblastoma tumors. In light of all these findings, we propose that DeltaNp73alpha not only acts as an inhibitor of p53/TAp73 functions in neuroblastoma tumors, but also cooperates with wt-p53 in playing a physiological role through the activation of BTG2TIS21/PC3 gene expression.

Adenoviridae↗
PubMed2004

Genome-wide analysis of gene expression in neuroblastomas detected by mass screening.

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood and the third most common pediatric cancer. Although numerous factors including patient age, disease stage and genetic abnormalities have been shown to be predictive of outcome, the mechanisms responsible for the highly variable clinical behavior of this tumor remain largely unknown. In order to gain new insights into the biology of this tumor, we performed microarray analysis and compared the gene expression patterns of NB detected by mass screening, characterized by highly probable spontaneous regression, versus stage 4 NB with poor prognosis. The bioinformatics analysis revealed a set of 19 discriminatory genes that may play a significant role in the natural progression of the disease. Validation of these results and further mechanistic studies would shed new light on the biology of tumor progression, and provide new tools to predict clinical outcome in children with NB.

Child, Preschool↗
PubMed2004

Oncogenic cooperation between H-Twist and N-Myc overrides failsafe programs in cancer cells.

N-Myc oncogene amplification is a frequent event in neuroblastoma and is strongly correlated with advanced disease stage and treatment failure. Similarly to c-Myc oncogenic activation, N-Myc deregulation promotes both cell proliferation and p53-dependent apoptosis by sensitizing cells to a variety of insults. Intriguingly, p53 mutations are uncommon in neuroblastomas, strongly suggesting that an alternative cooperating event circumvents this safeguard against oncogene-driven neoplasia. By performing a pangenomic cDNA microarray analysis, we demonstrate that human Twist is constantly overexpressed in N-Myc-amplified neuroblastomas. H-Twist overexpression is responsible for the inhibition of the ARF/p53 pathway involved in the Myc-dependent apoptotic response. This oncogenic cooperation of two key regulators of embryogenesis causes cell transformation and malignant outgrowth.

Animals↗
PubMed2003

Chfr inactivation is not associated to chromosomal instability in colon cancers.

Numerous observations suggest that chromosome instability is caused by mitotic abnormalities such as errors in the partitioning of chromosomes. Chfr was recently defined as a central component of a new mitotic checkpoint that delays chromosome condensation in response to mitotic stress. Chfr was shown to be frequently inactivated in several human neoplasms, including colon, lung and esophageal cancers. To test whether Chfr inactivation may lead or participate to chromosomal instability (CIN), we analysed the genetic and epigenetic status of the gene in a large panel of primary colon and breast cancers, as well as in colon and breast cancer cell lines displaying either a microsatellite instability or a CIN. Our results confirm that Chfr is frequently inactivated in colon cancers, through a mechanism of hypermethylation of the promoter sequences. In contrast, the loss of Chfr expression appears to be a rare event in breast cancers. Furthermore, our data demonstrate that Chfr inactivation is not associated with CIN in these frequent types of human cancers.

Cell Cycle Proteins↗
PubMed2003

Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions.

BACKGROUND: Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive. RESULTS: We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy. CONCLUSION: Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.

Benzothiazoles↗
PubMed2003

Alterations of anaphase-promoting complex genes in human colon cancer cells.

Ubiquitin-mediated proteolysis of cell cycle regulators is a major element of the cell cycle control. The anaphase-promoting complex (APC/C) is a large multisubunit ubiquitin-protein ligase required for the ubiquitination and degradation of G1 and mitotic checkpoint regulators. APC/C-dependent proteolysis regulates cyclin levels in G1, and triggers the separation of sister chromatids at the metaphase-anaphase transition and the destruction of mitotic cyclins at the end of mitosis. Furthermore, it was recently shown that APC/C regulates the degradation of crucial regulators of signal transduction pathways. We report here gene alterations in several components of this complex in human colon cancer cells, including APC6/CDC16 and APC8/CDC23 which are known to be key function elements. The experimental expression of a truncation mutant of APC8/CDC23 subunit (CDC23DeltaTPR) leads to abnormal levels of APC/C targets such as cyclin B1 and disturbs the cell cycle progression of colon epithelial cells through mitosis. Overall, these data support the hypothesis of a deleterious role of these mutations during colorectal carcinogenesis.

Anaphase-Promoting Complex-Cyclosome↗