Biomedical subjects

Cédric Malicet

Publications and source records attributed to Cédric Malicet.

9 recordsLinked to original sources

PubMed2006

p8 and prothymosin alpha: unity is strength.

p8 and prothymosin alpha are two natively unstructured proteins with anti-apoptotic activity. We showed that their interaction results in the formation of a one-to-one heterodimer complex with stable structure. To test whether the heterodimer bears the function previously attributed to both proteins, we monitored the consequences on apoptosis of modulating in vitro the concentrations of both proteins. Overexpression was obtained by transfection of appropriate vectors and inhibition by using specific siRNAs. In all conditions inhibition of apoptosis correlated with the level of the partner with lowest concentration, demonstrating that the anti-apoptotic effect previously attributed to each proteins was in fact borne by the p8/ProTalpha complex, the two proteins, being individually inactive. These results show that the function attributed to a natively unfolded protein might actually belong to a multi-protein complex in which the protein of interest is engaged.

Apoptosis↗

PubMed2006

Cell growth-dependent subcellular localization of p8.

p8 is a stress-induced protein, biochemically related to the architectural factor HMG-I/Y, overexpressed in many cancers and required for tumor expansion. The molecular mechanisms by which p8 may exert its effect in aspects of growth is unknown. Using immunocytochemistry, we found that p8 presents nuclear localization in sub-confluent cells, but it localizes throughout the whole cell in high density grown cells. Cells arrested in Go/G1, either by serum deprivation or by hydroxyurea treatment, show a nucleo-cytoplasmic localization of p8, whether in the rest of the cell cycle stages of actively dividing cells the localization is nuclear. A comparison of p8 sequences from human to fly predicts a conserved bipartite nuclear localization sequence (NLS). The putative NLS has been demonstrated to be functional, since nuclear import is energy dependent (inhibited by sodium azide plus 2-deoxyglucose), and fusion proteins GFP-p8 and GFP-NLSp8 localize to the nucleus, whereas GFP-p8NLSmut in which with Lys 65, 69, 76, and 77 mutated to Ala localized to the whole cell. p8 localization does not involve the CRM1 transporter, since it is insensitive to leptomycin B. Inhibitors of MAPK pathways did not affect p8 subcellular localization. The inhibition of deacetylation with Trichostatin A promotes cytoplasmic accumulation of p8. The results suggest that p8 growth stage-dependent localization is regulated by acetylation, that p8 is not free within the cell but forming part of a complex and that it may exert a role in both subcellular localizations.

Acetylation↗

PubMed2006

Regulation of apoptosis by the p8/prothymosin alpha complex.

p8 is a small-stress protein involved in several cellular functions including apoptosis. To identify its putative partners, we screened a HeLa cDNA library by using the two-hybrid technique and found that p8 binds the antiapoptotic protein prothymosin alpha (ProTalpha). Fluorescence spectroscopy, circular dichroism, and NMR spectroscopy showed that p8 and ProTalpha formed a complex. Binding resulted in important changes in the secondary and tertiary structures of the proteins. Because p8 and ProTalpha form a complex, they could act in concert to regulate the apoptotic cascade. We induced apoptosis in HeLa cells by staurosporine treatment and monitored the effects of knocking down p8 and/or ProTalpha or overexpressing p8 and/or ProTalpha on caspase 3/7 and 9 activities and on cell death. Transfecting ProTalpha or p8 small interfering RNAs increased the activities of both caspases and the number of apoptotic nuclei. However, transfecting both small interfering RNAs resulted in no further increase. Overexpressing p8 or ProTalpha did not alter caspase activities, whereas overexpressing both resulted in a significant reduction of caspase activities. These results strongly suggest that the antiapoptotic response of HeLa cells upon staurosporine treatment requires expression of both p8 and ProTalpha.

Apoptosis↗

PubMed2006

p8 is a new target of gemcitabine in pancreatic cancer cells.

Gemcitabine is the only available chemotherapeutic treatment of pancreatic cancers. It is, however, moderately effective, showing a tumor response rate of only 12%. The aim of this work was to identify new pathways involved in the resistance of pancreatic cancer cells to gemcitabine, in the hope of developing new adjuvant strategies to enhance its therapeutic efficacy. Comparison of gene expression patterns of five human pancreatic cancer cell lines showing different degrees of resistance to gemcitabine revealed specific overexpression of several genes in the most resistant. One of them encoded the antiapoptotic p8 protein. We found that (a) knocking down p8 expression in gemcitabine-resistant cells promoted cell death and increased caspase-3 activity; (b) forced overexpression of p8 in gemcitabine-sensitive cells increased their resistance to gemcitabine-induced apoptosis; and (c) gemcitabine down-regulated p8 mRNA expression. These results suggest that, in pancreatic cancer cells, a large part of gemcitabine-induced apoptosis results from the inhibition of the constitutive antiapoptotic activity of p8. Hence, targeting the p8-associated pathway could be a new adjuvant therapy improving the response of patients with pancreatic cancer to gemcitabine treatment.

Antimetabolites, Antineoplastic↗

PubMed2005

Interaction of the stress protein p8 with Jab1 is required for Jab1-dependent p27 nuclear-to-cytoplasm translocation.

p8 is an 80 amino-acid polypeptide identified because of its remarkable over-expression in the stressed pancreas. This protein, apparently devoid of enzymatic activity, is a powerful regulator of several intracellular pathways, suggesting that it has to interact with several molecular partners to modulate their activity. We used two-hybrid screening of a pre-transformed human testes cDNA library to identify some of these partners. One of them was the multifunctional protein Jab1, its interaction with p8 being confirmed by His6-pull down and co-immunoprecipitation assays. In addition, we could show that the two proteins co-localized in the cell. Our functional data demonstrate that Jab1 requires direct interaction with p8 to induce the translocation of p27 from nucleus to cytoplasm and its subsequent degradation. Experiments showing that the knock-down of p8 expression results in a strong inhibition of Jab1 activity confirmed that the mechanism by which Jab1 promotes cell growth by decreasing p27 level is p8-dependent.

Active Transport, Cell Nucleus↗

PubMed2005

Gene expression profiling of tumours derived from rasV12/E1A-transformed mouse embryonic fibroblasts to identify genes required for tumour development.

BACKGROUND: In cancer, cellular transformation is followed by tumour development. Knowledge on the mechanisms of transformation, involving activation of proto-oncogenes and inactivation of tumour-suppressor genes has considerably improved whereas tumour development remains poorly understood. An interesting way of gaining information on tumour progression mechanisms would be to identify genes whose expression is altered during tumour formation. We used the Affymetrix-based DNA microarray technology to analyze gene expression profiles of tumours derived from rasV12/E1A-transformed mouse embryo fibroblasts in order to identify the genes that could be involved in tumour development. RESULTS: Among the 12,000 genes analyzed in this study, only 489 showed altered expression during tumour development, 213 being up-regulated and 276 down-regulated. The genes differentially expressed are involved in a variety of cellular functions, including control of transcription, regulation of mRNA maturation and processing, regulation of protein translation, activation of interferon-induced genes, intracellular signalling, apoptosis, cell growth, angiogenesis, cytoskeleton, cell-to-cell interaction, extracellular matrix formation, metabolism and production of secretory factors. CONCLUSIONS: Some of the genes identified in this work, whose expression is altered upon rasV12/E1A transformation of MEFs, could be new cancer therapeutic targets.

Adenovirus E1A Proteins↗

PubMed2005

Inactivation of stress protein p8 increases murine carbon tetrachloride hepatotoxicity via preserved CYP2E1 activity.

The p8 protein is a transcription factor that regulates the expression of genes involved in cell defense against the adverse effects of stress. Its expression is strongly, rapidly, and transiently induced in most cells on exposure to various stress agents. This study assessed the role of p8 in the response of the liver to CCl(4)-induced injury. We found that p8 was indeed overexpressed in the liver after CCl(4) administration. Hepatic injury following CCl(4) injection was monitored in wild-type and p8(-/-) mice. Serum alanine and aspartate aminotransferase activities were higher and peaked earlier in p8(-/-) mice than in wild-type mice, which is in agreement with the observation of significantly larger areas of necrosis in p8(-/-) liver. Absence of p8 expression is therefore associated with increased liver sensitivity to CCl(4). In fact, CCl(4) toxicity is mediated by derivatives generated by its conversion by the enzyme CYP2E1. It is known that CYP2E1 is downregulated in the liver during the first hours following CCl(4) administration as part of a self-defense mechanism. We found that CYP2E1 downregulation was significantly delayed in p8(-/-) liver compared with wild-type liver, allowing increased production of toxic CCl(4) derivatives. In conclusion, inactivation of the p8 gene increases liver sensitivity to CCl(4), as it appears to delay the triggering of CYP2E1 downregulation. The p8 protein is therefore an important element of hepatocyte stress response.

Animals↗

PubMed2003

p8 inhibits the growth of human pancreatic cancer cells and its expression is induced through pathways involved in growth inhibition and repressed by factors promoting cell growth.

BACKGROUND: p8 is a stress-induced protein with multiple functions and biochemically related to the architectural factor HMG-I/Y. We analyzed the expression and function of p8 in pancreatic cancer-derived cells. METHODS: Expression of p8 was silenced in the human pancreatic cancer cell lines Panc-1 and BxPc-3 by infection with a retrovirus expressing p8 RNA in the antisense orientation. Cell growth was measured in control and p8-silenced cells. Influence on p8 expression of the induction of intracellular pathways promoting cellular growth or growth arrest was monitored. RESULTS: p8-silenced cells grew more rapidly than control cells transfected with the empty retrovirus. Activation of the Ras-->Raf-->MEK-->ERK and JNK intracellular pathways down-regulated p8 expression. In addition, the MEK1/2 inhibitor U0126 and the JNK inhibitor SP600125 up-regulates expression of p8. Conversely, p38 or TGFbeta-1 induced p8 expression whereas the specific p38 inhibitor SB203580 down-regulated p8 expression. Finally, TGFbeta-1 induction was in part mediated through p38. CONCLUSIONS: p8 inhibits the growth of human pancreatic cancer cells. p8 expression is induced through pathways involved in growth inhibition and repressed by factors that promote cell growth. These results suggest that p8 belongs to a pathway regulating the growth of pancreatic cancer cells.

Anthracenes↗

PubMed2003

Gene expression profiling by DNA microarray analysis in mouse embryonic fibroblasts transformed by rasV12 mutated protein and the E1A oncogene.

BACKGROUND: Ras is an area of intensive biochemical and genetic studies and characterizing downstream components that relay ras-induced signals is clearly important. We used a systematic approach, based on DNA microarray technology to establish a first catalog of genes whose expression is altered by ras and, as such, potentially involved in the regulation of cell growth and transformation. RESULTS: We used DNA microarrays to analyze gene expression profiles of rasV12/E1A-transformed mouse embryonic fibroblasts. Among the approximately 12,000 genes and ESTs analyzed, 815 showed altered expression in rasV12/E1A-transformed fibroblasts, compared to control fibroblasts, of which 203 corresponded to ESTs. Among known genes, 202 were up-regulated and 410 were down-regulated. About one half of genes encoding transcription factors, signaling proteins, membrane proteins, channels or apoptosis-related proteins was up-regulated whereas the other half was down-regulated. Interestingly, most of the genes encoding structural proteins, secretory proteins, receptors, extracellular matrix components, and cytosolic proteins were down-regulated whereas genes encoding DNA-associated proteins (involved in DNA replication and reparation) and cell growth-related proteins were up-regulated. These data may explain, at least in part, the behavior of transformed cells in that down-regulation of structural proteins, extracellular matrix components, secretory proteins and receptors is consistent with reversion of the phenotype of transformed cells towards a less differentiated phenotype, and up-regulation of cell growth-related proteins and DNA-associated proteins is consistent with their accelerated growth. Yet, we also found very unexpected results. For example, proteases and inhibitors of proteases as well as all 8 angiogenic factors present on the array were down-regulated in transformed fibroblasts although they are generally up-regulated in cancers. This observation suggests that, in human cancers, proteases, protease inhibitors and angiogenic factors could be regulated through a mechanism disconnected from ras activation. CONCLUSIONS: This study established a first catalog of genes whose expression is altered upon fibroblast transformation by rasV12/E1A. This catalog is representative of the genome but not exhaustive, because only one third of expressed genes was examined. In addition, contribution to ras signaling of post-transcriptional and post-translational modifications was not addressed. Yet, the information gathered should be quite useful to future investigations on the molecular mechanisms of oncogenic transformation.

Adenovirus E1A Proteins↗