G1 phase arrest by the phosphatidylinositol 3-kinase inhibitor LY 294002 is correlated to up-regulation of p27Kip1 and inhibition of G1 CDKs in choroidal melanoma cells

AbstractWe have investigated the effect of the flavonoid derivative LY 294002, a potent and selective phosphatidylinositol 3-kinase inhibitor, on cell cycle progression in human choroidal melanoma cells. We demonstrate that LY 294002 induces a specific G1 block in asynchronously growing cells leading to an almost complete inhibition of cell proliferation after three days of treatment. When melanoma cells are released from a nocodazole-induced G2/M block, LY 294002 is shown to delay and greatly restrain the G1/S transition. The inhibitor is able to exert its action as long as it is added during the G1 progression and before the cells enter in S phase. We report that the LY 294002-induced G1 arrest is closely correlated to inhibition of CDK4 and CDK2 activities leading to the impairment of pRb phosphorylation which normally occurs during G1 progression. While the inhibition of CDK4 may be attributed at least in part to the decline in CDK4 protein level, CDK2 activity reduction is rather due to the up-regulation of the CDK inhibitor p27Kip1 and to its increased association to CDK2

The interaction between the measles virus nucleoprotein and the Interferon Regulator Factor 3 relies on a specific cellular environment

<p>Abstract</p> <p>Background</p> <p>The genome of measles virus consists of a non-segmented single-stranded RNA molecule of negative polarity, which is encapsidated by the viral nucleoprotein (N) within a helical nucleocapsid. The N protein possesses an intrinsically disordered C-terminal domain (aa 401–525, N_TAIL) that is exposed at the surface of the viral nucleopcapsid. Thanks to its flexible nature, N_TAILinteracts with several viral and cellular partners. Among these latter, the Interferon Regulator Factor 3 (IRF-3) has been reported to interact with N, with the interaction having been mapped to the regulatory domain of IRF-3 and to N_TAIL. This interaction was described to lead to the phosphorylation-dependent activation of IRF-3, and to the ensuing activation of the pro-immune cytokine RANTES gene.</p> <p>Results</p> <p>After confirming the reciprocal ability of IRF-3 and N to be co-immunoprecipitated in 293T cells, we thoroughly investigated the N_TAIL-IRF-3 interaction using a recombinant, monomeric form of the regulatory domain of IRF-3. Using a large panel of spectroscopic approaches, including circular dichroism, fluorescence spectroscopy, nuclear magnetic resonance and electron paramagnetic resonance spectroscopy, we failed to detect any direct interaction between IRF-3 and either full-length N or N_TAILunder conditions where these latter interact with the C-terminal X domain of the viral phosphoprotein. Furthermore, such interaction was neither detected in <it>E. coli </it>nor in a yeast two hybrid assay.</p> <p>Conclusion</p> <p>Altogether, these data support the requirement for a specific cellular environment, such as that provided by 293T human cells, for the N_TAIL-IRF-3 interaction to occur. This dependence from a specific cellular context likely reflects the requirement for a human or mammalian cellular co-factor.</p

A new molecular breast cancer subclass defined from a large scale real-time quantitative RT-PCR study

BACKGROUND: Current histo-pathological prognostic factors are not very helpful in predicting the clinical outcome of breast cancer due to the disease's heterogeneity. Molecular profiling using a large panel of genes could help to classify breast tumours and to define signatures which are predictive of their clinical behaviour. METHODS: To this aim, quantitative RT-PCR amplification was used to study the RNA expression levels of 47 genes in 199 primary breast tumours and 6 normal breast tissues. Genes were selected on the basis of their potential implication in hormonal sensitivity of breast tumours. Normalized RT-PCR data were analysed in an unsupervised manner by pairwise hierarchical clustering, and the statistical relevance of the defined subclasses was assessed by Chi2 analysis. The robustness of the selected subgroups was evaluated by classifying an external and independent set of tumours using these Chi2-defined molecular signatures. RESULTS: Hierarchical clustering of gene expression data allowed us to define a series of tumour subgroups that were either reminiscent of previously reported classifications, or represented putative new subtypes. The Chi2 analysis of these subgroups allowed us to define specific molecular signatures for some of them whose reliability was further demonstrated by using the validation data set. A new breast cancer subclass, called subgroup 7, that we defined in that way, was particularly interesting as it gathered tumours with specific bioclinical features including a low rate of recurrence during a 5 year follow-up. CONCLUSION: The analysis of the expression of 47 genes in 199 primary breast tumours allowed classifying them into a series of molecular subgroups. The subgroup 7, which has been highlighted by our study, was remarkable as it gathered tumours with specific bioclinical features including a low rate of recurrence. Although this finding should be confirmed by using a larger tumour cohort, it suggests that gene expression profiling using a minimal set of genes may allow the discovery of new subclasses of breast cancer that are characterized by specific molecular signatures and exhibit specific bioclinical features

Profilage d'expression génique des cancers du sein (classification moléculaire et signature prédictive de la récurrence sous Tamoxifène)

MONTPELLIER-BU Médecine UPM (341722108) / SudocMONTPELLIER-BU Médecine (341722104) / SudocSudocFranceF

Interaction between the C-terminal domains of N and P proteins of measles virus investigated by NMR.

International audienceIn this paper we investigate the interaction between the C-terminal domains of the measles virus phosphoprotein (XD) and nucleoprotein (N(TAIL)) by using nuclear magnetic resonance chemical shift perturbation experiments. Using both N(TAIL) constructs and peptides, we show that contrary to the conserved Box2 region (N(489-506)), the C-terminal region of N(TAIL) (N(513-525)) does not directly interact with XD, and yet affects binding to XD. We tentatively propose a model where the C-terminus of N(TAIL) would stabilize the N(TAIL)-XD complex either via a functional coupling with N(489-506) or by reducing the entropic penalty associated to the binding-coupled-to-folding process