Nrf2 links epidermal barrier function with antioxidant defense.

The skin provides an efficient permeability barrier and protects from microbial invasion and oxidative stress. Here, we show that these essential functions are linked through the Nrf2 transcription factor. To test the hypothesis that activation of Nrf2 provides skin protection under stress conditions, we determined the consequences of pharmacological or genetic activation of Nrf2 in keratinocytes. Surprisingly, mice with enhanced Nrf2 activity in keratinocytes developed epidermal thickening, hyperkeratosis and inflammation resembling lamellar ichthyosis. This resulted from upregulation of the cornified envelope proteins small proline-rich proteins (Sprr) 2d and 2h and of secretory leukocyte peptidase inhibitor (Slpi), which we identified as novel Nrf2 targets in keratinocytes. Since Sprrs are potent scavengers of reactive oxygen species and since Slpi has antimicrobial activities, their upregulation contributes to Nrf2's protective function. However, it also caused corneocyte fragility and impaired desquamation, followed by alterations in the epidermal lipid barrier, inflammation and overexpression of mitogens that induced keratinocyte hyperproliferation. These results identify an unexpected role of Nrf2 in epidermal barrier function, which needs to be considered for pharmacological use of Nrf2 activators.

TAK1 is required for survival of mouse fibroblasts treated with TRAIL, and does so by NF-kappaB dependent induction of cFLIPL.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known as a "death ligand"-a member of the TNF superfamily that binds to receptors bearing death domains. As well as causing apoptosis of certain types of tumor cells, TRAIL can activate both NF-kappaB and JNK signalling pathways. To determine the role of TGF-beta-Activated Kinase-1 (TAK1) in TRAIL signalling, we analyzed the effects of adding TRAIL to mouse embryonic fibroblasts (MEFs) derived from TAK1 conditional knockout mice. TAK1-/- MEFs were significantly more sensitive to killing by TRAIL than wild-type MEFs, and failed to activate NF-kappaB or JNK. Overexpression of IKK2-EE, a constitutive activator of NF-kappaB, protected TAK1-/- MEFs against TRAIL killing, suggesting that TAK1 activation of NF-kappaB is critical for the viability of cells treated with TRAIL. Consistent with this model, TRAIL failed to induce the survival genes cIAP2 and cFlipL in the absence of TAK1, whereas activation of NF-kappaB by IKK2-EE restored the levels of both proteins. Moreover, ectopic expression of cFlipL, but not cIAP2, in TAK1-/- MEFs strongly inhibited TRAIL-induced cell death. These results indicate that cells that survive TRAIL treatment may do so by activation of a TAK1-NF-kappaB pathway that drives expression of cFlipL, and suggest that TAK1 may be a good target for overcoming TRAIL resistance.

Targeting mTORC2 inhibits colon cancer cell proliferation in vitro and tumor formation in vivo

The mammalian target of rapamycin (mTOR), which exists in two functionally distinct complexes, mTORC1 and mTORC2 plays an important role in tumor growth. Whereas the role of mTORC1 has been well characterized in this process, little is known about the functions of mTORC2 in cancer progression. In this study, we explored the specific role of mTORC2 in colon cancer using a short hairpin RNA expression system to silence the mTORC2-associated protein rictor. We found that downregulation of rictor in HT29 and LS174T colon cancer cells significantly reduced cell proliferation. Knockdown of rictor also resulted in a G1 arrest as observed by cell cycle analysis. We further observed that LS174T cells deficient for rictor failed to form tumors in a nude mice xenograft model. Taken together, these results show that the inhibition of mTORC2 reduces colon cancer cell proliferation in vitro and tumor xenograft formation in vivo. They also suggest that specifically targeting mTORC2 may provide a novel treatment strategy for colorectal cancer.

Small RNA as global regulator of carbon catabolite repression in Pseudomonas aeruginosa.

In the metabolically versatile bacterium Pseudomonas aeruginosa, the RNA-binding protein Crc is involved in catabolite repression of a range of degradative genes, such as amiE (encoding aliphatic amidase). We found that a CA-rich sequence (termed CA motif) in the amiE translation initiation region was important for Crc binding. The small RNA CrcZ (407 nt) containing 5 CA motifs was able to bind the Crc protein with high affinity and to remove it from amiE mRNA in vitro. Overexpression of crcZ relieved catabolite repression in vivo, whereas a crcZ mutation pleiotropically prevented the utilization of several carbon sources. The sigma factor RpoN and the CbrA/CbrB two-component system, which is known to maintain a healthy carbon-nitrogen balance, were necessary for crcZ expression. During growth on succinate, a preferred carbon source, CrcZ expression was low, resulting in catabolite repression of amiE and other genes under Crc control. By contrast, during growth on mannitol, a poor carbon source, elevated CrcZ levels correlated with relief of catabolite repression. During growth on glucose, an intermediate carbon source, CrcZ levels and amiE expression were intermediate between those observed in succinate and mannitol media. Thus, the CbrA-CbrB-CrcZ-Crc system allows the bacterium to adapt differentially to various carbon sources. This cascade also regulated the expression of the xylS (benR) gene, which encodes a transcriptional regulator involved in benzoate degradation, in an analogous way, confirming this cascade's global role.

Study of a sialyltransferase ST3GAL6, in adipogenesis and obesity

RésuméL'obésité et les maladies métaboliques qui lui sont associées tels que le diabète ou les maladies cardiovasculaires ont un impact épidémiologique croissant. Ainsi, les mécanismes moléculaires se produisant dans le tissu adipeux en expansion font l'objet de nombreuses investigations. Dans ce contexte, nous nous sommes particulièrement intéressés à l'adipogénèse, le procédé permettant la formation d'adipocytes matures et fonctionnels. Le gène St3gal6 code pour une enzyme appelée β-galactosidase a2,3-sialyltransferase 6 et participant à la voie de glycosylation. Cette protéine appartient à la famille des a2,3- sialyltransferases dont la fonction principale est de transférer un acide sialique à l'extrémité de chaînes glycosidiques présentes sur les glycoprotéines et les glycolipides. Dans une précédente étude de transcriptomique réalisée chez la souris, St3gal6 a été décrit comme un gène dont l'expression est augmentée dans le tissu adipeux blanc d'animaux en surpoids et dont l'expression est normalisée après une perte de poids. Afin d'étudier le rôle potentiel de St3gal6 dans le développement du tissu adipeux, nous nous sommes intéressés à la régulation de son expression en cas d'obésité ainsi qu'à ses effets sur l'adipogénèse. Nous avons d'abord montré que St3gal6 s'exprime aussi bien dans le tissu adipeux blanc que dans le tissu adipeux brun. Puis nous avons confirmé dans deux différents modèles animaux que l'expression de St3gal6 dans le tissu adipeux était augmentée en cas d'obésité. Nous avons aussi observé in vitro une induction de St3gal6 dans des adipocytes traités par des cytokines pro-inflammatoires sécrétées dans le tissu adipeux d'individus obèses. Enfin, parmi les six membres que compte la famille des a2,3-sialyltransferases, St3gal6 est celui dont l'expression est la plus significativement induite en situation d'obésité. En outre, au cours de la différenciation des adipocytes blancs et bruns, l'expression de St3gal6 est augmentée et son inhibition réduit le potentiel de maturation des adipocytes qui accumulent moins de lipides. A l'inverse, la surexpression de St3gal6 dans des préadipocytes blancs augmente leur taux de différenciation in vitro; la formation de gouttelettes lipidiques et l'expression de genes spécifiques de l'adipocyte mature sont accrues. Enfin, le traitement d'adipocytes blancs in vitro avec un inhibiteur pharmacologique des a2,3-sialyltransferases ou une sialidase clivant les résidus sialylés montre qu'un défaut de a2,3-sialylation affectant les adipocytes diminue leur potentiel adipogénique. Par conséquent, ces résultats suggèrent que St3gal6 est impliqué dans la voie de différenciation des adipocytes et que cette a2,3-sialylation joue un rôle dans le remodelage du tissu adipeux induit par l'obésité.AbstractIn order to better understand molecular events occurring in obesity and leading to its associated complications, we were interested in the biology of adipose tissue and particularly in the study of adipogenesis, the process by which new mature adipocytes develop and accumulate lipids.The β-galactosidase a2,3-sialyltransferase 6 (St3gal6) gene encodes for an enzyme involved in post-translational protein glycosylation. Thereby, St3gal6 enzyme belongs to the a2,3sialyltransferase family whose function is to add sialic acids at outer position on glycosidic chain of glycoproteins or glycolipids. Previously, in mouse, St3gal6 has been described as a gene whose expression in white adipose tissue is increased in overweighted animals and normalized after weight loss. Therefore, we have assumed that St3gal6 may play a role in adipose tissue development and in tissue remodelling triggered by obesity. First we show that St3gal6 is expressed in white but also in brown adipose tissue. St3gal6 upregulation upon weight gain was confirmed in two mouse models of obesity namely diet- induced and genetically-induced obesity. We also report that St3gal6 is induced by pro¬inflammatory cytokines known to be oversecreted in adipose tissue during obesity. Furthermore, St3gal6 is the a2,3-sialyltransferase whose expression is more markedly induced in adipose tissue. In addition, we demonstrate that St3gl6 expression is progressively increased in late stages of white and brown adipogenesis while St3gal6 knockdown inhibits adipocyte differentiation in vitro. Conversely, St3gal6 overexpression in a white preadipocyte cell line increases lipid accumulation during differentiation process and enhances gene expression of mature white adipocyte markers. Finally, using an a2-3 sialyltransferase inhibitor and a sialidase treatment on white adipocyte cell line, we observe that a decreased a2,3-sialylation impairs adipocyte differentiation in vitro. Altogether, these result suggest that St3gal6 plays a role in adipogenesis and in tissue remodelling associated with obesity likely through its enzymatic activity of a2,3-sialylation. Thus, a2,3-sialylation appears as a novel pathway of interest whose precise molecular mechanisms remain to be elucidated in the context of adipose tissue development and adipocyte functions.

Phenotypic switching in Pseudomonas brassicacearum involves GacS- and GacA-dependent Rsm small RNAs.

The plant-beneficial bacterium Pseudomonas brassicacearum forms phenotypic variants in vitro as well as in planta during root colonization under natural conditions. Transcriptome analysis of typical phenotypic variants using microarrays containing coding as well as noncoding DNA fragments showed differential expression of several genes relevant to secondary metabolism and of the small RNA (sRNA) genes rsmX, rsmY, and rsmZ. Naturally occurring mutations in the gacS-gacA system accounted for phenotypic switching, which was characterized by downregulation of antifungal secondary metabolites (2,4-diacetylphloroglucinol and cyanide), indoleacetate, exoenzymes (lipase and protease), and three different N-acyl-homoserine lactone molecules. Moreover, in addition to abrogating these biocontrol traits, gacS and gacA mutations resulted in reduced expression of the type VI secretion machinery, alginate biosynthesis, and biofilm formation. In a gacA mutant, the expression of rsmX was completely abolished, unlike that of rsmY and rsmZ. Overexpression of any of the three sRNAs in the gacA mutant overruled the pleiotropic changes and restored the wild-type phenotypes, suggesting functional redundancy of these sRNAs. In conclusion, our data show that phenotypic switching in P. brassicacearum results from mutations in the gacS-gacA system.

Characterization of PHO1 and PHO1;H1 gene function in Arabidopsis thaliana

Summary Phosphorus is one of the major macronutrients required for plant growth and development. Plant roots acquire phosphorus as inorganic phosphate (Pi), which is further distributed to the shoot, via the transpiration stream and root pressure, where Pi is imported again into cells. PHO1 in Arabidopsis has been identified as a protein involved in the loading of Pi into the root xylem. PHO1 does not have any homology to described Pi transporters including the Pht1 family of H+/ Pi cotransporters. PHO1 bears two domains, SPX and EXS domains, previously identified in Saccharomyces cerevisiae proteins involved in Pi transport and/or sensing, or in sorting proteins to endomembranes. Phylogenetic analysis of the PHO1 gene family revealed the presence of three clusters, with PHO1 and PHO1;H1 forming one cluster. The biological significance behind this cluster was demonstrated by the complementation of the pho1 mutant with only PHO1 and PHO1;H1, of all the PHO1 family members, when expressed under the PHO1 promoter. PHO1 has been shown to be expressed mostly in the root vascular cylinder and at low level in the shoot. PHO1;H1 had a different expression pattern, being expressed in both root and shoot vascular cylinder to the same level, with the levels in leaves increasing with the leaf maturity, suggesting additional role of PHO1;H1 in the Pi mobilization in leaves. In order to further explore the role of PHO1, Pi dynamics was studied on plants expressing PHO1 at different levels compared to the wild type: PHO1 overexpressors, PHO1 underexpressors and the pho1 mutant. Overexpression of the PHO1 protein in the shoot vascular tissue was shown to lead to increased Pi efflux out of the leaf cells and Pi accumulation in the shoot xylem apoplast compared to wild type, confirming the hypothesized role of PHO1 in xylem loading with Pi. The overexpression of PHO1 in the shoot was responsible far both changed Pi dynamic and stunted growth of PHO1 overexpressors, as shown by grafting experiments between wild type and PHO1 overexpressor. We found a ca. 2 fold decrease of shoot phosphorus and a 5-10 fold decrease in vacuolar Pi content in the PHO1 underexpressors and the pho1 null mutant compared to wild type, consistent with the role of PHO1 in the transfer of Pi from the root to the shoot. Shoot Pi deficiency results in a poor growth of the pho1 mutant. Grafting experiments between pho1 and wild type confirmed that both Pi deficiency and stunt growth of the pho1 mutant were dependent on the pho1 root, further supporting the importance of PHO1 in the root xylem loading with Pi. The pho1 mutant and the PHO1 underexpressors accumulated 8-15 fold more Pi in the root relative to wild type. In contrast to the pho1 mutant, the growth of PHO1 underexpressors was not impaired by the low shoat Pi content. This finding suggests that either PHO1 protein or root Pi concentration is important in Pi signaling and development of Pi deficiency symptoms leading to reduced growth. Résumé Le phosphore est l'un des nutriments essentiels à la croissance et au développement des plantes. Les racines absorbent le phosphore sous forme de phosphate inorganique (Pi) qui est dirigé, par la transpiration et la pression de la racine, vers les feuilles où le phosphate est acquis par les cellules. La protéine PHO1 a été démontrée indispensable au chargement du Pi dans le xylème des racines d'Arabidopsis. PHO1 ne démontre pas d'homologie aux transporteurs de Pi connus, incluant la famille Pht1 de cotransporteurs H+/Pi qui ont comme fonction le transport du phosphate à l'intérieur de la cellule. PHO1 contient deux domaines, SPX et EXS, aussi présents dans des protéines de Saccharomyces cerevisiae impliquées dans le transport ou la perception du phosphate, ou dans la localisation des protéines vers différentes membranes. Le génome d'Arabidopsis contient onze gènes homologues à PHO1. Neuf de ces homologues sont répartis en trois groupes. PHO1 et PHO1;H1 forment un de ces groupes. Nos travaux ont démontré que seuls PHO1;H1 et PHO1, sous contrôle du promoteur PHO1, peuvent complémenter le mutant pho1. PHO1 est exprimé principalement dans le cylindre vasculaire de la racine et faiblement dans la partie aérienne. Le degré d'expression de PHO1;H1 est similaire dans le cylindre vasculaire de la racine et des feuilles. Ceci suggère que PHO1;H1 est aussi impliqué dans la mobilisation du Pi dans les feuilles, en plus de son rôle dans le transfert du Pi dans le xylème des racines. Afin de mieux explorer le rôle de PHO1, la dynamique du phosphate a été observée dans trois lignées de plantes transgéniques: un sur-expresseur de PHO1, un sous-expresseur de PHO1 et le mutant pho1. La sur-expression de PHO1 dans le tissue vasculaire des feuilles a provoqué l'efflux du Pi vers l'espace apoplastic du xylème, ce qui confirme le rôle de PHO1 dans le chargement du Pi dans le xylème. La sur-expressìon de PHO1 dans la rosette est responsable d'un changement de la dynamique du Pi et de la diminution de la croissance, ce qui fut démontré par une expérience de greffe de la rosette du sur-expresseur de PHO1 sur les racines du sauvage. On a observé pour le sous-expresseur de PHO1 et le mutant pho1 une diminution du phosphore d'environ 2 fais au niveau des feuilles, et une diminution de 5-10 fois du Pi dans les vacuoles des feuilles, par rapport au sauvage. Ceci confirme le rôle proposé de PHO1 dans le transfert du Pi des racines aux feuilles. La carence de Pi chez pho1 implique une diminution de la taille de la rosette. Pour expliquer ce phénotype une autre expérience de greffe démontra que la cause de ce changement provenait des racines. Ceci renforce l'hypothèse de l'importance du rôle de PHO1 dans le xylème de la racine pour le chargement du Pi. Le mutant phot et le sous-expresseur de PHO1 accumulent 8-15 fois plus de Pi dans leurs racines comparé au sauvage. Cependant, contrairement au phot mutant, le sous-expresseur de PHO1 avait une croissance comparable au sauvage malgré le niveau bas du Pi dans les feuilles. Ceci suggère que la taille de la rosette lors d'une carence en Pi chez Arabidopsis serait la conséquence d'un changement de concentration de Pi dans les racines ou d'une influence de la protéine PHO1.

Targeting the JNK Signaling Pathway Potentiates the Antiproliferative Efficacy of Rapamycin in LS174T Colon Cancer Cells.

Background. Targeting the mTOR signaling pathway with rapamycin in cancer therapy has been less successful than expected due in part to the removal of a negative feedback loop resulting in the over-activation of the PI3K/Akt signaling pathway. As the c-Jun N-terminal kinase (JNK) signaling pathway has been found to be a functional target of PI3K, we investigate the role of JNK in the anticancer efficacy of rapamycin.Materials and Methods. The colon cancer cell line LS174T was treated with rapamycin and JNK phosphorylation was analyzed by Western Blot. Overexpression of a constitutively negative mutant of JNK in LS174T cells or treatment of LS174T cells with the JNK inhibitor SP600125 were used to determine the role of JNK in rapamycin-mediated tumor growth inhibition.Results. Treatment of LS174T cells with rapamycin resulted in the phosphorylation of JNK as observed by Western Blot. The expression of a negative mutant of JNK in LS174T cells or treatment of LS174T cells with SP600125 enhanced the antiproliferative effects of rapamycin. In addition, in vivo, the antitumor activity of rapamycin was potentiated on LS174T tumor xenografts that expressed the dominant negative mutant of JNK.Conclusions. Taken together, these results show that rapamycin-induced JNK phosphorylation and activation reduces the antitumor efficacy of rapamycin in LS174T cells. (C) 2011 Elsevier Inc. All rights reserved.

Lentiviral delivery of the human wild-type tau protein mediates a slow and progressive neurodegenerative tau pathology in the rat brain.

Most models for tauopathy use a mutated form of the Tau gene, MAPT, that is found in frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) and that leads to rapid neurofibrillary degeneration (NFD). Use of a wild-type (WT) form of human Tau protein to model the aggregation and associated neurodegenerative processes of Tau in the mouse brain has thus far been unsuccessful. In the present study, we generated an original "sporadic tauopathy-like" model in the rat hippocampus, encoding six Tau isoforms as found in humans, using lentiviral vectors (LVs) for the delivery of a human WT Tau. The overexpression of human WT Tau in pyramidal neurons resulted in NFD, the morphological characteristics and kinetics of which reflected the slow and sporadic neurodegenerative processes observed in sporadic tauopathies, unlike the rapid neurodegenerative processes leading to cell death and ghost tangles triggered by the FTDP-17 mutant Tau P301L. This new model highlights differences in the molecular and cellular mechanisms underlying the pathological processes induced by WT and mutant Tau and suggests that preference should be given to animal models using WT Tau in the quest to understand sporadic tauopathies.

BAFF AND APRIL: a tutorial on B cell survival.

BAFF, a member of the TNF family, is a fundamental survival factor for transitional and mature B cells. BAFF overexpression leads to an expanded B cell compartment and autoimmunity in mice, and elevated amounts of BAFF can be found in the serum of autoimmune patients. APRIL is a related factor that shares receptors with BAFF yet appears to play a different biological role. The BAFF system provides not only potential insight into the development of autoreactive B cells but a relatively simple paradigm to begin considering the balancing act between survival, growth, and death that affects all cells.

Posttranscriptional repression of GacS/GacA-controlled genes by the RNA-binding protein RsmE acting together with RsmA in the biocontrol strain Pseudomonas fluorescens CHA0.

In the plant-beneficial soil bacterium Pseudomonas fluorescens CHA0, the production of biocontrol factors (antifungal secondary metabolites and exoenzymes) is controlled at a posttranscriptional level by the GacS/GacA signal transduction pathway involving RNA-binding protein RsmA as a key regulatory element. This protein is assumed to bind to the ribosome-binding site of target mRNAs and to block their translation. RsmA-mediated repression is relieved at the end of exponential growth by two GacS/GacA-controlled regulatory RNAs RsmY and RsmZ, which bind and sequester the RsmA protein. A gene (rsmE) encoding a 64-amino-acid RsmA homolog was identified and characterized in strain CHA0. Overexpression of rsmE strongly reduced the expression of target genes (hcnA, for a hydrogen cyanide synthase subunit; aprA, for the main exoprotease; and phlA, for a component of 2,4-diacetylphloroglucinol biosynthesis). Single null mutations in either rsmA or rsmE resulted in a slight increase in the expression of hcnA, aprA, and phlA. By contrast, an rsmA rsmE double mutation led to strongly increased and advanced expression of these target genes and completely suppressed a gacS mutation. Both the RsmE and RsmA levels increased with increasing cell population densities in strain CHA0; however, the amount of RsmA showed less variability during growth. Expression of rsmE was controlled positively by GacA and negatively by RsmA and RsmE. Mobility shift assays demonstrated specific binding of RsmE to RsmY and RsmZ RNAs. The transcription and stability of both regulatory RNAs were strongly reduced in the rsmA rsmE double mutant. In conclusion, RsmA and RsmE together account for maximal repression in the GacS/GacA cascade of strain CHA0.

Construction of Pseudomonas strains with improved biocontrol activity

Selected strains of fluorescent pseudomonads suppress various plant diseases caused by soil-borne pathogenic fungi, by a blend of several mechanisms including aggressive root colonization, antibiosis, competition for nutrients, induction of resistance in the plant, and enzymatic attack of the pathogen. These traits are amenable to genetic analysis and, therefore, to modification by genetic engineering. Biocontrol activities of Pseudomonas spp. have been enhanced in two ways: (i) by overexpression of traits known to involved in diseaese suppression, and (ii) by introduction of additional beneficial traits into strains having basal biocontrol activities. Under experimental conditions in microcosms, a number of genetically modified Pseudomonas strains have given promising results. It remains to be seen whether such strains will be superior to the best naturally occurring strains, applied singly or in combination, under greenhouse and field conditions.

Regulatory RNA as mediator in GacA/RsmA-dependent global control of exoproduct formation in Pseudomonas fluorescens CHA0.

In Pseudomonas fluorescens CHA0, an antagonist of root-pathogenic fungi, the GacS/GacA two-component system tightly controls the expression of antifungal secondary metabolites and exoenzymes at a posttranscriptional level, involving the RNA-binding protein and global regulator of secondary metabolism RsmA. This protein was purified from P. fluorescens, and RNA bound to it was converted to cDNA, which served as a probe to isolate the corresponding chromosomal locus, rsmZ. This gene encoded a regulatory RNA of 127 nucleotides and a truncated form lacking 35 nucleotides at the 3' end. Expression of rsmZ depended on GacA, increased with increasing population density, and was stimulated by the addition of a solvent-extractable extracellular signal produced by strain CHA0 at the end of exponential growth. This signal appeared to be unrelated to N-acyl-homoserine lactones. A conserved upstream element in the rsmZ promoter, but not the stress sigma factor RpoS, was involved in rsmZ expression. Overexpression of rsmZ effectively suppressed the negative effect of gacS and gacA mutations on target genes, i.e., hcnA (for hydrogen cyanide synthase) and aprA (for the major exoprotease). Mutational inactivation of rsmZ resulted in reduced expression of these target genes in the presence of added signal. Overexpression of rsmA had a similar, albeit stronger negative effect. These results support a model in which GacA upregulates the expression of regulatory RNAs, such as RsmZ of strain CHA0, in response to a bacterial signal. By a titration effect, RsmZ may then alleviate the repressing activity of RsmA on the expression of target mRNAs.

p27 and Skp2 immunoreactivity and its clinical significance with endocrine and chemo-endocrine treatments in node-negative early breast cancer.

BACKGROUND: Low p27 and high Skp2 immunoreactivity are associated with a poor prognosis and other poor prognostic features including resistant phenotypes and antiestrogen drug resistance. We investigated these proteins in two International Breast Cancer Study Group trials studying node-negative early breast cancer. PATIENTS AND METHODS: Trial VIII compared chemotherapy followed by goserelin with either modality alone in premenopausal patients. Trial IX compared chemotherapy followed by tamoxifen with tamoxifen alone in postmenopausal patients. Central Pathology Office assessed p27 and Skp2 expression in the primary tumor by immunohistochemistry among 1631 (60%) trial patients. RESULTS: p27 and Skp2 were inversely related; 13% of tumors expressed low p27 and high Skp2. Low p27 and high Skp2 were associated with unfavorable prognostic factors including larger size and higher grade tumors, absence of estrogen receptor and progesterone receptor, human epidermal growth factor receptor 2 overexpression and high Ki-67 (each P < 0.05). Low p27 and high Skp2 were not associated with disease-free survival (P = 0.42 and P = 0.48, respectively). The relative effects of chemo-endocrine versus endocrine therapy were similar regardless of p27 or Skp2. CONCLUSIONS: We confirm the association of low p27 and high Skp2 with other poor prognostic features, but found no predictive or prognostic value, and therefore do not recommend routine determination of p27 and Skp2 for node-negative breast cancer.

Role of the glyoxalase system in astrocyte-mediated neuroprotection.

The glyoxalase system is the most important pathway for the detoxification of methylglyoxal (MG), a highly reactive dicarbonyl compound mainly formed as a by-product of glycolysis. MG is a major precursor of advanced glycation end products (AGEs), which are associated with several neurodegenerative disorders. Although the neurotoxic effects of MG and AGEs are well characterized, little is known about the glyoxalase system in the brain, in particular with regards to its activity in different neural cell types. Results of the present study reveal that both enzymes composing the glyoxalase system [glyoxalase-1 (Glo-1) and Glo-2] were highly expressed in primary mouse astrocytes compared with neurons, which translated into higher enzymatic activity rates in astrocytes (9.9- and 2.5-fold, respectively). The presence of a highly efficient glyoxalase system in astrocytes was associated with lower accumulation of AGEs compared with neurons (as assessed by Western blotting), a sixfold greater resistance to MG toxicity, and the capacity to protect neurons against MG in a coculture system. In addition, Glo-1 downregulation using RNA interference strategies resulted in a loss of viability in neurons, but not in astrocytes. Finally, stimulation of neuronal glycolysis via lentiviral-mediated overexpression of 6-phosphofructose-2-kinase/fructose-2,6-bisphosphatase-3 resulted in increased MG levels and MG-modified proteins. Since MG is largely produced through glycolysis, this suggests that the poor capacity of neurons to upregulate their glycolytic flux as compared with astrocytes may be related to weaker defense mechanisms against MG toxicity. Accordingly, the neuroenergetic specialization taking place between these two cell types may serve as a protective mechanism against MG-induced neurotoxicity.