WNT-5A, but not matrix metalloproteinase 3 or beta-catenin protein, expression is related to early stages of lip carcinogenesis

Background: Oncogenic Wnt/beta-catenin signaling occurs in numerous types of cancers, but little is known about the role of the Wnt protein family member, WNT-5A, in lip carcinogenesis. The aim of this study was to investigate WNT-5A, beta-catenin, and matrix metalloproteinase (MMP)-3 protein expression in actinic cheilitis (AC), and lip squamous cell carcinoma (LSCC). Methods: Twenty-one cases of AC, and fifty-one cases of LSCC were analyzed, with normal lip mucosa used as a control. Qualitative and semi-quantitative analyses of WNT-5A, beta-catenin, and MMP-3 immunostaining pattern and cellular distribution were performed. Results: WNT-5A was observed in more than 50% of the cells, scattered in all layers of AC, in contrast to the absence of immunostaining in normal lip mucosa. AC presented a higher level of WNT-5A expression than LSCC (P = 0.0289, Fisher test), while MMP-3 immunoexpression was statistically more significant in LSCC than in AC (P = 0.0285, Fisher test). Immunolabeling of beta-catenin protein was differentially distributed between samples; the majority of AC cases (61.90%) demonstrated a membranous-cytoplasmic pattern, while a considerable number of LSCC cases (29.41%) revealed a cytoplasmic pattern, instead of the usual membranous pattern. Conclusions: The present results suggest that WNT-5A may be an important marker during initial events of AC malignant transformation, in which non-canonical and canonical Wnt/beta-catenin signaling pathways could be involved. Additionally, WNT-5A might recruit other events in LSCC, such as MMP-3 protein synthesis, as its presence is increased in established malignant processes without beta-catenin dependency.

Dual trafficking of Slit3 to mitochondria and cell surface demonstrates novel localization for Slit protein

Drosophila slit is a secreted protein involved in midline patterning. Three vertebrate orthologs of the fly slit gene, Slit1, 2, and 3, have been isolated. Each displays overlapping, but distinct, patterns of expression in the developing vertebrate central nervous system, implying conservation of function. However, vertebrate Slit genes are also expressed in nonneuronal tissues where their cellular locations and functions are unknown. In this study, we characterized the cellular distribution and processing of mammalian Slit3 gene product, the least evolutionarily conserved of the vertebrate Slit genes, in kidney epithelial cells, using both cellular fractionation and immunolabeling. Slit3, but not Slit2, was predominantly localized within the mitochondria. This localization was confirmed using immunoelectron microscopy in cell lines and in mouse kidney proximal tubule cells. In confluent epithelial monolayers, Slit3 was also transported to the cell surface. However, we found no evidence of Slit3 proteolytic processing similar to that seen for Slit2. We demonstrated that Slit3 contains an NH2-terminal mitochondrial localization signal that can direct a reporter green fluorescent protein to the mitochondria. The equivalent region from Slit1 cannot elicit mitochondrial targeting. We conclude that Slit3 protein is targeted to and localized at two distinct sites within epithelial cells: the mitochondria, and then, in more confluent cells, the cell surface. Targeting to both locations is driven by specific NH2-terminal sequences. This is the first examination of Slit protein localization in nonneuronal cells, and this study implies that Slit3 has potentially unique functions not shared by other Slit proteins.

Quantitative evaluation of altered hepatic spaces and membrane transport in fibrotic rat liver

Four animal models were used to quantitatively evaluate hepatic alterations in this study: (1) a carbon tetrachloride control group (phenobarbital treatment only), (2) a CCl4-treated group (phenobarbital with CCl4 treatment), (3) an alcohol-treated group (liquid diet with alcohol treatment), and (4) a pair-fed alcohol control group (liquid diet only). At the end of induction, single-pass perfused livers were used to conduct multiple indicator dilution (MID) studies. Hepatic spaces (vascular space, extravascular albumin space, extravascular sucrose space, and cellular distribution volume) and water hepatocyte permeability/surface area product were estimated from nonlinear regression of outflow concentration versus time profile data. The hepatic extraction ratio of H-3-taurocholate was determined by the nonparametric moments method. Livers were then dissected for histopathologic analyses (e.g., fibrosis index, number of fenestrae). In these 4 models, CCl4-treated rats were found to have the smallest vascular space, extravascular albumin space, H-3-taurocholate extraction, and water hepatocyte permeability/surface area product but the largest extravascular sucrose space and cellular distribution volume. In addition, a linear relationship was found to exist between histopathologic analyses (fibrosis index or number of fenestrae) and hepatic spaces. The hepatic extraction ratio of H-3-taurocholate and water hepatocyte permeability/surface area product also correlated to the severity of fibrosis as defined by the fibrosis index. In conclusion, the multiple indicator dilution data obtained from the in situ perfused rat liver can be directly related to histopathologic analyses.

TRAIP is a regulator of the spindle assembly checkpoint.

Accurate chromosome segregation during mitosis is temporally and spatially coordinated by fidelity-monitoring checkpoint systems. Deficiencies in these checkpoint systems can lead to chromosome segregation errors and aneuploidy, and promote tumorigenesis. Here, we report that the TRAF-interacting protein (TRAIP), a ubiquitously expressed nucleolar E3 ubiquitin ligase important for cellular proliferation, is localized close to mitotic chromosomes. Its knockdown in HeLa cells by RNA interference (RNAi) decreased the time of early mitosis progression from nuclear envelope breakdown (NEB) to anaphase onset and increased the percentages of chromosome alignment defects in metaphase and lagging chromosomes in anaphase compared with those of control cells. The decrease in progression time was corrected by the expression of wild-type but not a ubiquitin-ligase-deficient form of TRAIP. TRAIP-depleted cells bypassed taxol-induced mitotic arrest and displayed significantly reduced kinetochore levels of MAD2 (also known as MAD2L1) but not of other spindle checkpoint proteins in the presence of nocodazole. These results imply that TRAIP regulates the spindle assembly checkpoint, MAD2 abundance at kinetochores and the accurate cellular distribution of chromosomes. The TRAIP ubiquitin ligase activity is functionally required for the spindle assembly checkpoint control.

Compressed collagen gel: a novel scaffold for human bladder cells.

Collagen is highly conserved across species and has been used extensively for tissue regeneration; however, its mechanical properties are limited. A recent advance using plastic compression of collagen gels to achieve much higher concentrations significantly increases its mechanical properties at the neo-tissue level. This controlled, cell-independent process allows the engineering of biomimetic scaffolds. We have evaluated plastic compressed collagen scaffolds seeded with human bladder smooth muscle cells inside and urothelial cells on the gel surface for potential urological applications. Bladder smooth muscle and urothelial cells were visualized using scanning electron microscopy, conventional histology and immunohistochemistry; cell viability and proliferation were also quantified for 14 days in vitro. Both cell types tested proliferated on the construct surface, forming dense cell layers after 2 weeks. However, smooth muscle cells seeded within the construct, assessed with the Alamar blue assay, showed lower proliferation. Cellular distribution within the construct was also evaluated, using confocal microscopy. After 14 days of in vitro culture, 30% of the smooth muscle cells were found on the construct surface compared to 0% at day 1. Our results provide some evidence that cell-seeded plastic compressed collagen has significant potential for bladder tissue regeneration, as these materials allow efficient cell seeding inside the construct as well as cell proliferation.

Solubility of cytoskeletal proteins in immunohistochemistry and the influence of fixation.

For accurate and quantitative immunohistochemical localization of antigens it is crucial to know the solubility of tissue proteins and their degree of loss during processing. In this study we focused on the solubility of several cytoskeletal proteins in cat brain tissue at various ages and their loss during immunohistochemical procedures. We further examined whether fixation affected either solubility or immunocytochemical detectability of several cytoskeletal proteins. An assay was designed to measure the solubility of cytoskeletal proteins in cryostat sections. Quantity and quality of proteins lost or remaining in tissue were measured and analyzed by electrophoresis and immunoblots. Most microtubule proteins were found to be soluble in unfixed and alcohol fixed tissues. Furthermore, the microtubule proteins remaining in the tissue had a changed cellular distribution. In contrast, brain spectrin and all three neurofilament subunits were insoluble and remained in the tissue, allowing their immunocytochemical localization in alcohol-fixed tissue. Synapsin I, a protein associated with the spectrin cytoskeleton, was soluble, and aldehyde fixation is advised for its immunohistochemical localization. With aldehyde fixation, the immunoreactivity of some antibodies against neurofilament proteins was reduced in axons unveiling novel immunogenic sites in nuclei that may represent artifacts of fixation. In conclusion, protein solubility and the effects of fixation are influential factors in cytoskeletal immunohistochemistry, and should be considered before assessments for a quantitative distribution are made.

Analysis of susceptibility to human immunodeficiency virus 1 (HIV-1) by comparative genetics

Summary [résumé français voir ci-dessous] From the beginning of the 20th century the world population has been confronted with the human immune deficiency virus 1 (HIV-1). This virus has the particularity to mutate fast, and could thus evade and adapt to the human host. Our closest evolutionary related organisms, the non-human primates, are less susceptible to HIV-1. In a broader sense, primates are differentially susceptible to various retrovirus. Species specificity may be due to genetic differences among primates. In the present study we applied evolutionary and comparative genetic techniques to characterize the evolutionary pattern of host cellular determinants of HIV-1 pathogenesis. The study of the evolution of genes coding for proteins participating to the restriction or pathogenesis of HIV-1 may help understanding the genetic basis of modern human susceptibility to infection. To perform comparative genetics analysis, we constituted a collection of primate DNA and RNA to allow generation of de novo sequence of gene orthologs. More recently, release to the public domain of two new primate complete genomes (bornean orang-utan and common marmoset) in addition of the three previously available genomes (human, chimpanzee and Rhesus monkey) help scaling up the evolutionary and comparative genome analysis. Sequence analysis used phylogenetic and statistical methods for detecting molecular adaptation. We identified different selective pressures acting on host proteins involved in HIV-1 pathogenesis. Proteins with HIV-1 restriction properties in non-human primates were under strong positive selection, in particular in regions of interaction with viral proteins. These regions carried key residues for the antiviral activity. Proteins of the innate immunity presented an evolutionary pattern of conservation (purifying selection) but with signals of relaxed constrain if we compared them to the average profile of purifying selection of the primate genomes. Large scale analysis resulted in patterns of evolutionary pressures according to molecular function, biological process and cellular distribution. The data generated by various analyses served to guide the ancestral reconstruction of TRIM5a a potent antiviral host factor. The resurrected TRIM5a from the common ancestor of Old world monkeys was effective against HIV-1 and the recent resurrected hominoid variants were more effective against other retrovirus. Thus, as the result of trade-offs in the ability to restrict different retrovirus, human might have been exposed to HIV-1 at a time when TRIM5a lacked the appropriate specific restriction activity. The application of evolutionary and comparative genetic tools should be considered for the systematical assessment of host proteins relevant in viral pathogenesis, and to guide biological and functional studies. Résumé La population mondiale est confrontée depuis le début du vingtième siècle au virus de l'immunodéficience humaine 1 (VIH-1). Ce virus a un taux de mutation particulièrement élevé, il peut donc s'évader et s'adapter très efficacement à son hôte. Les organismes évolutivement le plus proches de l'homme les primates nonhumains sont moins susceptibles au VIH-1. De façon générale, les primates répondent différemment aux rétrovirus. Cette spécificité entre espèces doit résider dans les différences génétiques entre primates. Dans cette étude nous avons appliqué des techniques d'évolution et de génétique comparative pour caractériser le modèle évolutif des déterminants cellulaires impliqués dans la pathogenèse du VIH- 1. L'étude de l'évolution des gènes, codant pour des protéines impliquées dans la restriction ou la pathogenèse du VIH-1, aidera à la compréhension des bases génétiques ayant récemment rendu l'homme susceptible. Pour les analyses de génétique comparative, nous avons constitué une collection d'ADN et d'ARN de primates dans le but d'obtenir des nouvelles séquences de gènes orthologues. Récemment deux nouveaux génomes complets ont été publiés (l'orang-outan du Bornéo et Marmoset commun) en plus des trois génomes déjà disponibles (humain, chimpanzé, macaque rhésus). Ceci a permis d'améliorer considérablement l'étendue de l'analyse. Pour détecter l'adaptation moléculaire nous avons analysé les séquences à l'aide de méthodes phylogénétiques et statistiques. Nous avons identifié différentes pressions de sélection agissant sur les protéines impliquées dans la pathogenèse du VIH-1. Des protéines avec des propriétés de restriction du VIH-1 dans les primates non-humains présentent un taux particulièrement haut de remplacement d'acides aminés (sélection positive). En particulier dans les régions d'interaction avec les protéines virales. Ces régions incluent des acides aminés clé pour l'activité de restriction. Les protéines appartenant à l'immunité inné présentent un modèle d'évolution de conservation (sélection purifiante) mais avec des traces de "relaxation" comparé au profil général de sélection purifiante du génome des primates. Une analyse à grande échelle a permis de classifier les modèles de pression évolutive selon leur fonction moléculaire, processus biologique et distribution cellulaire. Les données générées par les différentes analyses ont permis la reconstruction ancestrale de TRIM5a, un puissant facteur antiretroviral. Le TRIM5a ressuscité, correspondant à l'ancêtre commun entre les grands singes et les groupe des catarrhiniens, est efficace contre le VIH-1 moderne. Les TRIM5a ressuscités plus récents, correspondant aux ancêtres des grands singes, sont plus efficaces contre d'autres rétrovirus. Ainsi, trouver un compromis dans la capacité de restreindre différents rétrovirus, l'homme aurait été exposé au VIH-1 à une période où TRIM5a manquait d'activité de restriction spécifique contre celui-ci. L'application de techniques d'évolution et de génétique comparative devraient être considérées pour l'évaluation systématique de protéines impliquées dans la pathogenèse virale, ainsi que pour guider des études biologiques et fonctionnelles

Cell locations for AQP1, AQP4 and 9 in the non-human primate brain.

The presence of three water channels (aquaporins, AQP), AQP1, AQP4 and AQP9 were observed in normal brain and several rodent models of brain pathologies. Little is known about AQP distribution in the primate brain and its knowledge will be useful for future testing of drugs aimed at preventing brain edema formation. We studied the expression and cellular distribution of AQP1, 4 and 9 in the non-human primate brain. The distribution of AQP4 in the non-human primate brain was observed in perivascular astrocytes, comparable to the observation made in the rodent brain. In contrast with rodent, primate AQP1 is expressed in the processes and perivascular endfeet of a subtype of astrocytes mainly located in the white matter and the glia limitans, possibly involved in water homeostasis. AQP1 was also observed in neurons innervating the pial blood vessels, suggesting a possible role in cerebral blood flow regulation. As described in rodent, AQP9 mRNA and protein were detected in astrocytes and in catecholaminergic neurons. However additional locations were observed for AQP9 in populations of neurons located in several cortical areas of primate brains. This report describes a detailed study of AQP1, 4 and 9 distributions in the non-human primate brain, which adds to the data already published in rodent brains. This relevant species differences have to be considered carefully to assess potential drugs acting on AQPs non-human primate models before entering human clinical trials.

MAP2 Isoforms in Developing Cat Cerebral Cortex and Corpus Callosum.

The microtubule-associated protein MAP2 was studied in the developing cat visual cortex and corpus callosum. Biochemically, no MAP2a was detectable in either structure during the first postnatal month; adult cortex revealed small amounts of MAP2a. MAP2b was abundant in cortical tissue during the first postnatal month and decreased in concentration towards adulthood; it was barely detectable in corpus callosum at all ages. MAP2c was present in cortex and corpus callosum at birth; in cortex it consisted of three proteins of similar molecular weights between 65 and 70 kD. The two larger, phosphorylated forms disappeared after postnatal day 28, the smaller form after day 39. In corpus callosum, MAP2c changed from a phosphorylated to an unphosphorylated variant during the first postnatal month and then disappeared. Immunocytochemical experiments revealed MAP2 in cell bodies and dendrites of neurons in all cortical layers, from birth onwards. In corpus callosum, in the first month after birth, a little MAP2, possibly MAP2c, was detectable in axons. The present data indicate that MAP2 isoforms differ in their cellular distribution, temporal appearance and structural association, and that their composition undergoes profound changes during the period of axonal stabilization and dendritic maturation.

Extracellular microvesicles from astrocytes contain functional glutamate transporters: regulation by protein kinase C and cell activation.

Glutamate transport through astrocytic excitatory amino-acid transporters (EAAT)-1 and EAAT-2 is paramount for neural homeostasis. EAAT-1 has been reported in secreted extracellular microvesicles (eMV, such as exosomes) and because the protein kinase C (PKC) family controls the sub-cellular distribution of EAATs, we have explored whether PKCs drive EAATs into eMV. Using rat primary astrocytes, confocal immunofluorescence and ultracentrifugation on sucrose gradient we here report that PKC activation by phorbol myristate acetate (PMA) reorganizes EAAT-1 distribution and reduces functional [(3)H]-aspartate reuptake. Western-blots show that EAAT-1 is present in eMV from astrocyte conditioned medium, together with NaK ATPase and glutamine synthetase all being further increased after PMA treatment. However, nanoparticle tracking analysis reveals that PKC activation did not change particle concentration. Functional analysis indicates that eMV have the capacity to reuptake [(3)H]-aspartate. In vivo, we demonstrate that spinal astrocytic reaction induced by peripheral nerve lesion (spared nerve injury, SNI) is associated with a phosphorylation of PKC δ together with a shift of EAAT distribution ipsilaterally. Ex vivo, spinal explants from SNI rats release eMV with an increased content of NaK ATPase, EAAT-1 and EAAT-2. These data indicate PKC and cell activation as important regulators of EAAT-1 incorporation in eMV, and raise the possibility that microvesicular EAAT-1 may exert extracellular functions. Beyond a putative role in neuropathic pain, this phenomenon may be important for understanding neural homeostasis and a wide range of neurological diseases associated with astrocytic reaction as well as non-neurological diseases linked to eMV release.

Expression of the monocarboxylate transporter MCT1 in the adult human brain cortex.

Distribution of the monocarboxylate transporter MCT1 has been investigated in the cortex of normal adult human brain. Similarly to the glucose transporter GLUT1 55 kDa isoform, MCT1 was found to be strongly expressed on blood vessels in all cortical layers. In addition, laminar analysis revealed intense MCT1 expression in the neuropil of layer IV in primary auditory (AI) and visual (VI) areas, while this expression was more homogeneous in the non-primary auditory area STA. The cellular distribution shows that MCT1 is strongly expressed by glial cells often associated with blood vessels that were identified as astrocytes. The observed distribution of MCT1 supports the concept that, under certain circumstances, monocarboxylates could be provided as energy substrates to the adult human brain. Moreover, the distinct laminar pattern of MCT1 expression between primary and non-primary cortical areas may reflect different types of neuronal activity requiring adequate supply of specific energy substrates.

Neurofilament proteins in the postnatal rat hippocampus. Developmental expression and changes in experimental epilepsy

Neurofilament proteins (NFs) are the major components of the intermediate filaments of the neuronal cytoskeleton. The three different NF proteins; the low (NF-L), medium (NF-M),and dendrites.NF proteins play an important role in neuronal development, and plasticity,and seem to contribute to the pathophysiology of several diseases. However, the detailed expression patterns of NF proteins in the course of postnatal aturation, and in response to seizures in the rat have remained unknown. In this work, I have studied the developmental expression and cellular distribution of the three NF proteins in the rat hippocampus during the postnatal development. The reactivity of NF proteins in response to kainic acid (KA)-induced status epilepticus (SE)was studied in the hippocampus of 9-day-old rats, and using in vitro organotypic hippocampal slices cultures prepared from P6-7 rats. The results showed that NF-L and NF-M proteins are expressed already at the postnatal day 1, while the expression of NF-H mainly occurred during the second postnatal week. The immunoreactivity of NF proteins varied depending on the cell type and sub-cellular location in the hippocampus. In adult rats, KA-induced SE typically results in severe and permanent NF degradation. However, in our P9 rats KA-induced SE resulted in a transient increase in the expression of NF proteins during the first few hours but not degradation. No neuronal death or mossy fiber sprouting was observed at any time after SE. The in vitro studies with OHCs, which mimick the in vivo developing models where a local injection of KA is applied(e.g. intrahippocampal), indicated that NF proteins were rapidly degraded in response to KA treatment, this effect being effectively inhibited by the treatment with the AMPA receptor antagonist CNQX, and calpain inhibitor MDL-28170. These compounds also significantly ameliorated the KA-induced region-specific neuronal damage. The NMDA receptor antagonist and the L-type Ca2+ channel blocker did not have any significant effect. In conclusion, the results indicate that the developmental expression of NF in the rat hippocampus is differentially regulated and targeted in the different hippocampal cell types during the postnatal development. Furthermore, despite SE, the mechanisms leading to NF degradation and neuronal death are not activated in P9 rats unlike in adults. The reason for this remains unknown. The results in organotypic hippocampal cultures confirm the validity of this in vitro model to study development processes, and to perform pharmacological studies. The results also suggest that calpain proteases as interesting pharmacological targets to reduce neuronal damage after acute excitotoxic insults.

Mechanism of the transforming growth factor-β induction of fibronectin expression in hepatic stem-like cells

Transforming growth factor-β1 (TGF-β1) plays an important role in the fibrogenic process in the liver. The aim of the present study was to explore the action of TGF-β1 on fibronectin expression in rat hepatic stem-like cells and the underlying mechanisms. The level of fibronectin expression was determined in hepatic stem-like cells (WB cells) before and after TGF-β1 stimulation by RT-PCR and Western blot methods. Using immunogold transmission electron microscopy and the Western blot method, we observed the result of the expression and the distribution of cAMP, phosphorylated Smad3 and Smad7 before and after TGF-β1 treatment. The levels of fibronectin expression in both mRNA and protein increased 4- to 5-fold after TGF-β1 stimulation, reaching an optimum level after 8 h and then gradually falling back. Similarly, TGF-β1 stimulation resulted in an increase of cAMP in WB cells, peaking at 8 h. After treatment with TGF-β1 for 24 h, the expression of cAMP gradually decreased. In addition, we found that TGF-β1 treatment also contributed to the increased expression and to changes in cellular distribution of phosphorylated Smad3 (translocation from the cytoplasm to the nucleus) and Smad7 (translocation from the nucleus to the cytoplasm) in WB cells. The present study demonstrates that TGF-β is involved in the fibrogenic process in hepatic stem cells through up-regulation of fibronectin expression, and the mechanisms underlying this process may be associated with the activation of cAMP and Smad pathways.

The role of GAPDH in maintaining the functional state of the DNA repair enzyme APE1

Les sites apuriniques/apyrimidiniques (AP) sont des sites de l’ADN hautement mutagène. Les dommages au niveau de ces sites peuvent survenir spontanément ou être induits par une variété d’agents. Chez l’humain, les sites AP sont réparés principalement par APE1, une enzyme de réparation de l’ADN qui fait partie de la voie de réparation par excision de base (BER). APE1 est une enzyme multifonctionnelle; c’est une AP endonucléase, 3’-diestérase et un facteur redox impliqué dans l’activation des facteurs de transcription. Récemment, il a été démontré qu’APE1 interagit avec l’enzyme glycolytique GAPDH. Cette interaction induit l’activation d’APE1 par réduction. En outre, la délétion du gène GAPDH sensibilise les cellules aux agents endommageant l’ADN, induit une augmentation de formation spontanée des sites AP et réduit la prolifération cellulaire. A partir de toutes ces données, il était donc intéressant d’étudier l’effet de la délétion de GAPDH sur la progression du cycle cellulaire, sur la distribution cellulaire d’APE1 et d’identifier la cystéine(s) d’APE1 cible(s) de la réduction par GAPDH. Nos travaux de recherche ont montré que la déficience en GAPDH cause un arrêt du cycle cellulaire en phase G1. Cet arrêt est probablement dû à l’accumulation des dommages engendrant un retard au cours duquel la cellule pourra réparer son ADN. De plus, nous avons observé des foci nucléaires dans les cellules déficientes en GAPDH qui peuvent représenter des agrégats d’APE1 sous sa forme oxydée ou bien des focis de la protéine inactive au niveau des lésions d’ADN. Nous avons utilisé la mutagénèse dirigée pour créer des mutants (Cys en Ala) des sept cystéines d’APE1 qui ont été cloné dans un vecteur d’expression dans les cellules de mammifères. Nous émettons l’hypothèse qu’au moins un mutant ou plus va être résistant à l’inactivation par oxydation puisque l’alanine ne peut pas s’engager dans la formation des ponts disulfures. Par conséquent, on anticipe que l’expression de ce mutant dans les cellules déficientes en GAPDH pourrait restaurer une distribution cellulaire normale de APE1, libérerait les cellules de l’arrêt en phase G1 et diminuerait la sensibilité aux agents endommageant l’ADN. En conclusion, il semble que GAPDH, en préservant l’activité d’APE1, joue un nouveau rôle pour maintenir l’intégrité génomique des cellules aussi bien dans les conditions normales qu’en réponse au stress oxydatif.

Relationship between structure and photoactivity of porphyrins derived from protoporphyrin IX

Protoporphyrin (Pp IX) derivatives were prepared to study the relationship between photosensitizer structure and photoactivity, with an emphasis on understanding the role of membrane interactions in the efficiency of photosensitizers used in photodynamic therapy (PDT). The synthetic strategies described here aimed at changing protoporphyrin periferic groups, varying overall charge and oil/water partition, while maintaining their photochemical properties. Three synthetic routes were used: (1) modification of Pp IX at positions 3(1) and 8(1) by addition of alkyl amine groups of different lengths (compounds 2-5), (2) change of Pp IX at positions 13(3) and 17(3), generating alkyl amines (compounds 6 and 7), a phosphate amine (compound 8), and quarternary ammonium compounds (compounds 9 and 10), and (3) amine-alkylation of Hematoporphyrin IX (Hp IX) at positions 3(1), 8(1), 13(3) and 17(3) (compound 12). Strategy 1 leads to hydrophobic compounds with low photocytotoxicity. Strategy 2 leads to compounds 6-10 that have high levels of binding/incorporation in vesicles, mitochondria and cells, which are indicative of high bioavailability. Addition of the phosphate group (compound 8), generates an anionic compound that has low liposome and cell incorporation, plus low photocytotoxicity. Compound 12 has intermediate incorporation and photocytotoxic properties. Compound modification is also associated with changes in their sub-cellular localization: 30% of 8 (anionic) is found in mitochondria as compared to 95% of compound 10 (cationic). Photocytotoxicity was shown to be highly correlated with membrane affinity, which depends on the asymmetrical and amphiphilic characters of sens, as well as with sub-cellular localization.