Polychlorinated biphenyls (PCBs) are nongenotoxic hepatic tumor promoters. PCBs have been shown to cause oxidative stress, but the exact mechanism by which PCBs exert their tumor promoting activity is not clear. In our study, PCB-153, a non-coplanar congener, caused a transient increase in hepatic NF-B DNA binding activity and cell proliferation, while PCB-77, a coplanar congener, showed no effect. Our second study using a mouse model that was deficient in the p50 subunit of NF-kB (p50-/-) showed that NF-kB contributes to the changes in hepatocyte proliferation and apoptosis in response to PCB-153 treatment: a single dose of PCB-153 increased hepatic NF-B activity and cell proliferation in wild type mice, but not in the p50-/- mice; longer-term treatment with PCB-153 increased cell proliferation in p50-/- mice, but this increase was less than that in the wild type. In addition, p50-/- livers had more apoptosis than in the wild type, and PCB-153 inhibited apoptosis in the p50-/- livers. p50-/- livers had less cyclin D1 protein than the wild type, but that the mRNA levels were same. Bcl-xL protein was not changed by PCB-153, and wild type and p50-/- mice had the same level of Bcl-xL protein. In the third study, PCB-77 caused an increase in hepatic NF-kB DNA binding activity and cell proliferation during the promotion stage, and this increase was blocked by dietary supplementation of vitamin E, but the number and volume of placental glutathione S-transferase (PGST)-positive foci were slightly, though insignificantly, increased in the same animals. The apparent conflict could be due to different effect in different cells: high level vitamin E significantly inhibited PCB-77-induced cell proliferation in normal hepatocytes, while this inhibitory effect was much less in the PGST-positive hepatocytes. In conclusion, our studies show that a non-coplanar PCB can cause an increase in hepatic NF-kB DNA binding activity in rats and mice, and this increase contributes to the change in cell proliferation and apoptosis. Dietary vitamin E supplementation did not show protective effect on the formation of altered hepatic foci that were promoted by PCBs, although vitamin E supplementation decreased PCBs-induced hepatic NF-kB activation and cell proliferation.

This study investigated the interaction between polychlorinated biphenyls (PCBs) and selenium to explain the mechanism involved that could affect selenium metabolism and its anti-cancer property. PCBs congeners and mixtures were previously found to reduce hepatic Se and Se-dependent glutathione peroxidase activity. I hypothesized that certain PCB congeners affect selenium metabolism in the rat liver resulting in diminished antioxidant capacity of selenoproteins, which could alter the ability of Se to protect against PCBs induced tumor promotion. In the first study, the influence of 3,3,4,4-tetrachlorobiphenyl (PCB 77) on hepatic Se and glutathione peroxidase (GPx1) activity as well as cytochrome P450 1A1 induction was examined by employing a time-course study, which showed that PCB 77 significantly reduced the hepatic selenium level and GPx1 activity and that this effect was influenced by gender. The next study explored how PCB 77 could deplete hepatic selenium by determining selenium concentrations in different tissues, feces and urine. This study demonstrated that PCB-77 decreased hepatic Se by increased excretion of Se in urine but not in feces. Unlike glutathione peroxidase, thioredoxin reductase activity was not affected by PCB 77. The third study investigated the effect of selenium supplementation on the tumor promoting activity of PCB 77 and 2,2,4,4,5,5-hexaclorobiphenyl (PCB 153) using a 2-stage carcinogenesis model. Se supplementation did not diminish the induction of altered hepatic foci by coplanar PCB 77 or ortho-substituted PCB 153. Instead of protection, the number of foci per cubic centimeter and per liver among the PCB-77 treated rats was increased as the selenium dietary level increased. PCB 153 did not show the same selenium dose-response effect; nevertheless, selenium supplementation did not confer protection against foci development. On the other hand, supranutritional selenium reduced the mean focal volume. Supranutritional selenium or PCBs did not affect cell proliferation or thioredoxin reductase activity. Lastly, the use of the Zeeman graphite furnace atomic absorption spectrometry (GFAAS) method and closed microwave digestion technique for selenium determination of biological samples was compared with the neutron activation analysis and fluorometry methods. I found that GFAAS was not as reliable as the other methods.

La colibactine est une toxine largement distribuée chez Escherichia coli. Sa synthèse est assurée par des enzymes codés par un îlot génomique appelé pks. Elle provoque des cassures double brin de l'ADN, des mutations, d'important remaniements chromosomiques et favorise l'émergence de tumeurs intestinales en modèle murin. Par ailleurs, les E. coli producteurs colonisent fréquemment les tumeurs de patients atteints de cancer colorectal. Nos travaux montrent que les bactéries productrices de colibactine induisent la sénescence cellulaire et stimulent de façon indirect la prolifération cellulaire in vitro et la croissance tumorale in vivo. L'action pro-proliférative des cellules rendues sénescentes par les E. coli producteurs de colibactine est liée à la production du facteur de croissance HGF. L'étude de la signalisation cellulaire responsable montre l'implication du facteur de transcription c-Myc, l'activation de la transcription d'un microARN qui en ciblent la peptidase SENP1, et une modification de la SUMOylation des protéines de l'hôte, notamment p53, un effecteur connu de la sénescence cellulaire. Cette voie de signalisation et les transcripts codant HGF ont été analysés dans des tumeurs de patients atteints de cancer colorectal colonisés ou non par des E. coli producteurs de colibactine. Les résultats obtenus soutiennent les résultats obtenus in vitro et dans le modèle murin. L'ensemble suggère que les bactéries productrices de colibactine favorisent l'émergence d'un micro-environnement tumoral sénescent susceptible de favoriser la croissance tumorale via la sécrétion de HGF. En parallèle, nous avons caractérisé sur le plan structural et fonctionnel la protéine ClbP de l'îlot pks. Les résultats obtenus montrent que ClbP est une peptidase à serine active dont le site actif est extracytoplasmique et indispensable à l'activité biologique de l'îlot pks. Des inhibiteurs « drug-like » de ClbP ont été identifiés à l'aide d'approches structurales, biochimiques, cellulaires et microbiologiques. Ces molécules se lient au site actif de ClbP avec une affinité nanomolaire et bloquent les activités génotoxiques et pro-tumorales des E. coli producteurs de colibactine. ClbP constitue donc une cible thérapeutique potentielle permettant de bloquer les effets délétères des E. coli producteurs de colibactine. / The colibactin toxin is widely distributed in Escherichia coli. Its synthesis is performed by enzymes encoded by the genomic island pks. It causes DNA double-strand breaks, mutations, chromosomal rearrangements in host cells and contributes to tumorigenesis in a mouse model. In addition, colibactin-producing E. coli are frequently isolated from tumors of patients with colorectal cancer. Our work shows that colibactin-producing bacteria induce cellular senescence and, consequently, can indirectly stimulate cell proliferation in vitro and tumor growth in vivo. The pro-proliferative effect mediated by these senescent cells is due to the secretion of growth factors, in particular HGF. The cell signaling responsible for cellular senescence shows the involvement of the transcription factor c-Myc, the transcription of a microRNA targeting the peptidase SENP1, and a modification of protein SUMOylation, including p53, a well-known effector of cellular senescence. This signaling pathway and HGF-encoding transcripts were analyzed in tumors of patients with colorectal cancer colonized or not by colibactin-producing E. coli. The results support the findings obtained in vitro and in the mouse model. Taken together, the results suggest that, in tumors, colibactin-producing bacteria promote the emergence of a senescent microenvironment, which can stimulate tumor growth via the secretion of HGF. In parallel, we determined the structure and function of the pks-encoded protein ClbP. The results show that ClbP is an active serine peptidase, whose active site is extracytoplasmic and essential to the biological activity of pks island. "Drug-like" inhibitors of ClbP were identified using structural, biochemical, cellular and microbiological approaches. These molecules bind to the active site of ClbP with nanomolar affinity and block the genotoxic and pro-tumoral activities of colibactin-producing E. coli. ClbP is therefore a potential therapeutic target to block the deleterious effects of bacteria-producing colibactin.

Polychlorinated biphenyls (PCBs) are ubiquitious lipophilic environmental pollutants. At least some of the PCB congeners and mixtures are hepatic tumor promoters. The mechanisms are not fully understood and might be multifactorial Besides being the most abundant congener in the environment, 2,2,4,4,5,5-hexachlorobiphenyl (PCB-153), has been previously shown to increase hepatocyte proliferation 48h after exposure in rats. The goal of this study was to determine whether hepatic Kupffer cells are important in the promoting activity of PCBs. The hypothesis of this study was that modulation of Kupffer cell activity by PCBs may contribute to PCB-induced liver tumor promotion. The experimental approach consisted on three in vivo models (tumor promotion model and two short term exposure models) and one in vitro model. In the tumor promotion model, glycine inactivation of Kupffer cells did not significantly influence the promoting activity of PCB-77 (3,3,4,4-tetrachlorobiphenyl) or PCB-153. For the short term exposure model, we investigated the effect of Kupffer cell inactivation by glycine and the effect of Kupffer cell depletion on PCB-153s impact on hepatocyte proliferation. The oil used as a vehicle had no significant effect on any of the end points considered. Inhibition of Kupffer cells with glycine or the absence of Kupffer cells did not affect cell proliferation or NF-B activation after PCB treatment compared to the control. In vitro, PCB-153 increased DNA binding activity of NF-B in Kupffer cells but did not significantly increase the TNF- concentration in the medium. In conclusion, PCB-153 increased the number of preneoplastic foci per liver in the casein group but had no significant effect on cell proliferation, and Kupffer cells do not seem to play a role in hepatocyte proliferation.

Die Proteinkinase C (PKC) ist eine wichtige Signaltransduktionskomponente, deren Aktivierung die Expression zahlreicher Gene induziert und zur Zelldifferenzierung und Zellproliferation führt. Ein besonders hohes Expressionsniveau der PKC findet man in vielen Tumoren. So korreliert in malignen Gliazellen das Expressionsniveau der PKC mit deren Wachstumsgeschwindigkeit. Es wird angenommen, daß die aktivierte PKC eine wichtige Rolle in der Tumorpromotion hat. In der vorliegenden Arbeit wurde untersucht, ob in Astrozytomzellinien Gene zu finden sind, die nach PKC-Aktivierung durch den Phorbol-Ester TPA differentiell exprimiert werden. Zunächst wurden kultivierte Zellen der Astrozytomzellinie LN-405 mit TPA respektive dem PKC-Inhibitor Chelerythrin behandelt. Nach Gewinnung der mRNA aus der zuvor isolierten RNA wurden in einem mehrstufigem PCR-Verfahren (SSH) cDNA-Abschnitte gewonnen, die zu vermeintlich differentiell exprimierten Genen gehören. Diese cDNA-Abschnitte wurden in Plasmid-Vektoren eingefügt, kloniert und zur Bestimmung sequenziert. Um falsch positive Sequenzen zu erkennen, wurden die zuvor radioaktiv markierten cDNA-Abschnitte mit Northernblots hybridisiert. Gleichzeitig ließ sich so ein zeitabhängiger Anstieg der Expression nach PKC-Stimulation untersuchen. Durch den PCR-Select-Assay (SSH) konnten insgesamt 11 Gene gefunden werden, die sich in der radioaktiven Northernblot-Hybridisierung, als nach PKC-Aktivierung differentiell exprimiert, darstellen ließen. Dabei bestätigt der gefundene Zusammenhang zwischen PKC-Aktivierung und differentieller Exprimierung bei fünf der 11 Gene (IL-8, Calpain, Interferon-gamma Rezeptor 2, Methionin Adenosyltransferase, beta-2 adrenerger Rezeptor) Ergebnisse anderer Autoren, wobei dieser Zusammenhang nur bei zwei Genen (IL-8 und Calpain) auch in Astrozytom- bzw. Gliom-Zellen schon früher gezeigt werden konnte. Sechs Gene (M-Phase Phosphoprotein-1, ect2-Onkogen, ERM-Gen, Ornithin-Decarboxylase-Antizym 2, MHC-bindendes Protein 2, Sequenz aus Cosmid F0811) wurden in der vorliegenden Arbeit erstmalig als PKC-abhängig exprimiert beschrieben. Die gefundenen Gene haben auf verschiedene Funktionen der Zellen Einfluß. So beeinflussen sie die Regulation des Zellzyklus (MPP1, ect2-Oncogen), die Immunregulation (MBP-2, IL-8, Interferon-gamma Rezeptor 2), die Signaltransduktion (beta-2 AR), die Transkription (ERM-Gen), die Proteinsynthese (ODC-Antizym, MAT), die Wachstumskontrolle (ODC-Antizym) und die Regulation der PKC selbst (Calpain). Für fünf Gene läßt sich ein eindeutiger Zusammenhang mit der Tumorpromotion herstellen: IL-8 (Angioneogenese), MBP-2 (Immunsuppression), ERM-Gen (Transkriptionspromotion), MAT (allgemein fördernder Einfluß auf den Metabolismus) und ect2-Oncogen (Oncogen). / The protein kinase C (PKC) is one of the major signal transduction systems and its activation leads to the induction of the expression of several genes, to cell differentiation and cell proliferation. Very high expressed PKC are found in many tumors. In malignant glia cells the expression of PKC correlates with their proliferation rate. The PKC activity has an important role for the tumor promotion. The object of this paper, was to investigated, if there are genes differentialy expressed after activation of PKC through the phorbol-ester TPA in astrocytoma cell lines. The astrocytoma cell line LN-405 was incubated with TPA and the PKC-inhibitor chelerythrine respectively. After isolation of RNA and mRNA the suppression subtractive hybridization (SSH) was used to isolate differentially expressed cDNA fragments. These cDNA fragments were inserted into the T/A cloning vector, cloned and sequenced. To detect false positives the cDNA fragments were analysed with northern blot technique. Examined was also a time-dependent acceleration of expression after TPA treatment. 11 genes were detected by suppression subtractive hybridization, showing differentially expressed in the northern blot hybridization. Five of the genes were found differentially expressed after PKC activation before (IL-8, calpain, interferon gamma receptor 2, beta-2-adrenergic receptor, methionine adenosyltransferase alpha), two of these genes (IL-8, calpain) also in astrocytoma- and glioma-cells respectively. Six genes (M-phase phosphoprotein 1, ect2-onkogene, erm gene, ornithine decarboxylase antizyme 2, MHC binding protein 2, sequence from Cosmid F0811) were described as PKC dependent expressed for the first time. The genes detected influence several cell functions. They are involved in cell-cycle regulation (MPP1, ect2-oncogene), immuneregulation (MBP-2, IL-8, interferon gamma receptor 2), signal transduction (beta 2 adrenergic receptor), transcription (erm-gene), synthesis of proteins (ODC-antizyme 2, MAT), growth control (ODC-antizyme) and regulation of PKC (Calpain). Five genes show a clear connection to tumor promotion: IL-8 (angioneogenesis), MPB-2 (immunesuppression), erm gene (promotion of transcription), MAT (promotion of metabolism) and ect2-oncogene (oncogene).